Comparative MedicineCopyright 2004by the American Association for Laboratory Animal Science

Vol 54 No 2April 2004

128

OverviewNatural and Experimental Helicobacter Infections

Mark T Whary DVM PhD and James G Fox DVM

Received 21704 Revision requested 21904 Accepted 31804Division of Comparative Medicine Massachusetts Institute of Technology Cam-bridge Massachusetts

Corresponding author M T Whary Division of Comparative Medicine 77Massachusetts Ave Bldg 16-825A Cambridge Massachusetts 02139

Helicobacter pylori was first identified in 1982 (163) as an in-fectious cause of chronic active gastritis and since then has beenassociated with peptic ulcer gastric adenocarcinoma and mu-cosa-associated lymphoid tissue (MALT) lymphoma in humans(162) The Helicobacter genus now includes at least 26 formallynamed species with additional novel species in the process of be-ing characterized (Fig 1) Beyond the impact of H pylori on hu-man health (13) considerable research has focused onHelicobacter spp isolated from humans and animals and the as-sociated natural diseases that range from subclinical inflamma-tion to cancer in the gastrointestinal tract and liver Identificationand characterization of novel Helicobacter spp associated withclinical disease in their natural host have provided opportunitiesto investigate idiopathic disease syndromes through experimen-tal Helicobacter infection of animal models particularly rodentsNatural and experimental Helicobacter infections reproduciblymimic important features of human disease and are yielding newinformation on human health issues such as gastritis and its pro-gression to gastric atrophy and cancer idiopathic hepatitis in-cluding cholecystitis and the development of hepatocellularcarcinoma bacteremia in immunodeficient humans and inflam-matory bowel diseases (IBD) such as Crohnrsquos disease and ulcer-ative colitis (201) This review is an update of a prior publicationon the role of Helicobacter spp in newly recognized gastrointesti-nal tract diseases of animals (85)

The Helicobacter GenusTropism of the genus Helicobacter ranges from the stomach

cecum and colon to the liver or genital tract of mammals andbirds (Table 1) Essentially every species that has been system-atically examined to date has one or more helicobacters in itsgastrointestinal flora Some Helicobacter species have limitedhost range (ie H pylori in humans and nonhuman primates)whereas others such as H cinaedi can infect humans but arefound more commonly in various animal reservoirs including

Current information about Helicobacter infections in humans and various domestic wild and research animalspecies that have been used or have the potential to be used as animal models of human disease is presented TheHelicobacter genus now includes at least 26 formally named species with additional novel species in the process ofbeing characterized The natural history host range with zoonosis potential pathology and diagnostic techniques arepresented along with examples of how Helicobacter infection has interfered with unrelated in vivo research Currentrecommendations for deriving and managing helicobacter-free animal colonies for research are provided

hamsters nonhuman primates dogs cats and foxes (85) Allknown gastric and enterohepatic Helicobacter infections appearto persist for the life of the host Chronic infection is commonlyassociated with subclinical disease in immunocompetent hostsSignificant innate humoral and cell-mediated immune re-sponses appear unable to clear infection and paradoxically con-tribute to lesions particularly in immunologically dysregulatedhosts Helicobacter infections in animals and their zoonotic po-tential remain to be clarified For example endemic H pyloriinfection in a colony of purpose-bred research cats is thought tohave originated with a human caretaker (117 118) otherwisethe risk of H pylori transmission from cats and dogs to humansappears minimal Helicobacters of the same species of gastricspirals (ie lsquoH heilmanniirsquo) that have colonized humans (45244 250) have also colonized their dogs or cats people that workon farms with livestock may also be infected with lsquoHheilmanniirsquo Other less well-characterized gastric spiral organ-isms potentially could infect humans Intestinal Helicobacterspecies (H canis H cinaedi H pullorum H canadensis and lsquoHrappinirsquo) have been isolated from immunosuppressed and im-munocompetent patients with septicemia enteritis and procti-tis as well as from animal reservoirs (71 85) Helicobacter bilisisolated from dogs cats mice gerbils and rats has been identi-fied in human gallbladder and hepatobiliary cancer by use ofmolecular techniques (79 165) Except for H pylori evidenceimplicating Helicobacter spp as a cause of human disease islimited but growing Many investigators have based their studyconclusions on molecular techniques because helicobacters aretechnically difficult to culture Thus there may be more frequentcauses of Helicobacter-associated human disease than is cur-rently appreciated

The International Committee of Systematic Bacteriology Sub-committee on the Taxonomy of Campylobacter and Related Bac-teria has established minimum requirements for the formaldescription of new species of the genus Helicobacter (41) Formalnaming of a putative new species or subspecies must be based onphenotypic and genotypic examination of at least five strains tocharacterize the range of potential variation among independentisolates The Helicobacter genus has grown to include 26 formallynamed species (Fig 1 Table 1) and includes a diverse group of

Pages 128-158

129

potentially emerging pathogens in multiple speciesmdashhumansnonhuman primates cats dogs ferrets swine sheep cattlebirds mice rats hamsters gerbils and a variety of wildlife suchas exotic cats foxes woodchucks and even dolphins whales andseals Helicobacters that have been provisionally named such asH bovis are listed as lsquoH bovisrsquo throughout this text

Helicobacter spp are gram-negative bacteria that are speciatedby morphology (curved to spiral to fusiform in shape variation inflagella and presence or absence of periplasmic fibers) growthrequirements (microaerobic to anaerobic optimal growth at 37andor 42degC) biochemical profiles (such as oxidase catalase andurease production) antibiotic susceptibility and sequencing ofconserved 16S rRNA (Table 2) Many of these characteristic fea-tures are shared in common with Campylobacter spp and mayexplain early misidentification of H pylori as C pyloridis (164)Despite availability of advanced molecular techniques compari-son of near-complete 16S ribosomal DNA sequences does not al-ways provide conclusive evidence for species level identificationand can be misleading (249) For example Helicobacternemestrinae (ATCC 49396T) believed to be a novel gastrichelicobacter isolated from a pigtailed macaque was later recog-nized to be a strain of H pylori by use of sequence analysis of 7housekeeping and 2 flagellin genes that clustered together withsequences from 20 or more H pylori isolates from diverse sources(235) Data also indicated that lsquoH westmeadiirsquo is a strain of Hcinaedi and that Helicobacter sp strain Mainz also should be spe-ciated as H cinaedi (249) In addition to 16S rRNA sequencinghybridization experiments between DNAs from strains of related

taxa of Campylobacter Wolinella and Helicobacter spp and 23SrRNAs from reference strains have been used to revise taxonomicclassification of these bacteria (248) Others have recommendedthat 16S rDNA sequencing is preferable to 23S rDNA analysis(144) Additional identifying information can be gained fromwhole-cell protein electrophoresis fatty acid analysis plasmidprofile determination mass spectrometry DNA-DNA hybridiza-tion restriction fragment length polymorphism (RFLP) analysisand random amplified polymorphic DNA (RAPD) fingerprintinganalysis (36 139 177)

Helicobacter spp have co-evolved with their hosts and othercommensal microbiota of the gastrointestinal tract to reach ho-meostatic equilibrium that minimizes the risk of clinical diseasein most of their natural hosts Dependent on the host range andtissue tropism of individual Helicobacter sp clinical disease de-velops not only in immunocompromised humans and animalsbut also in a subset of immunocompetent individuals Risk fac-tors for clinical disease in otherwise clinically normal hosts pre-sumably include environmental factors and host-pathogengenetic polymorphisms Even within the grouping of gastrichelicobacters the genetic diversity of individual strains rangesfrom high in H pylori (4 13) to low in strains of H mustelae(241) This diversity reflects the adaptation of the helicobacter toits natural tissue tropism within a host and potentially to othermicrobiota particularly in the case of enterohepatichelicobacters a topic of great research interest The importanceof genetic and environmental predisposition is well illustratedby the concurrent increase in use of genetically modified mouse

Figure 1 Dendogram demonstrating genetic relationship among Helicobacter species

Natural and experimental Helicobacter infections

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130

models and associated diseases that have been attributed tonatural Helicobacter infection

Rodent Helicobacter InfectionsOverview Helicobacter spp colonize the cecum and colon of

mice and other rodents and have been associated with liver dis-ease in select strains of mice and rats Naturally acquired infec-tions are persistent with long-term shedding of the organism infeces Endemic infections are common unless specific steps aretaken to prevent introduction and dissemination Helicobacter-associated disease is dependent on interaction among host fac-tors of age sex genetics and immune competence and bacterialvirulence factors that either are known or are suspected to influ-ence tissue tropism and host immune responses Naturally ac-quired Helicobacter infections have been identified in all of thecommonly used laboratory animal rodent species (mice rats ger-bils hamsters) as well as in wild rodents Helicobacter hepaticusan enterohepatic helicobacter in mice was the first murinehelicobacter discovered as a confounding factor in long-term car-cinogenesis studies (80 115 256) and is known to causetyphlocolitis (Table 3) and hepatocellular adenomas and carcino-mas in susceptible mouse strains (Table 4) Helicobacterhepaticus and the gastric helicobacters that experimentally colo-

nize mice (H felis H pylori ) and gerbils (H pylori) are examplesof Helicobacter-associated disease models that are strengtheningthe link between chronic infections the associated tissue damagefrom chronic inflammation and the progression of dysplastic le-sions to cancer in the liver and gastrointestinal tract (Table 4)

Natural Helicobacter infections in mice Helicobacterhepaticus a bacterium with a spiral shape and bipolar singlesheathed flagella was isolated from the liver of mice with chronicactive hepatitis (80) The bacteria also colonized the cecal and co-lonic mucosae of mice (Fig 2) The bacterium grows at 37degC un-der microaerobic and anaerobic conditions rapidly hydrolyzesurea is catalase and oxidase positive reduces nitrate to nitriteand is resistant to cephalothin and metronidazole (80)Helicobacter hepaticus was the first enterohepatic helicobacteridentified in mice that was associated with chronic active hepati-tis in susceptible mouse strains including AJCr BALBCAnNCrSJLNCr B6C3F1 SCIDNCr and C3HHeNCr mice (80 256)The AJCr mouse an immunocompetent strain and SCIDNCran immunodeficient strain appear to be among the most hepati-tis-prone strains identified to date with liver lesions that becomeprogressively more severe with age (87 97 152) Helicobacterhepaticus-associated hepatitis and progression to hepatocellularcarcinoma in AJCr mice is most prevalent after postinfection

Table 1 Host range and tissue tropism of Helicobacter species

Helicobacter taxon Source(s) Primary site Secondary site

H acinonychis Cheetah tiger StomachH aurati Hamster Stomach intestineH bilis Mouse dog rat cat gerbil human Intestine Liver (mouse) gallbladder (human)H bizzozeroniia Human cat dog cheetah nonhuman primate wild rat StomachlsquoH bovisrsquo Cattle AbomasumH canadensis Human wild geese IntestineH canis Human cat dog Intestine Liver (dog)H cetorum Dolphin whale Stomach H cholecystus Hamster Gallbladder H cinaedi Human hamster macaque dog Intestine Blood brain joint (human) liver (macaque)H felis Cat dog human StomachH fennelliae Human IntestineH ganmani Mouse Intestine H hepaticus Mouse gerbil IntestineH marmotae Woodchuck cat Intestine Liver (woodchuck)H mesocricetorum Hamster Intestine lsquoH muricolarsquo Korean wild mice IntestineH muridarum Mouse rat Intestine Stomach (mouse)H mustelae Ferret mink StomachH pametensis Wild birds swine IntestineH pullorum Human chicken Intestine Liver (chicken)H pylori Human nonhuman primate cat StomachlsquoH rappinirsquob Human cat dog mouse sheep swine Intestine Placentafetusliver (sheep) blood (humans)H rodentium Mouse rat IntestineH salomonis Dog StomachlsquoH suisrsquo Swine Stomach lsquoH suncusrsquo House musk shrew StomachH trogontum Rat IntestineH typhlonius Mouse rat Intestine H winghamensis Human Intestine

Unnamed novel helicobacters Source Primary site Reference GenBank or MIT accessions

AF333338 AF333339 Rhesus macaque Intestine (84)AF107494 Cotton-top tamarin Intestine (207)MIT 97-6535 Ferret Liver (104)MIT 266-1 Ferret Intestine (90)AF336947 Cat Intestine (211)MIT 01-5926 Sea otter Intestine Unpublished (Fox)MIT 03-5765 Sea lion Intestine Unpublished (Fox)AFAY203898 AFAY203899 Harp seal Stomach (124)AF320621 Mouse Intestine liver (217) MIT 94-022 Mouse Intestine Unpublished (Fox)

aLikely the same as lsquoH heilmanniirsquo lsquoH heilmanniirsquo (formerly Gastrospirillum hominis) has the same phenotype as listed here for H bizzozeronii Only a single lsquoHheilmanniirsquo strain has been isolated by use of culture bFormerly regarded as lsquoFlexispira rappinirsquo now subgrouped into ten taxa

131

month 18 and for unknown reasons males are predisposed tomore severe lesions (87)

Mice of the SCIDNCr strain that were naturally infected withH hepaticus developed hepatitis proliferative typhlitis and coli-tis (152) Lesions in the liver of SCID mice consisted of Kupffer

Ito and oval cell hyperplasia along with multifocal to coalescingcoagulative hepatocyte necrosis Numerous Warthin-Starry-posi-tive bacteria were observed in the parenchyma and there wereminimal to mild accumulations of monocytic cells and neutro-phils Proliferative typhlitis was characterized by moderate to

Table 2 Biochemical profile growth characteristics and flagellar morphology of Helicobacter species

Helicobacter Catalase Nitrate Alkaline Urease Indoxyl acetate γ-Glutamyl Growth Resistance tob Flagellataxon production reduction phosphatase hydrolysis transferase At 42degC With 1 Nalidixic acid Cephalothin

glycine

H acinonychis + - + + - + - - R S BipolarH aurati + - - + + + + - S R BipolarH bilis + + - + - + + + R R BipolarH bizzozeroniic + + + + + + + - R S BipolarlsquoH bovisrsquo ND ND ND + ND ND ND ND ND ND NDH canadensis + +- - - + - + + R R MonoBipolarH canis - - + - + + + - S I BipolarH cetorum + - - + - + + ND I S BipolarH cholecystus + + + - - - + + I R MonopolarH cinaedi + + - - - - - + S I BipolarH felis + + + + - + + - R S BipolarH fennelliae + - + - + - - + S S BipolarH ganmani + + - - - ND - - R S BipolarH hepaticus + + - + + - - + R R BipolarH marmotae + - + + - - - + R R BipolarH mesocricetorum + + + - ND - + - S R BipolarH muridarum + - + + + + - - R R BipolarH mustelae + + + + + + + - S R PeritrichousH pametensis + + + - - - + + S S BipolarH pullorum + + - - - ND + - R S MonopolarH pylori + - + + - + - - R S MonopolarlsquoH rappinirsquo +- - - + - + + - R R BipolarH rodentium + + - - - - + + R R BipolarH salomonis + + + + + + - ND R S BipolarlsquoH suisrsquo ND ND ND ND ND ND ND ND ND ND BipolarlsquoH suncusrsquo + + + + ND ND - ND R R BipolarH trogontum + + - + - + + ND R R BipolarH winghamensis - - - - + ND - + R R Bipolar

ND = not determined R = resistant S = susceptible I = indeterminate

Table 4 Summary of key rodent models of Helicobacter gastrointestinal and liver cancera

Rodent Infective agent transgene Tumor Comment

C57BL6 mice H felis Gastric adenocarcinoma Natural feline pathogen but lacks vacA and PAI

INS-GAS FVB mice H felis and H pylori Gastric adenocarcinoma Constitutive hypergastrinemia promotes tumorigenesis

Mongolian gerbil H pylori Gastric adenocarcinoma Closely mimics human disease long time course and few reagents

BALBc mice Several Helicobacter spp Gastric MALT lymphoma Usually requires 18-24 months

Genetically engineered mice ldquoEndogenous microbiotardquo Large intestinal carcinoma Bacteria in endogenous microbiota models IL10-- mice especially on 129Sv or H hepaticus (cecum plusmn colon) not well defined H hepaticus reliably induces disease

background

Lymphocyte-deficient mice H hepaticus Large intestinal carcinoma Often used for adoptive transfer studies H hepaticus inducesSCID or Rag-- especially on (cecum plusmn colon) tumors in untreated Rag2-- mice

129Sv background

AJCr and other mice H hepaticus Hepatocellular carcinoma Natural murine pathogen induces chronic active hepatitis and HCC

aTable adapted from Rogers and Fox (201)MALT = Mucosa-associated lymphoid tissue HCC = hepatocellular carcinoma

Table 3 Helicobacter-associated typhlocolitis in mice

Genetic status of mice Type of defect Pathology

CD45RBhigh-reconstituted scids Reconstitution with naiumlve CD4+T cell TyphlocolitisTCR α--β -- defined flora Abnormal T cell receptors TyphlocolitisScid ICR defined flora Lack T and B cells TyphlocolitisIL-10-- Lack IL-10 TyphlocolitisRAG2-- Lack T and B cells Typhlocolitis colon cancerIL-7--RAG2-- Lack IL-7 T and B cells NoneAJCr Normal Typhlitis Swiss Webster gnotobiotic Normal EnterocolitisNF-κB (p50-- p65+-) Lack NF-κB Typhlocolitismdr1a-- Lack P-glycoprotein Typhlocolitis

IL = Interleukin

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

132

marked mucosal epithelial cell hyperplasia with mild monocyticand neutrophilic infiltration In contrast infected immunocompe-tent AJCr mice which develop a substantial immune response toH hepaticus associated with prominent multifocal mononuclearcell infiltrates in the liver (262) typically have low numbers of Hhepaticus observable at the periphery of foci of inflammation andcolonizing the biliary canaliculi (Fig 3) Therefore the successrate for recovery of H hepaticus in culture from infected AJCrmice is high when the cecum and colon are sampled but is lowfrom liver specimens

Helicobacter hepaticus infection in AJCr mice has been charac-terized on a longitudinal basis through 18 months of age (87)Helicobacter hepaticus colonization persisted in the large intes-tine and liver and was associated with chronic proliferative hepa-titis and liver cancer and in some mice chronic typhlitis (87)Infected AJCr mice developed sustained serum IgG antibody re-sponses to H hepaticus and high serum enzyme activities indica-tive of hepatocellular injury To fulfill Kochrsquos postulatescolonization of germfree mice by H hepaticus was induced andthe mice went on to develop chronic hepatitis some mice devel-oped enterocolitis (97)

A clinical syndrome of typhlocolitis with and without rectalprolapse or concomitant hepatitis has been described in a varietyof inbred strains of immunocompromised mice infected with Hhepaticus (Table 3) (66 254) In immune dysregulated micemononuclear inflammatory cells infiltrate the mucosa and sub-mucosa of the cecum and colon Epithelial cell hyperplasia can besubstantial with frequent villous to papillomatous folds extend-ing into the lumen and a high mitotic index in the crypts Num-bers of goblet cells are variably decreased in the proliferativemucosa Helicobacter hepaticus infection has been associatedwith development of large intestinal adenocarcinoma in RAG2--

mice on a 129SvEv background (59)

Diagnostic Methods for H hepaticusCulture Diagnostic specimens (feces cecal scrapings tissues)

should be stored at ndash70degC in brain heart infusion or Brucellabroth containing 30 glycerol For culture from the gastrointesti-nal tract specimens are homogenized in 1 ml of phosphate-buff-ered saline (PBS) and are filtered through a 045-microm filter toeliminate competitive commensals For larger helicobacters suchas H bilis or H trogontum a 065-microm filter is used to aid in iso-lation Filtrate from gastrointestinal tract specimens is culturedon blood agar supplemented with 1 trimethoprim vancomycinand polymyxin and specimens from the liver are plated directlyonto blood agar Plates are incubated at 37degC (or 42degC for otherHelicobacter spp) under microaerobic conditions in vented jars(90 N2 5 H2 and 5 CO2 alternatively a 801010 mixture ofthe same gases can be used) for 3 to 7 days Plates should be keptfor 21 days before concluding there is no growth Growth of Hhepaticus occurs as a mucoid film or lawn but not in distinct colo-nies on plates Bacterial numbers can be expanded by inocula-tion into Brucella broth containing 5 fetal bovine serum andincubation for 24 to 48 h on a rotary shaker Many Helicobacterspecies require moist agar for efficient growth

Figure 2 Electron micrograph of H hepaticus Notice the spiral mor-phology as well as the single bipolar flagella (A) in the colon crypt (B)and within the biliary canaliculi of the liver (C) Bars = 05 microm Repro-duced with permission from Dr Schauer and Dr Solnick (231)

Figure 3 (A) Photomicrograph of a section of the liver from an AJmouse infected with H hepaticus Notice characteristic lesions associ-ated with chronic active hepatitis including cholangiohepatitis (upperleft white arrow) interface hepatitis (lower right black arrow) hepato-cellular dysplasia (black arrowhead) and lobular inflammation (cen-tral upper white arrow) HampE stain magnification 20times (B) Inset depictsWarthin-Starry-positive organisms with morphology consistent with Hhepaticus in the liver Magnification 100times

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

129

potentially emerging pathogens in multiple speciesmdashhumansnonhuman primates cats dogs ferrets swine sheep cattlebirds mice rats hamsters gerbils and a variety of wildlife suchas exotic cats foxes woodchucks and even dolphins whales andseals Helicobacters that have been provisionally named such asH bovis are listed as lsquoH bovisrsquo throughout this text

Helicobacter spp are gram-negative bacteria that are speciatedby morphology (curved to spiral to fusiform in shape variation inflagella and presence or absence of periplasmic fibers) growthrequirements (microaerobic to anaerobic optimal growth at 37andor 42degC) biochemical profiles (such as oxidase catalase andurease production) antibiotic susceptibility and sequencing ofconserved 16S rRNA (Table 2) Many of these characteristic fea-tures are shared in common with Campylobacter spp and mayexplain early misidentification of H pylori as C pyloridis (164)Despite availability of advanced molecular techniques compari-son of near-complete 16S ribosomal DNA sequences does not al-ways provide conclusive evidence for species level identificationand can be misleading (249) For example Helicobacternemestrinae (ATCC 49396T) believed to be a novel gastrichelicobacter isolated from a pigtailed macaque was later recog-nized to be a strain of H pylori by use of sequence analysis of 7housekeeping and 2 flagellin genes that clustered together withsequences from 20 or more H pylori isolates from diverse sources(235) Data also indicated that lsquoH westmeadiirsquo is a strain of Hcinaedi and that Helicobacter sp strain Mainz also should be spe-ciated as H cinaedi (249) In addition to 16S rRNA sequencinghybridization experiments between DNAs from strains of related

taxa of Campylobacter Wolinella and Helicobacter spp and 23SrRNAs from reference strains have been used to revise taxonomicclassification of these bacteria (248) Others have recommendedthat 16S rDNA sequencing is preferable to 23S rDNA analysis(144) Additional identifying information can be gained fromwhole-cell protein electrophoresis fatty acid analysis plasmidprofile determination mass spectrometry DNA-DNA hybridiza-tion restriction fragment length polymorphism (RFLP) analysisand random amplified polymorphic DNA (RAPD) fingerprintinganalysis (36 139 177)

Helicobacter spp have co-evolved with their hosts and othercommensal microbiota of the gastrointestinal tract to reach ho-meostatic equilibrium that minimizes the risk of clinical diseasein most of their natural hosts Dependent on the host range andtissue tropism of individual Helicobacter sp clinical disease de-velops not only in immunocompromised humans and animalsbut also in a subset of immunocompetent individuals Risk fac-tors for clinical disease in otherwise clinically normal hosts pre-sumably include environmental factors and host-pathogengenetic polymorphisms Even within the grouping of gastrichelicobacters the genetic diversity of individual strains rangesfrom high in H pylori (4 13) to low in strains of H mustelae(241) This diversity reflects the adaptation of the helicobacter toits natural tissue tropism within a host and potentially to othermicrobiota particularly in the case of enterohepatichelicobacters a topic of great research interest The importanceof genetic and environmental predisposition is well illustratedby the concurrent increase in use of genetically modified mouse

Figure 1 Dendogram demonstrating genetic relationship among Helicobacter species

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

130

models and associated diseases that have been attributed tonatural Helicobacter infection

Rodent Helicobacter InfectionsOverview Helicobacter spp colonize the cecum and colon of

mice and other rodents and have been associated with liver dis-ease in select strains of mice and rats Naturally acquired infec-tions are persistent with long-term shedding of the organism infeces Endemic infections are common unless specific steps aretaken to prevent introduction and dissemination Helicobacter-associated disease is dependent on interaction among host fac-tors of age sex genetics and immune competence and bacterialvirulence factors that either are known or are suspected to influ-ence tissue tropism and host immune responses Naturally ac-quired Helicobacter infections have been identified in all of thecommonly used laboratory animal rodent species (mice rats ger-bils hamsters) as well as in wild rodents Helicobacter hepaticusan enterohepatic helicobacter in mice was the first murinehelicobacter discovered as a confounding factor in long-term car-cinogenesis studies (80 115 256) and is known to causetyphlocolitis (Table 3) and hepatocellular adenomas and carcino-mas in susceptible mouse strains (Table 4) Helicobacterhepaticus and the gastric helicobacters that experimentally colo-

nize mice (H felis H pylori ) and gerbils (H pylori) are examplesof Helicobacter-associated disease models that are strengtheningthe link between chronic infections the associated tissue damagefrom chronic inflammation and the progression of dysplastic le-sions to cancer in the liver and gastrointestinal tract (Table 4)

Natural Helicobacter infections in mice Helicobacterhepaticus a bacterium with a spiral shape and bipolar singlesheathed flagella was isolated from the liver of mice with chronicactive hepatitis (80) The bacteria also colonized the cecal and co-lonic mucosae of mice (Fig 2) The bacterium grows at 37degC un-der microaerobic and anaerobic conditions rapidly hydrolyzesurea is catalase and oxidase positive reduces nitrate to nitriteand is resistant to cephalothin and metronidazole (80)Helicobacter hepaticus was the first enterohepatic helicobacteridentified in mice that was associated with chronic active hepati-tis in susceptible mouse strains including AJCr BALBCAnNCrSJLNCr B6C3F1 SCIDNCr and C3HHeNCr mice (80 256)The AJCr mouse an immunocompetent strain and SCIDNCran immunodeficient strain appear to be among the most hepati-tis-prone strains identified to date with liver lesions that becomeprogressively more severe with age (87 97 152) Helicobacterhepaticus-associated hepatitis and progression to hepatocellularcarcinoma in AJCr mice is most prevalent after postinfection

Table 1 Host range and tissue tropism of Helicobacter species

Helicobacter taxon Source(s) Primary site Secondary site

H acinonychis Cheetah tiger StomachH aurati Hamster Stomach intestineH bilis Mouse dog rat cat gerbil human Intestine Liver (mouse) gallbladder (human)H bizzozeroniia Human cat dog cheetah nonhuman primate wild rat StomachlsquoH bovisrsquo Cattle AbomasumH canadensis Human wild geese IntestineH canis Human cat dog Intestine Liver (dog)H cetorum Dolphin whale Stomach H cholecystus Hamster Gallbladder H cinaedi Human hamster macaque dog Intestine Blood brain joint (human) liver (macaque)H felis Cat dog human StomachH fennelliae Human IntestineH ganmani Mouse Intestine H hepaticus Mouse gerbil IntestineH marmotae Woodchuck cat Intestine Liver (woodchuck)H mesocricetorum Hamster Intestine lsquoH muricolarsquo Korean wild mice IntestineH muridarum Mouse rat Intestine Stomach (mouse)H mustelae Ferret mink StomachH pametensis Wild birds swine IntestineH pullorum Human chicken Intestine Liver (chicken)H pylori Human nonhuman primate cat StomachlsquoH rappinirsquob Human cat dog mouse sheep swine Intestine Placentafetusliver (sheep) blood (humans)H rodentium Mouse rat IntestineH salomonis Dog StomachlsquoH suisrsquo Swine Stomach lsquoH suncusrsquo House musk shrew StomachH trogontum Rat IntestineH typhlonius Mouse rat Intestine H winghamensis Human Intestine

Unnamed novel helicobacters Source Primary site Reference GenBank or MIT accessions

AF333338 AF333339 Rhesus macaque Intestine (84)AF107494 Cotton-top tamarin Intestine (207)MIT 97-6535 Ferret Liver (104)MIT 266-1 Ferret Intestine (90)AF336947 Cat Intestine (211)MIT 01-5926 Sea otter Intestine Unpublished (Fox)MIT 03-5765 Sea lion Intestine Unpublished (Fox)AFAY203898 AFAY203899 Harp seal Stomach (124)AF320621 Mouse Intestine liver (217) MIT 94-022 Mouse Intestine Unpublished (Fox)

aLikely the same as lsquoH heilmanniirsquo lsquoH heilmanniirsquo (formerly Gastrospirillum hominis) has the same phenotype as listed here for H bizzozeronii Only a single lsquoHheilmanniirsquo strain has been isolated by use of culture bFormerly regarded as lsquoFlexispira rappinirsquo now subgrouped into ten taxa

131

month 18 and for unknown reasons males are predisposed tomore severe lesions (87)

Mice of the SCIDNCr strain that were naturally infected withH hepaticus developed hepatitis proliferative typhlitis and coli-tis (152) Lesions in the liver of SCID mice consisted of Kupffer

Ito and oval cell hyperplasia along with multifocal to coalescingcoagulative hepatocyte necrosis Numerous Warthin-Starry-posi-tive bacteria were observed in the parenchyma and there wereminimal to mild accumulations of monocytic cells and neutro-phils Proliferative typhlitis was characterized by moderate to

Table 2 Biochemical profile growth characteristics and flagellar morphology of Helicobacter species

Helicobacter Catalase Nitrate Alkaline Urease Indoxyl acetate γ-Glutamyl Growth Resistance tob Flagellataxon production reduction phosphatase hydrolysis transferase At 42degC With 1 Nalidixic acid Cephalothin

glycine

H acinonychis + - + + - + - - R S BipolarH aurati + - - + + + + - S R BipolarH bilis + + - + - + + + R R BipolarH bizzozeroniic + + + + + + + - R S BipolarlsquoH bovisrsquo ND ND ND + ND ND ND ND ND ND NDH canadensis + +- - - + - + + R R MonoBipolarH canis - - + - + + + - S I BipolarH cetorum + - - + - + + ND I S BipolarH cholecystus + + + - - - + + I R MonopolarH cinaedi + + - - - - - + S I BipolarH felis + + + + - + + - R S BipolarH fennelliae + - + - + - - + S S BipolarH ganmani + + - - - ND - - R S BipolarH hepaticus + + - + + - - + R R BipolarH marmotae + - + + - - - + R R BipolarH mesocricetorum + + + - ND - + - S R BipolarH muridarum + - + + + + - - R R BipolarH mustelae + + + + + + + - S R PeritrichousH pametensis + + + - - - + + S S BipolarH pullorum + + - - - ND + - R S MonopolarH pylori + - + + - + - - R S MonopolarlsquoH rappinirsquo +- - - + - + + - R R BipolarH rodentium + + - - - - + + R R BipolarH salomonis + + + + + + - ND R S BipolarlsquoH suisrsquo ND ND ND ND ND ND ND ND ND ND BipolarlsquoH suncusrsquo + + + + ND ND - ND R R BipolarH trogontum + + - + - + + ND R R BipolarH winghamensis - - - - + ND - + R R Bipolar

ND = not determined R = resistant S = susceptible I = indeterminate

Table 4 Summary of key rodent models of Helicobacter gastrointestinal and liver cancera

Rodent Infective agent transgene Tumor Comment

C57BL6 mice H felis Gastric adenocarcinoma Natural feline pathogen but lacks vacA and PAI

INS-GAS FVB mice H felis and H pylori Gastric adenocarcinoma Constitutive hypergastrinemia promotes tumorigenesis

Mongolian gerbil H pylori Gastric adenocarcinoma Closely mimics human disease long time course and few reagents

BALBc mice Several Helicobacter spp Gastric MALT lymphoma Usually requires 18-24 months

Genetically engineered mice ldquoEndogenous microbiotardquo Large intestinal carcinoma Bacteria in endogenous microbiota models IL10-- mice especially on 129Sv or H hepaticus (cecum plusmn colon) not well defined H hepaticus reliably induces disease

background

Lymphocyte-deficient mice H hepaticus Large intestinal carcinoma Often used for adoptive transfer studies H hepaticus inducesSCID or Rag-- especially on (cecum plusmn colon) tumors in untreated Rag2-- mice

129Sv background

AJCr and other mice H hepaticus Hepatocellular carcinoma Natural murine pathogen induces chronic active hepatitis and HCC

aTable adapted from Rogers and Fox (201)MALT = Mucosa-associated lymphoid tissue HCC = hepatocellular carcinoma

Table 3 Helicobacter-associated typhlocolitis in mice

Genetic status of mice Type of defect Pathology

CD45RBhigh-reconstituted scids Reconstitution with naiumlve CD4+T cell TyphlocolitisTCR α--β -- defined flora Abnormal T cell receptors TyphlocolitisScid ICR defined flora Lack T and B cells TyphlocolitisIL-10-- Lack IL-10 TyphlocolitisRAG2-- Lack T and B cells Typhlocolitis colon cancerIL-7--RAG2-- Lack IL-7 T and B cells NoneAJCr Normal Typhlitis Swiss Webster gnotobiotic Normal EnterocolitisNF-κB (p50-- p65+-) Lack NF-κB Typhlocolitismdr1a-- Lack P-glycoprotein Typhlocolitis

IL = Interleukin

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

132

marked mucosal epithelial cell hyperplasia with mild monocyticand neutrophilic infiltration In contrast infected immunocompe-tent AJCr mice which develop a substantial immune response toH hepaticus associated with prominent multifocal mononuclearcell infiltrates in the liver (262) typically have low numbers of Hhepaticus observable at the periphery of foci of inflammation andcolonizing the biliary canaliculi (Fig 3) Therefore the successrate for recovery of H hepaticus in culture from infected AJCrmice is high when the cecum and colon are sampled but is lowfrom liver specimens

Helicobacter hepaticus infection in AJCr mice has been charac-terized on a longitudinal basis through 18 months of age (87)Helicobacter hepaticus colonization persisted in the large intes-tine and liver and was associated with chronic proliferative hepa-titis and liver cancer and in some mice chronic typhlitis (87)Infected AJCr mice developed sustained serum IgG antibody re-sponses to H hepaticus and high serum enzyme activities indica-tive of hepatocellular injury To fulfill Kochrsquos postulatescolonization of germfree mice by H hepaticus was induced andthe mice went on to develop chronic hepatitis some mice devel-oped enterocolitis (97)

A clinical syndrome of typhlocolitis with and without rectalprolapse or concomitant hepatitis has been described in a varietyof inbred strains of immunocompromised mice infected with Hhepaticus (Table 3) (66 254) In immune dysregulated micemononuclear inflammatory cells infiltrate the mucosa and sub-mucosa of the cecum and colon Epithelial cell hyperplasia can besubstantial with frequent villous to papillomatous folds extend-ing into the lumen and a high mitotic index in the crypts Num-bers of goblet cells are variably decreased in the proliferativemucosa Helicobacter hepaticus infection has been associatedwith development of large intestinal adenocarcinoma in RAG2--

mice on a 129SvEv background (59)

Diagnostic Methods for H hepaticusCulture Diagnostic specimens (feces cecal scrapings tissues)

should be stored at ndash70degC in brain heart infusion or Brucellabroth containing 30 glycerol For culture from the gastrointesti-nal tract specimens are homogenized in 1 ml of phosphate-buff-ered saline (PBS) and are filtered through a 045-microm filter toeliminate competitive commensals For larger helicobacters suchas H bilis or H trogontum a 065-microm filter is used to aid in iso-lation Filtrate from gastrointestinal tract specimens is culturedon blood agar supplemented with 1 trimethoprim vancomycinand polymyxin and specimens from the liver are plated directlyonto blood agar Plates are incubated at 37degC (or 42degC for otherHelicobacter spp) under microaerobic conditions in vented jars(90 N2 5 H2 and 5 CO2 alternatively a 801010 mixture ofthe same gases can be used) for 3 to 7 days Plates should be keptfor 21 days before concluding there is no growth Growth of Hhepaticus occurs as a mucoid film or lawn but not in distinct colo-nies on plates Bacterial numbers can be expanded by inocula-tion into Brucella broth containing 5 fetal bovine serum andincubation for 24 to 48 h on a rotary shaker Many Helicobacterspecies require moist agar for efficient growth

Figure 2 Electron micrograph of H hepaticus Notice the spiral mor-phology as well as the single bipolar flagella (A) in the colon crypt (B)and within the biliary canaliculi of the liver (C) Bars = 05 microm Repro-duced with permission from Dr Schauer and Dr Solnick (231)

Figure 3 (A) Photomicrograph of a section of the liver from an AJmouse infected with H hepaticus Notice characteristic lesions associ-ated with chronic active hepatitis including cholangiohepatitis (upperleft white arrow) interface hepatitis (lower right black arrow) hepato-cellular dysplasia (black arrowhead) and lobular inflammation (cen-tral upper white arrow) HampE stain magnification 20times (B) Inset depictsWarthin-Starry-positive organisms with morphology consistent with Hhepaticus in the liver Magnification 100times

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

Vol 54 No 2Comparative MedicineApril 2004

130

models and associated diseases that have been attributed tonatural Helicobacter infection

Rodent Helicobacter InfectionsOverview Helicobacter spp colonize the cecum and colon of

mice and other rodents and have been associated with liver dis-ease in select strains of mice and rats Naturally acquired infec-tions are persistent with long-term shedding of the organism infeces Endemic infections are common unless specific steps aretaken to prevent introduction and dissemination Helicobacter-associated disease is dependent on interaction among host fac-tors of age sex genetics and immune competence and bacterialvirulence factors that either are known or are suspected to influ-ence tissue tropism and host immune responses Naturally ac-quired Helicobacter infections have been identified in all of thecommonly used laboratory animal rodent species (mice rats ger-bils hamsters) as well as in wild rodents Helicobacter hepaticusan enterohepatic helicobacter in mice was the first murinehelicobacter discovered as a confounding factor in long-term car-cinogenesis studies (80 115 256) and is known to causetyphlocolitis (Table 3) and hepatocellular adenomas and carcino-mas in susceptible mouse strains (Table 4) Helicobacterhepaticus and the gastric helicobacters that experimentally colo-

nize mice (H felis H pylori ) and gerbils (H pylori) are examplesof Helicobacter-associated disease models that are strengtheningthe link between chronic infections the associated tissue damagefrom chronic inflammation and the progression of dysplastic le-sions to cancer in the liver and gastrointestinal tract (Table 4)

Natural Helicobacter infections in mice Helicobacterhepaticus a bacterium with a spiral shape and bipolar singlesheathed flagella was isolated from the liver of mice with chronicactive hepatitis (80) The bacteria also colonized the cecal and co-lonic mucosae of mice (Fig 2) The bacterium grows at 37degC un-der microaerobic and anaerobic conditions rapidly hydrolyzesurea is catalase and oxidase positive reduces nitrate to nitriteand is resistant to cephalothin and metronidazole (80)Helicobacter hepaticus was the first enterohepatic helicobacteridentified in mice that was associated with chronic active hepati-tis in susceptible mouse strains including AJCr BALBCAnNCrSJLNCr B6C3F1 SCIDNCr and C3HHeNCr mice (80 256)The AJCr mouse an immunocompetent strain and SCIDNCran immunodeficient strain appear to be among the most hepati-tis-prone strains identified to date with liver lesions that becomeprogressively more severe with age (87 97 152) Helicobacterhepaticus-associated hepatitis and progression to hepatocellularcarcinoma in AJCr mice is most prevalent after postinfection

Table 1 Host range and tissue tropism of Helicobacter species

Helicobacter taxon Source(s) Primary site Secondary site

H acinonychis Cheetah tiger StomachH aurati Hamster Stomach intestineH bilis Mouse dog rat cat gerbil human Intestine Liver (mouse) gallbladder (human)H bizzozeroniia Human cat dog cheetah nonhuman primate wild rat StomachlsquoH bovisrsquo Cattle AbomasumH canadensis Human wild geese IntestineH canis Human cat dog Intestine Liver (dog)H cetorum Dolphin whale Stomach H cholecystus Hamster Gallbladder H cinaedi Human hamster macaque dog Intestine Blood brain joint (human) liver (macaque)H felis Cat dog human StomachH fennelliae Human IntestineH ganmani Mouse Intestine H hepaticus Mouse gerbil IntestineH marmotae Woodchuck cat Intestine Liver (woodchuck)H mesocricetorum Hamster Intestine lsquoH muricolarsquo Korean wild mice IntestineH muridarum Mouse rat Intestine Stomach (mouse)H mustelae Ferret mink StomachH pametensis Wild birds swine IntestineH pullorum Human chicken Intestine Liver (chicken)H pylori Human nonhuman primate cat StomachlsquoH rappinirsquob Human cat dog mouse sheep swine Intestine Placentafetusliver (sheep) blood (humans)H rodentium Mouse rat IntestineH salomonis Dog StomachlsquoH suisrsquo Swine Stomach lsquoH suncusrsquo House musk shrew StomachH trogontum Rat IntestineH typhlonius Mouse rat Intestine H winghamensis Human Intestine

Unnamed novel helicobacters Source Primary site Reference GenBank or MIT accessions

AF333338 AF333339 Rhesus macaque Intestine (84)AF107494 Cotton-top tamarin Intestine (207)MIT 97-6535 Ferret Liver (104)MIT 266-1 Ferret Intestine (90)AF336947 Cat Intestine (211)MIT 01-5926 Sea otter Intestine Unpublished (Fox)MIT 03-5765 Sea lion Intestine Unpublished (Fox)AFAY203898 AFAY203899 Harp seal Stomach (124)AF320621 Mouse Intestine liver (217) MIT 94-022 Mouse Intestine Unpublished (Fox)

aLikely the same as lsquoH heilmanniirsquo lsquoH heilmanniirsquo (formerly Gastrospirillum hominis) has the same phenotype as listed here for H bizzozeronii Only a single lsquoHheilmanniirsquo strain has been isolated by use of culture bFormerly regarded as lsquoFlexispira rappinirsquo now subgrouped into ten taxa

131

month 18 and for unknown reasons males are predisposed tomore severe lesions (87)

Mice of the SCIDNCr strain that were naturally infected withH hepaticus developed hepatitis proliferative typhlitis and coli-tis (152) Lesions in the liver of SCID mice consisted of Kupffer

Ito and oval cell hyperplasia along with multifocal to coalescingcoagulative hepatocyte necrosis Numerous Warthin-Starry-posi-tive bacteria were observed in the parenchyma and there wereminimal to mild accumulations of monocytic cells and neutro-phils Proliferative typhlitis was characterized by moderate to

Table 2 Biochemical profile growth characteristics and flagellar morphology of Helicobacter species

Helicobacter Catalase Nitrate Alkaline Urease Indoxyl acetate γ-Glutamyl Growth Resistance tob Flagellataxon production reduction phosphatase hydrolysis transferase At 42degC With 1 Nalidixic acid Cephalothin

glycine

H acinonychis + - + + - + - - R S BipolarH aurati + - - + + + + - S R BipolarH bilis + + - + - + + + R R BipolarH bizzozeroniic + + + + + + + - R S BipolarlsquoH bovisrsquo ND ND ND + ND ND ND ND ND ND NDH canadensis + +- - - + - + + R R MonoBipolarH canis - - + - + + + - S I BipolarH cetorum + - - + - + + ND I S BipolarH cholecystus + + + - - - + + I R MonopolarH cinaedi + + - - - - - + S I BipolarH felis + + + + - + + - R S BipolarH fennelliae + - + - + - - + S S BipolarH ganmani + + - - - ND - - R S BipolarH hepaticus + + - + + - - + R R BipolarH marmotae + - + + - - - + R R BipolarH mesocricetorum + + + - ND - + - S R BipolarH muridarum + - + + + + - - R R BipolarH mustelae + + + + + + + - S R PeritrichousH pametensis + + + - - - + + S S BipolarH pullorum + + - - - ND + - R S MonopolarH pylori + - + + - + - - R S MonopolarlsquoH rappinirsquo +- - - + - + + - R R BipolarH rodentium + + - - - - + + R R BipolarH salomonis + + + + + + - ND R S BipolarlsquoH suisrsquo ND ND ND ND ND ND ND ND ND ND BipolarlsquoH suncusrsquo + + + + ND ND - ND R R BipolarH trogontum + + - + - + + ND R R BipolarH winghamensis - - - - + ND - + R R Bipolar

ND = not determined R = resistant S = susceptible I = indeterminate

Table 4 Summary of key rodent models of Helicobacter gastrointestinal and liver cancera

Rodent Infective agent transgene Tumor Comment

C57BL6 mice H felis Gastric adenocarcinoma Natural feline pathogen but lacks vacA and PAI

INS-GAS FVB mice H felis and H pylori Gastric adenocarcinoma Constitutive hypergastrinemia promotes tumorigenesis

Mongolian gerbil H pylori Gastric adenocarcinoma Closely mimics human disease long time course and few reagents

BALBc mice Several Helicobacter spp Gastric MALT lymphoma Usually requires 18-24 months

Genetically engineered mice ldquoEndogenous microbiotardquo Large intestinal carcinoma Bacteria in endogenous microbiota models IL10-- mice especially on 129Sv or H hepaticus (cecum plusmn colon) not well defined H hepaticus reliably induces disease

background

Lymphocyte-deficient mice H hepaticus Large intestinal carcinoma Often used for adoptive transfer studies H hepaticus inducesSCID or Rag-- especially on (cecum plusmn colon) tumors in untreated Rag2-- mice

129Sv background

AJCr and other mice H hepaticus Hepatocellular carcinoma Natural murine pathogen induces chronic active hepatitis and HCC

aTable adapted from Rogers and Fox (201)MALT = Mucosa-associated lymphoid tissue HCC = hepatocellular carcinoma

Table 3 Helicobacter-associated typhlocolitis in mice

Genetic status of mice Type of defect Pathology

CD45RBhigh-reconstituted scids Reconstitution with naiumlve CD4+T cell TyphlocolitisTCR α--β -- defined flora Abnormal T cell receptors TyphlocolitisScid ICR defined flora Lack T and B cells TyphlocolitisIL-10-- Lack IL-10 TyphlocolitisRAG2-- Lack T and B cells Typhlocolitis colon cancerIL-7--RAG2-- Lack IL-7 T and B cells NoneAJCr Normal Typhlitis Swiss Webster gnotobiotic Normal EnterocolitisNF-κB (p50-- p65+-) Lack NF-κB Typhlocolitismdr1a-- Lack P-glycoprotein Typhlocolitis

IL = Interleukin

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

132

marked mucosal epithelial cell hyperplasia with mild monocyticand neutrophilic infiltration In contrast infected immunocompe-tent AJCr mice which develop a substantial immune response toH hepaticus associated with prominent multifocal mononuclearcell infiltrates in the liver (262) typically have low numbers of Hhepaticus observable at the periphery of foci of inflammation andcolonizing the biliary canaliculi (Fig 3) Therefore the successrate for recovery of H hepaticus in culture from infected AJCrmice is high when the cecum and colon are sampled but is lowfrom liver specimens

Helicobacter hepaticus infection in AJCr mice has been charac-terized on a longitudinal basis through 18 months of age (87)Helicobacter hepaticus colonization persisted in the large intes-tine and liver and was associated with chronic proliferative hepa-titis and liver cancer and in some mice chronic typhlitis (87)Infected AJCr mice developed sustained serum IgG antibody re-sponses to H hepaticus and high serum enzyme activities indica-tive of hepatocellular injury To fulfill Kochrsquos postulatescolonization of germfree mice by H hepaticus was induced andthe mice went on to develop chronic hepatitis some mice devel-oped enterocolitis (97)

A clinical syndrome of typhlocolitis with and without rectalprolapse or concomitant hepatitis has been described in a varietyof inbred strains of immunocompromised mice infected with Hhepaticus (Table 3) (66 254) In immune dysregulated micemononuclear inflammatory cells infiltrate the mucosa and sub-mucosa of the cecum and colon Epithelial cell hyperplasia can besubstantial with frequent villous to papillomatous folds extend-ing into the lumen and a high mitotic index in the crypts Num-bers of goblet cells are variably decreased in the proliferativemucosa Helicobacter hepaticus infection has been associatedwith development of large intestinal adenocarcinoma in RAG2--

mice on a 129SvEv background (59)

Diagnostic Methods for H hepaticusCulture Diagnostic specimens (feces cecal scrapings tissues)

should be stored at ndash70degC in brain heart infusion or Brucellabroth containing 30 glycerol For culture from the gastrointesti-nal tract specimens are homogenized in 1 ml of phosphate-buff-ered saline (PBS) and are filtered through a 045-microm filter toeliminate competitive commensals For larger helicobacters suchas H bilis or H trogontum a 065-microm filter is used to aid in iso-lation Filtrate from gastrointestinal tract specimens is culturedon blood agar supplemented with 1 trimethoprim vancomycinand polymyxin and specimens from the liver are plated directlyonto blood agar Plates are incubated at 37degC (or 42degC for otherHelicobacter spp) under microaerobic conditions in vented jars(90 N2 5 H2 and 5 CO2 alternatively a 801010 mixture ofthe same gases can be used) for 3 to 7 days Plates should be keptfor 21 days before concluding there is no growth Growth of Hhepaticus occurs as a mucoid film or lawn but not in distinct colo-nies on plates Bacterial numbers can be expanded by inocula-tion into Brucella broth containing 5 fetal bovine serum andincubation for 24 to 48 h on a rotary shaker Many Helicobacterspecies require moist agar for efficient growth

Figure 2 Electron micrograph of H hepaticus Notice the spiral mor-phology as well as the single bipolar flagella (A) in the colon crypt (B)and within the biliary canaliculi of the liver (C) Bars = 05 microm Repro-duced with permission from Dr Schauer and Dr Solnick (231)

Figure 3 (A) Photomicrograph of a section of the liver from an AJmouse infected with H hepaticus Notice characteristic lesions associ-ated with chronic active hepatitis including cholangiohepatitis (upperleft white arrow) interface hepatitis (lower right black arrow) hepato-cellular dysplasia (black arrowhead) and lobular inflammation (cen-tral upper white arrow) HampE stain magnification 20times (B) Inset depictsWarthin-Starry-positive organisms with morphology consistent with Hhepaticus in the liver Magnification 100times

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

131

month 18 and for unknown reasons males are predisposed tomore severe lesions (87)

Mice of the SCIDNCr strain that were naturally infected withH hepaticus developed hepatitis proliferative typhlitis and coli-tis (152) Lesions in the liver of SCID mice consisted of Kupffer

Ito and oval cell hyperplasia along with multifocal to coalescingcoagulative hepatocyte necrosis Numerous Warthin-Starry-posi-tive bacteria were observed in the parenchyma and there wereminimal to mild accumulations of monocytic cells and neutro-phils Proliferative typhlitis was characterized by moderate to

Table 2 Biochemical profile growth characteristics and flagellar morphology of Helicobacter species

Helicobacter Catalase Nitrate Alkaline Urease Indoxyl acetate γ-Glutamyl Growth Resistance tob Flagellataxon production reduction phosphatase hydrolysis transferase At 42degC With 1 Nalidixic acid Cephalothin

glycine

H acinonychis + - + + - + - - R S BipolarH aurati + - - + + + + - S R BipolarH bilis + + - + - + + + R R BipolarH bizzozeroniic + + + + + + + - R S BipolarlsquoH bovisrsquo ND ND ND + ND ND ND ND ND ND NDH canadensis + +- - - + - + + R R MonoBipolarH canis - - + - + + + - S I BipolarH cetorum + - - + - + + ND I S BipolarH cholecystus + + + - - - + + I R MonopolarH cinaedi + + - - - - - + S I BipolarH felis + + + + - + + - R S BipolarH fennelliae + - + - + - - + S S BipolarH ganmani + + - - - ND - - R S BipolarH hepaticus + + - + + - - + R R BipolarH marmotae + - + + - - - + R R BipolarH mesocricetorum + + + - ND - + - S R BipolarH muridarum + - + + + + - - R R BipolarH mustelae + + + + + + + - S R PeritrichousH pametensis + + + - - - + + S S BipolarH pullorum + + - - - ND + - R S MonopolarH pylori + - + + - + - - R S MonopolarlsquoH rappinirsquo +- - - + - + + - R R BipolarH rodentium + + - - - - + + R R BipolarH salomonis + + + + + + - ND R S BipolarlsquoH suisrsquo ND ND ND ND ND ND ND ND ND ND BipolarlsquoH suncusrsquo + + + + ND ND - ND R R BipolarH trogontum + + - + - + + ND R R BipolarH winghamensis - - - - + ND - + R R Bipolar

ND = not determined R = resistant S = susceptible I = indeterminate

Table 4 Summary of key rodent models of Helicobacter gastrointestinal and liver cancera

Rodent Infective agent transgene Tumor Comment

C57BL6 mice H felis Gastric adenocarcinoma Natural feline pathogen but lacks vacA and PAI

INS-GAS FVB mice H felis and H pylori Gastric adenocarcinoma Constitutive hypergastrinemia promotes tumorigenesis

Mongolian gerbil H pylori Gastric adenocarcinoma Closely mimics human disease long time course and few reagents

BALBc mice Several Helicobacter spp Gastric MALT lymphoma Usually requires 18-24 months

Genetically engineered mice ldquoEndogenous microbiotardquo Large intestinal carcinoma Bacteria in endogenous microbiota models IL10-- mice especially on 129Sv or H hepaticus (cecum plusmn colon) not well defined H hepaticus reliably induces disease

background

Lymphocyte-deficient mice H hepaticus Large intestinal carcinoma Often used for adoptive transfer studies H hepaticus inducesSCID or Rag-- especially on (cecum plusmn colon) tumors in untreated Rag2-- mice

129Sv background

AJCr and other mice H hepaticus Hepatocellular carcinoma Natural murine pathogen induces chronic active hepatitis and HCC

aTable adapted from Rogers and Fox (201)MALT = Mucosa-associated lymphoid tissue HCC = hepatocellular carcinoma

Table 3 Helicobacter-associated typhlocolitis in mice

Genetic status of mice Type of defect Pathology

CD45RBhigh-reconstituted scids Reconstitution with naiumlve CD4+T cell TyphlocolitisTCR α--β -- defined flora Abnormal T cell receptors TyphlocolitisScid ICR defined flora Lack T and B cells TyphlocolitisIL-10-- Lack IL-10 TyphlocolitisRAG2-- Lack T and B cells Typhlocolitis colon cancerIL-7--RAG2-- Lack IL-7 T and B cells NoneAJCr Normal Typhlitis Swiss Webster gnotobiotic Normal EnterocolitisNF-κB (p50-- p65+-) Lack NF-κB Typhlocolitismdr1a-- Lack P-glycoprotein Typhlocolitis

IL = Interleukin

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

132

marked mucosal epithelial cell hyperplasia with mild monocyticand neutrophilic infiltration In contrast infected immunocompe-tent AJCr mice which develop a substantial immune response toH hepaticus associated with prominent multifocal mononuclearcell infiltrates in the liver (262) typically have low numbers of Hhepaticus observable at the periphery of foci of inflammation andcolonizing the biliary canaliculi (Fig 3) Therefore the successrate for recovery of H hepaticus in culture from infected AJCrmice is high when the cecum and colon are sampled but is lowfrom liver specimens

Helicobacter hepaticus infection in AJCr mice has been charac-terized on a longitudinal basis through 18 months of age (87)Helicobacter hepaticus colonization persisted in the large intes-tine and liver and was associated with chronic proliferative hepa-titis and liver cancer and in some mice chronic typhlitis (87)Infected AJCr mice developed sustained serum IgG antibody re-sponses to H hepaticus and high serum enzyme activities indica-tive of hepatocellular injury To fulfill Kochrsquos postulatescolonization of germfree mice by H hepaticus was induced andthe mice went on to develop chronic hepatitis some mice devel-oped enterocolitis (97)

A clinical syndrome of typhlocolitis with and without rectalprolapse or concomitant hepatitis has been described in a varietyof inbred strains of immunocompromised mice infected with Hhepaticus (Table 3) (66 254) In immune dysregulated micemononuclear inflammatory cells infiltrate the mucosa and sub-mucosa of the cecum and colon Epithelial cell hyperplasia can besubstantial with frequent villous to papillomatous folds extend-ing into the lumen and a high mitotic index in the crypts Num-bers of goblet cells are variably decreased in the proliferativemucosa Helicobacter hepaticus infection has been associatedwith development of large intestinal adenocarcinoma in RAG2--

mice on a 129SvEv background (59)

Diagnostic Methods for H hepaticusCulture Diagnostic specimens (feces cecal scrapings tissues)

should be stored at ndash70degC in brain heart infusion or Brucellabroth containing 30 glycerol For culture from the gastrointesti-nal tract specimens are homogenized in 1 ml of phosphate-buff-ered saline (PBS) and are filtered through a 045-microm filter toeliminate competitive commensals For larger helicobacters suchas H bilis or H trogontum a 065-microm filter is used to aid in iso-lation Filtrate from gastrointestinal tract specimens is culturedon blood agar supplemented with 1 trimethoprim vancomycinand polymyxin and specimens from the liver are plated directlyonto blood agar Plates are incubated at 37degC (or 42degC for otherHelicobacter spp) under microaerobic conditions in vented jars(90 N2 5 H2 and 5 CO2 alternatively a 801010 mixture ofthe same gases can be used) for 3 to 7 days Plates should be keptfor 21 days before concluding there is no growth Growth of Hhepaticus occurs as a mucoid film or lawn but not in distinct colo-nies on plates Bacterial numbers can be expanded by inocula-tion into Brucella broth containing 5 fetal bovine serum andincubation for 24 to 48 h on a rotary shaker Many Helicobacterspecies require moist agar for efficient growth

Figure 2 Electron micrograph of H hepaticus Notice the spiral mor-phology as well as the single bipolar flagella (A) in the colon crypt (B)and within the biliary canaliculi of the liver (C) Bars = 05 microm Repro-duced with permission from Dr Schauer and Dr Solnick (231)

Figure 3 (A) Photomicrograph of a section of the liver from an AJmouse infected with H hepaticus Notice characteristic lesions associ-ated with chronic active hepatitis including cholangiohepatitis (upperleft white arrow) interface hepatitis (lower right black arrow) hepato-cellular dysplasia (black arrowhead) and lobular inflammation (cen-tral upper white arrow) HampE stain magnification 20times (B) Inset depictsWarthin-Starry-positive organisms with morphology consistent with Hhepaticus in the liver Magnification 100times

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

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Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

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Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

Vol 54 No 2Comparative MedicineApril 2004

132

marked mucosal epithelial cell hyperplasia with mild monocyticand neutrophilic infiltration In contrast infected immunocompe-tent AJCr mice which develop a substantial immune response toH hepaticus associated with prominent multifocal mononuclearcell infiltrates in the liver (262) typically have low numbers of Hhepaticus observable at the periphery of foci of inflammation andcolonizing the biliary canaliculi (Fig 3) Therefore the successrate for recovery of H hepaticus in culture from infected AJCrmice is high when the cecum and colon are sampled but is lowfrom liver specimens

Helicobacter hepaticus infection in AJCr mice has been charac-terized on a longitudinal basis through 18 months of age (87)Helicobacter hepaticus colonization persisted in the large intes-tine and liver and was associated with chronic proliferative hepa-titis and liver cancer and in some mice chronic typhlitis (87)Infected AJCr mice developed sustained serum IgG antibody re-sponses to H hepaticus and high serum enzyme activities indica-tive of hepatocellular injury To fulfill Kochrsquos postulatescolonization of germfree mice by H hepaticus was induced andthe mice went on to develop chronic hepatitis some mice devel-oped enterocolitis (97)

A clinical syndrome of typhlocolitis with and without rectalprolapse or concomitant hepatitis has been described in a varietyof inbred strains of immunocompromised mice infected with Hhepaticus (Table 3) (66 254) In immune dysregulated micemononuclear inflammatory cells infiltrate the mucosa and sub-mucosa of the cecum and colon Epithelial cell hyperplasia can besubstantial with frequent villous to papillomatous folds extend-ing into the lumen and a high mitotic index in the crypts Num-bers of goblet cells are variably decreased in the proliferativemucosa Helicobacter hepaticus infection has been associatedwith development of large intestinal adenocarcinoma in RAG2--

mice on a 129SvEv background (59)

Diagnostic Methods for H hepaticusCulture Diagnostic specimens (feces cecal scrapings tissues)

should be stored at ndash70degC in brain heart infusion or Brucellabroth containing 30 glycerol For culture from the gastrointesti-nal tract specimens are homogenized in 1 ml of phosphate-buff-ered saline (PBS) and are filtered through a 045-microm filter toeliminate competitive commensals For larger helicobacters suchas H bilis or H trogontum a 065-microm filter is used to aid in iso-lation Filtrate from gastrointestinal tract specimens is culturedon blood agar supplemented with 1 trimethoprim vancomycinand polymyxin and specimens from the liver are plated directlyonto blood agar Plates are incubated at 37degC (or 42degC for otherHelicobacter spp) under microaerobic conditions in vented jars(90 N2 5 H2 and 5 CO2 alternatively a 801010 mixture ofthe same gases can be used) for 3 to 7 days Plates should be keptfor 21 days before concluding there is no growth Growth of Hhepaticus occurs as a mucoid film or lawn but not in distinct colo-nies on plates Bacterial numbers can be expanded by inocula-tion into Brucella broth containing 5 fetal bovine serum andincubation for 24 to 48 h on a rotary shaker Many Helicobacterspecies require moist agar for efficient growth

Figure 2 Electron micrograph of H hepaticus Notice the spiral mor-phology as well as the single bipolar flagella (A) in the colon crypt (B)and within the biliary canaliculi of the liver (C) Bars = 05 microm Repro-duced with permission from Dr Schauer and Dr Solnick (231)

Figure 3 (A) Photomicrograph of a section of the liver from an AJmouse infected with H hepaticus Notice characteristic lesions associ-ated with chronic active hepatitis including cholangiohepatitis (upperleft white arrow) interface hepatitis (lower right black arrow) hepato-cellular dysplasia (black arrowhead) and lobular inflammation (cen-tral upper white arrow) HampE stain magnification 20times (B) Inset depictsWarthin-Starry-positive organisms with morphology consistent with Hhepaticus in the liver Magnification 100times

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

133

Polymerase chain reaction (PCR) analysis The PCRmethods for H hepaticus and other rodent helicobacters havebeen described (6 198 210) Samples for PCR analysis should becollected aseptically and stored at ndash20degC prior to DNA extractionPolymerase chain reaction amplification of bacterial DNA ex-tracted from feces can be performed using the QIAamp Tissue Kit(Qiagen Inc Valencia Calif) and following the kit protocol for theisolation of nucleic acids from blood The PCR assay for detectionof Helicobacter DNA in feces permits serial monitoring of indi-vidual or groups of laboratory rodents Cecal scraping specimensobtained at necropsy are extracted for PCR analysis using theBoehringer-Mannheim High Pure PCR Template Preparation Kit(Roche Molecular Biochemicals Indianapolis Ind) and followingthe kit protocol for the isolation of nucleic acids from tissuePrimer sequences specific for H hepaticus have been published5rsquo-GCA TTT GAA ACT GTT ACT CTG-3rsquo (C68) and 5rsquo-CTG TTTTCA AGC TCC CC-3rsquo (C69) yields an amplification product of 414basepairs (bp) (210) Helicobacter hepaticus has three ureasestructural genes (ureA ureB and ureC) that are highly conservedand are homologous to those of the gastric Helicobacter speciesAn RFLP assay using the nucleotide sequence of the ureAB genesequence can be used in PCR and RFLP assays for diagnosis ofH hepaticus (214)

A RFLP analysis also has been used to differentiate betweenenterohepatic helicobacters including H hepaticus H bilis andH muridarum (198 212) A fluorogenic nuclease PCR assay thateliminates post-PCR processing provided sensitive specific andhigh-throughput capacity for detection of Helicobacter spp Hhepaticus H bilis and H typhlonius in fecal DNA samples fromrodents (47) Because H hepaticus does not grow in discrete colo-nies in vitro standard limiting dilution methods for bacterialquantification of H hepaticus are difficult to interpret A real-time PCR assay has been developed to quantify H hepaticus re-covered from feces or cecal scraping specimens using primers forthe H hepaticus cdtB gene and mouse 18S rRNA (107)

Serologic testing To our knowledge commercial serologictesting for Helicobacter infection of rodents is not available Ex-perimentally measurement of Helicobacter-specific serum IgG ormucosal IgA antibody responses by use of an ELISA can be doneusing a bacterial sonicate (97) membrane digest preparations(156 262) or recombinant antigen (62 138 155) Presence of in-fection is readily supported by positive serologic ELISA resultsfor Helicobacter antigens and has been documented to be an as-say with sensitivity exceeding 90 although with variable speci-ficity particularly if mice are infected concurrently with multipleHelicobacter spp (260)

Histologic examination Warthin-Starry or Steiner silverstains are used to localize H hepaticus in the liver parenchymaand biliary system or gastric helicobacters in the stomach Silverstaining of the large intestine is unrewarding because manyother enteric organisms will also be visualized by use of a silverstain In the liver of infected mice bacteria appear localized be-tween hepatocytes Severity of the hepatic lesions appears to beassociated with the abundance of bacteria in SCID mice howeverthe opposite is true in immunocompetent mice For example ininfected AJ mice the organisms can be difficult to find in theliver (Fig 3) Transmission electron microscopy has revealed spi-ral bacteria with morphology consistent with H hepaticus withinbile canaliculi (Fig 2) Helicobacter hepaticus in tissues can alsobe localized by use of immunofluorescence with polyclonal rabbit

anti-H hepaticus sera (88)Genomic analysis and diversity of H hepaticus Pulsed-

field gel electrophoresis (PFGE) was used to establish genomicdiversity in 11 strains of H hepaticus obtained from geographi-cally distant locations in the United States and Europe (206) Iso-lates from three of four independent US sources had similarPFGE patterns suggesting relative genomic conservation as wellas diversity The balance of 7 isolates from various areas in Eu-rope differed significantly in PFGE patterns from those of the USisolates and from one another Use of DNA fingerprinting analy-sis could be helpful in epidemiologic studies of H hepaticus andthis assay highlights the potential strain diversity within Hhepaticus

The genetic relationship among H hepaticus C jejuni and Hpylori was initially examined by use of comparative sequenceanalysis of an ordered cosmid library (106) The complete se-quence of H hepaticus has since been published (234) Theanalysis focused on comparison among H hepaticus H pyloriand C jejuni providing insights into the diverse mechanisms oftissue tropism and pathogenesis of these three pathogensHelicobacter hepaticus has a genome of 1799146 bp predictedto encode 1875 proteins approximately 50 have homologuesin H pylori Importantly H hepaticus lacks almost all H pylori-specific colonization and virulence factors including adhesinsvacuolating cytotoxin A (vacA) and the cytotoxin-associatedgene pathogenicity island (cagPAI) Helicobacter hepaticus hasorthologues of C jejuni adhesin PEB1 and the cytolethal dis-tending toxin (CDT) Experimentally induced infection with anisogenic stain of H hepaticus lacking CDT did not promotetyphlocolitis in IL-10-- mice to the extent caused by wild-type Hhepaticus suggesting a role for CDT in inflammation (266)Helicobacter hepaticus has a novel genomic island and putativepathogenicity island encoding the basic components of a type-IVsecretion system as well as other virulence protein homologuesUse of whole-genome microarray analysis indicated that this is-land is lacking in many isolates of H hepaticus (234)Helicobacter hepaticus predictably induces colitis hepatitis andhepatocellular carcinoma in susceptible strains of mice Someinvestigators have reported lack of expected morbidity and le-sion development in H hepaticus infection studies (44) Suchdifferences in outcome may be related to the genetic diversity il-lustrated by these genomic studies

Helicobacter bilis First isolated from bile liver and intes-tine of aged inbred mice in 1995 (98) H bilis has since been iso-lated from dogs rats and gerbils As additional evidence thatthe wide host range of H bilis potentially includes humans Hbilis was detected by use of PCR analysis and was identified byuse of 16S rRNA analysis in gallbladder and bile specimensfrom Chileans with chronic cholecystitis (79) and was associ-ated with biliary tract and gallbladder cancers in two high-riskpopulations Japanese and Thai (165) Helicobacter bilis is afusiform bacterium encased in periplasmic fibers with three to14 bipolar sheathed flagella Similar to H trogontum of rats Hbilis grows at 37 and 42degC under microaerobic conditions and isurease catalase and oxidase positive Similar to H hepaticusinfection H bilis infection of mice is associated with moderateto severe proliferative typhlitis and chronic active hepatitis inimmunocompromised mice (101) Coinfection with H bilis andH rodentium was associated with diarrhea in a breeding colonyof SCIDTrp53-- mice (215) A third of the colony developed mu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

Vol 54 No 2Comparative MedicineApril 2004

134

coid watery or severe hemorrhagic diarrhea with mortalityHelicobacter bilis and H rodentium were isolated by use ofmicroaerobic culture of feces or cecal specimens from affectedmice and their identity was confirmed by use of PCR analysisSentinel Swiss mice exposed to bedding from cages containingaffected mice acquired the infection but did not develop clinicalsigns of disease Lesions consisted of rectal prolapse and prolif-erative typhlocolitis with focal ulcers in the cecum colon andrectum Treatment for 2 weeks with food wafers containing 15 mgof amoxicillin 069 mg of metronidazole and 0185 mg of bismuthper mouse per day previously documented to eradicate Hhepaticus in immunocompetent mice resolved the diarrhea butdid not eliminate H bilis or H rodentium infection Experimen-tally induced infection of 4-week-old defined flora TacIcrHa(ICR)-scidfDF mice with H bilis alone reproducibly caused similardisease (216)

Polymerase chain reaction analysis has been more sensitivethan culture or serologic testing at determining H bilis infectionstatus in experimental studies particularly during the firstmonth after inoculation because seroconversion can take severalmonths (126) Efforts to improve serologic sensitivity and speci-ficity have included identifying an immunodominant H bilis-specific antigen by testing sera from infected mice against a Hbilis genomic DNA expression library (62) Immunoreactiveclones contained sequences that encoded a predicted 167-kDaprotein that identified H bilis- but not H hepaticus-infectedmice by use of an ELISA Immunoblot (western blot) reactivityof sera from H bilis-infected mice against select bands in Hbilis lysates was high in comparison with no reaction forimmunoblots prepared from H hepaticus H muridarum orunrelated bacterial lysates However primers for sequenceswithin the coding region of the 167-kda protein amplified DNAfrom H hepaticus and H muridarum indicating that these spe-cies may express homologous antigens Recombinant proteinfragments of the P167 protein were compared with H bilismembrane extracts using an ELISA protocol to determine theseroconversion status of 76 mice naturally infected with H bilisor H hepaticus alone or with an unspeciated Helicobacter sp(138) Assay sensitivity was highest for the membrane extractsbut specificity of the assay was greatly improved using the re-combinant P167 fragment proteins

Characterization of nine immunogenic polypeptides proteins inouter membrane preparations of H bilis indicated that isolatesfrom a variety of sources (mouse dog rat and gerbil) had similarprotein profiles that were distinct from those of H pylori (105)Immunoblot cross-reactivity was limited to their flagellins Ter-minal sequences of the two membrane proteins did not have ho-mology with protein sequences available in public databasesindicating that H bilis has a conserved unique outer membraneprotein profile that is distinct from that of H pylori

Helicobacter rodentium Helicobacter rodentium was thefirst urease-negative Helicobacter species isolated from labora-tory mice (213) Helicobacter rodentium is spiral shaped with bi-polar single nonsheathed flagella which is a structural featureshared with H canadensis (74) and H winghamensis (166) iso-lated from humans with gastroenteritis with H pullorum iso-lated from humans and birds (233) and with H ganmani frommice (199) Helicobacter rodentium grows at 37 and 42degC undermicroaerobic and anaerobic conditions is urease-negative and isonly weakly positive for catalase and oxidase In immunocompe-

tent mice H rodentium appears nonpathogenic as part of com-mensal intestinal flora but to our knowledge pathogenic poten-tial of H rodentium has not been systematically studiedCoinfection with H rodentium and H bilis was implicated in anoutbreak of diarrhea and typhlocolitis in a colony of SCID mice(215) Antibiotic therapy helped resolve clinical disease but failedto eradicate the helicobacters

Helicobacter ganmani Helicobacter ganmani sp nov anurease-negative anaerobe was isolated from the intestines oflaboratory mice at four animal facilities (199) Isolates were un-usual in that they only grew anaerobically at 37degC and were inca-pable of growth under microaerobic conditions Like Hrodentium isolates had single bipolar unsheathed flagella andwere urease negative Helicobacter ganmani is positive for oxi-dase and reduces nitrate to nitrite but does not hydrolyze hippu-rate or indoxyl acetate and can be cultured on charcoal agar withresistance to cephalothin Analysis of 16S rDNA sequence indi-cates that H rodentium is the most closely related species with982 similarity Analysis of whole-cell proteins from 9 isolatesby use of sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) differentiated H ganmani from H rodentiumand other Helicobacter spp

Helicobacter typhlonius Helicobacter typhlonius an ure-ase-negative helicobacter was isolated from colonies of laboratorymice independently by personnel at two laboratories (82 100102) and has been detected in rat feces by use of PCR analysis(153) Typhlocolitis in CB-17 SCID mice was observed but incontrast to that in mice infected with H hepaticus (152) or Hbilis (101) lesions of chronic active hepatitis in CB-17 SCID micewere not detected in mice inoculated with H typhlonius (102)The IL-10-- mice naturally infected with a helicobacter later iden-tified as H typhlonius developed diarrhea perianal ulcerationintestinal bleeding and rectal prolapse (82) Helicobacter-free IL-10-- mice were experimentally challenged with H typhlonius andtyphlocolitis was reproduced (82) Helicobacter typhlonius isclosely related to H hepaticus and has similar morphology withspiral shape and bipolar sheathed flagella A large interveningsequence in its 16S rRNA gene and biochemical differences in-cluding being urease negative distinguishes H typhlonius fromH hepaticus Helicobacter typhlonius infection of research miceappears to the less common than infection with H hepaticus andH rodentium and its prevalence is similar to the lower preva-lence of H bilis in colonies of mice in academic institutions (260)

Helicobacter muridarum Helicobacter muridarum wasfirst isolated from the intestinal mucosa of rats and mice (149190) Natural infection of immunocompetent rodents has notbeen associated with clinical disease Although the natural nichefor H muridarum is the large intestine H muridarum has beenassociated with gastritis in mice some of which had developedgastric atrophy that presumably promoted colonization of Hmuridarum in the less-acidic stomach (193) In a T-cell transfermodel of IBD in CB17 SCID mice monoassociation with Hmuridarum provoked typhlocolitis on receipt of CD45RBhi CD4+

T cells from conventionally reared congenic BALBc mice (134)After cell transfer CB17 SCID mice monoassociated with Hmuridarum developed typhlocolitis within 5 to 6 weeks com-pared with 8 to 12 weeks in conventionally housed cohorts

lsquoHelicobacter muricolarsquo A novel enteric helicobacter wasisolated from the cecum and feces of Korean wild mice (Mus mus-culus molossinus) (264) This isolate was provisionally named lsquoH

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

135

muricolarsquo on the basis of phenotypic characteristics and 16SrRNA gene sequence analysis lsquoHelicobacter muricolarsquo is a mi-croaerophilic helicobacter with a pair of nonsheathed bipolar fla-gella and is urease catalase and oxidase positive reduces nitrateto nitrite does not hydrolyze hippurate and is susceptible to na-lidixic acid and resistant to cephalothin

lsquoHelicobacter rappinirsquo A novel bacterium with fusiformmorphology periplasmic fibers and bipolar tufts of sheathed fla-gella was identified in the intestinal mucosae of laboratory mice(208) The isolate grew under microaerobic conditions and wasurease positive On the basis of 16S rRNA gene sequence analy-sis the organism was identified as lsquoFlexispira rappinirsquo and isnow referred to as lsquoH rappinirsquo

Experimental Helicobacter Infection ofMice

Mouse models of Helicobacter-associated hepatitis andhepatic cancer In experimental studies of H hepaticus infec-tion in AJCr mice the development of Helicobacter-associatedlesions particularly hepatitis has been associated with the con-comitant development of a significant serum IgG and Th1 cell-mediated immune response to H hepaticus antigens (262) whichis consistent with the robust response of IL10-- mice to Hhepaticus infection (145 146) Significant amounts of mucosalIgA systemic IgG and T-cell responses develop early after infec-tion but are ineffective in preventing chronic infection or devel-opment of lesions

The importance of host genetics is indicated by strain suscepti-bility to liver disease in mice The predisposition of AJCr mice todevelop H hepaticus-associated hepatitis contrasts with mild orno disease in C57BL6 mice (129 258) Colonization by Hhepaticus in the cecum of experimentally infected AJCr andC57BL6 mice was quantified by use of real-time PCR analysiswith primers for the H hepaticus cdtB gene and mouse 18SrRNA (258) Colonization and lesions were evaluated after 6months of H hepaticus infection Quantitative PCR analysis ofcecal specimens indicated that H hepaticus colonized C57BL6mice to a greater extent than it colonized AJCr mice Consistentwith prior reports AJCr mice developed more severe parenchy-mal necrosis portal inflammation and phlebitis in the liver com-pared with mild disease observed in infected C57BL6 miceThus hepatitis in AJCr mice caused by H hepaticus infection isassociated with significantly lower colonization by H hepaticusin the cecum compared with higher colonization in hepatitis-re-sistant C57BL6 mice Host responses of AJCr mice that limitcecal colonization with H hepaticus may have important roles inthe pathogenesis of hepatic lesions

To examine the genetic basis for susceptibility to H hepaticus-induced liver disease disease-susceptible AJCr mice and dis-ease-resistant C57BL6 mice were compared using 9 AXBrecombinant inbred (RI) mouse strains (129) After 14 months ofinfection with H hepaticus determination of colonization by Hhepaticus in the liver and cecum and morphometric evaluation ofthe liver lesions were performed to quantify and correlate the se-verity of inflammation apoptosis and proliferation Significantvariation between RI strains in severity of hepatitis suggested amultigenic basis of disease resistance Quantitative trait analy-sis using linear regression suggested possible linkage to loci onmouse chromosome 19 Hepatocellular and biliary epithelialapoptosis and proliferation indices including proliferation of oval

cells were markedly increased and correlated with severity of in-flammation Prevalence of hepatic neoplasia also was increasedin susceptible RI strains (129)

Both H hepaticus and H bilis are members of theenterohepatic group of murine helicobacters and are ureasepositive a purported virulence factor in common with that of Hpylori that depends on urease activity to survive in the acidicenvironment of the stomach (24) Urease was found to be criticalfor colonization of the ferret stomach with H mustelae (3) andin vivo complementation of ureB restores the ability of isogenicH pylori urease mutants to colonize mice (53) Helicobacter py-lori urease has also been documented to stimulate substantialincreases in macrophage inducible nitric oxide synthase (iNOS)expression and nitric oxide (NO) production implicating it inNO-dependent mucosal damage and carcinogenesis (109) Incontrast to gastric helicobacters all of which are urease-positivethe functional role of urease in enterohepatic helicobacters isnot clear because the natural niche of the cecum and colon is notacidic and many members of the enterohepatic group ofhelicobacters are urease negative The urease gene of Hhepaticus has been cloned and found to contain a homologue ofeach gene in the H pylori urease cluster (7) Isogenic urease mu-tants of H hepaticus were compared with the wild-type parentalstrain for viability in a low pH environment in vitro and for abil-ity to colonize outbred Swiss Webster mice (22) The urease mu-tants were more susceptible to acid killing and failed to infectoutbred Swiss Webster mice by use of oral gavage or C57BL6mice by use of intraperitoneal administration These results in-dicate that urease activity is essential for intestinal infection byH hepaticus and that failure to colonize is not attributable toacid susceptibility because parenteral dosing also failed to infectmice

A urease-negative Helicobacter sp isolated from a wild mouseinduced proliferative typhlocolitis in defined flora SCID mice(TacICRHascidfRFscid) that was accompanied by severe hem-orrhagic diarrhea weight loss phlebothrombosis and hepatitis(217) Infected AJ mice did not develop clinical signs of diseasebut had mild to moderate proliferative typhlocolitis and moder-ate to marked cholangiohepatitis at 7 and 24 weeks after infec-tion This same novel Helicobacter isolate was used to examinethe ontogeny of a characteristic lesion of H hepaticus-inducedhepatitis in the AJ mouse (219) Helicobacter hepaticus-associ-ated hepatitis is characterized by random distribution of multi-focal accumulation of mononuclear inflammatory cells aroundsmall intralobular hepatic venules These foci of inflammatoryinfiltrates resemble tertiary lymphoid development that hasbeen observed in other models of chronic autoimmunity or in-flammation and are thought to arise by a process termed lym-phoid organ neogenesis The small high endothelial venules ininflammatory lesions in AJ mice infected with the novelhelicobacter were positive by use of immunohistochemicalanalysis for features typical of lymphoid organ neogenesis thatincluded peripheral node addressin and the mucosal addressincell adhesion molecule The chemokines SLC (CCL 21) and BLC(CXCL13) also were present as were B220+ B cells and thenaiumlve T-cell subset that expresses CD45loCD62Lhi These find-ings suggest that lymphoid aggregates observed in lesions ofHelicobacter-induced chronic active hepatitis arise through theprocess of lymphoid organ neogenesis

Results of early studies suggested a potential autoimmune-like

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

Vol 54 No 2Comparative MedicineApril 2004

136

mechanism for hepatitis because autoantibodies were detected tohsp70 which was expressed by H hepaticus and diseased hepa-tocytes (255) The mechanism by which the chronic inflammatoryreaction in the liver of H hepaticus-infected mice leads to canceris unknown A putative mutagen secreted by H hepaticus has notbeen detected using the Ames assay (19) Measurement of oxida-tive damage in liver DNA of infected mice has linked accumula-tion of 8-oxodeoxyguanosine with disease progression (225)Oxidative stress associated with inflammation can result in lipidperoxidation and the generation of malondialdehyde which inturn can react with deoxyguanosine in DNA resulting in the for-mation of adducts Adducts may cause mutations that ultimatelylead to liver carcinogenesis Increased amounts of adducts weredetected in the liver DNA of H hepaticus-infected AJCr micewith highest amounts found at 12 months (223) Although a lobepredisposition for H hepaticus-induced liver tumors has not beenreported to our knowledge adducts were particularly increasedin the caudate and median lobes with the left lobe having thelowest amount of adducts compared with the right and medianlobes Other promutagenic DNA lesions 7-methylguanine andO6-methylguanine indicative of nitrosation of endogenousamines by nitric oxide were not detected Analysis of carcinomasand adenomas taken from H hepaticus-infected AJCr mice didnot reveal mutations in the ras or p53 oncogenes (226) These re-sults suggest a non-genotoxic tumor promotion mechanism pos-sibly mediated by reactive oxygen species generated during theinflammatory response (19) Overexpression of epidermal growthfactor transforming growth factor α (TGF-α) cyclin D1 Cdk4and c-Myc is associated with cellular transformation and werefound to be up-regulated in the liver of H hepaticus-infected AJCrmice with highest expression in tumors (196)

Early exposure of AJCr mice to H hepaticus infection pro-moted the most severe hepatitis and preneoplastic changes in theliver particularly in male mice (200) Mouse pups were either ex-posed to H hepaticus by oral infection of the pregnant femaleandor weanlings born to infected and uninfected dams were ex-perimentally re-dosed with H hepaticus at 3 or 12 weeks of ageEffects of gestational exposure to H hepaticus were not signifi-cant but male AJ offspring infected by 3 weeks of age developedthe most severe hepatitis whereas those first infected at 12weeks of age were hepatitis resistant Interestingly comparedwith females males were most prone to severe disease as previ-ously reported (87) but males of the current study had a bimodalpattern of susceptibility The most severe lesions in males in-cluded lobular necrogranulomatous and interface (chronic active)hepatitis whereas females were more prone to develop portal(chronic persistent) hepatitis Hepatic bacterial load and precur-sor lesions of hepatocellular carcinoma including clear and ti-groid cell foci of altered hepatocytes were strongly associatedwith lobular hepatitis severity (Fig 3) (200)

Mouse Models of Helicobacter-AssociatedIBD-like Disease

Experimentally induced infection using H trogontumNational Institutes of Health (NIH) germ-free mice experimen-tally infected with H trogontum were persistently colonized atvarious levels of the gastrointestinal tract when necropsied atpostinoculation week 6 (173) All infected mice developed micro-scopically detected inflammation including gastritis in at leastone region of the bowel The predominant histologic change was a

moderate diffuse inflammatory infiltrate of mononuclear cells inthe lamina propria often accompanied by mild infiltration ofpolymorphonuclear neutrophilic leukocytes Two animals had fo-cal infiltration of inflammatory cells in the liver although bacte-ria could not be detected

The role of the immune response in either limiting colonizationor in contributing to the intensity of the inflammatory responseto Helicobacter infections that can progress to cancer in mousemodels has been widely investigated (201) Soon after the discov-ery of H hepaticus in 1991 case reports associated H hepaticusinfection with inflammatory bowel syndromes in nude and SCIDmice manifested as rectal prolapse accompanied by chronic pro-liferative typhlitis colitis and proctitis (254) Infected AJCr miceproduce predominantly IgG2a serum antibodies to H hepaticuswhich is consistent with a Th1 immune response as reported forhumans infected with H pylori and mice with H felis Spleenmononuclear cells from infected AJCr mice proliferate in vitro inresponse to H hepaticus antigens and produce more gamma in-terferon (IFN-γ) than interleukin (IL-) 4 or IL-5 also characteris-tic of a Th1 immune response (262) AJ mice may develop mild tomoderate typhlitis after chronic infection with H hepaticus Tocorrelate histologic changes with proinflammatory influencesfrom the infection cecal tissues from AJCr mice infected with Hhepaticus were compared for gene expression profiles to deter-mine potential differences prior to the onset of inflammation (onemonth) and after chronic inflammation was established at threemonths (174) One month after infection 25 genes were up-regu-lated and three were down-regulated in contrast to up-regulationof 31 and down-regulation of two genes at the 3-month timepoint A subset of proinflammatory genes including IFN-γ inter-feron gamma inducible protein (IP-10) macrophage inflamma-tory protein 1α (MIP 1α) and serum amyloid A1 were among theup-regulated genes

In murine models such as IL-10-- mice which develop Th1-mediated typhlocolitis some features of the pathogenesis associ-ated with human IBD are mimicked Epithelial hyperplasia andprogression to dysplasia with neoplastic sequelae have been asso-ciated with dysregulated responses to enteric flora including Hhepaticus in certain strains of mice (8 59 261) The most suscep-tible strains of IL-10-- mice develop severe spontaneoustyphlocolitis when maintained under conventional housing condi-tions The importance of genetic background has been high-lighted by evidence that severity of typhlocolitis in response toenteric flora varies with mouse strain Most severe disease devel-oped in IL-10-- 129SvEv and IL-10-- BALBc strains intermedi-ate severity was noted in the IL-10-- 129 times C57BL6J outbredcrosses and least-severe disease was observed in IL-10-- C57BL6J mice (8) The associated chronic inflammation and epithelialdysplasia progresses to colorectal adenocarcinoma in a high per-centage (60) of IL-10-- 129SvEv mice by 6 months of age (8)

The IL-10-- mice experimentally infected with H typhlonius(82) and most notably H hepaticus (146) develop IBD-like clini-cal signs of disease including diarrhea perianal ulceration intes-tinal bleeding and rectal prolapse (8 82 143) The cecocolicjunction and distal portion of the colon are most severely affectedwith mucosal ulceration and focal transmural inflammationcharacterized by extensive infiltration of the lamina propria withlymphocytes plasma cells macrophages and scattered neutro-phils The IL-10-- C57BL10J mice coinfected with the murinenematode Heligmosomoides polygyrus which induces a Th2 host

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

137

response and H hepaticus which induces a Th1 response devel-oped less severe typhlocolitis and epithelial hyperplasia than didIL-10-- mice infected with H hepaticus alone (261) The reductionin mucosal damage in the H polygyrusH hepaticus-coinfectedIL-10-- mice was associated with suppressed Th1 responses to Hhepaticus despite increased colonization of H hepaticus in thececum typical of a Th2 bias toward Helicobacter infection Theameliorating effect of parasitism on the mucosal damage associ-ated with H hepaticus in the IL-10-- mouse IBD model supportsprior findings that enteric parasitism can reduce Helicobacter-as-sociated gastric tissue damage (73)

Efforts to define the relevant contributions of innate immunityversus adaptive immunity to IBD have taken advantage of theability of H hepaticus to reproducibly cause IBD-like disease inimmunodeficient mice such as the SCID CB-17 strain (18 160) Tcell receptor knockouts (21) NF-kappa B-deficient mice (57) andRAG2-- mice (60) SCID mice develop typhlocolitis after adoptivetransfer of CD4+ T cells expressing high levels of CD45RB a pheno-type of naiumlve T cells Germfree SCID mice do not develop inflam-mation after cell transfer indicating that a response to theintestinal flora is key to the model When SCID mice were experi-mentally infected with H hepaticus development of typhlocolitiswas accelerated after reconstitution with CD45RBhighCD4+ T-cells(18) Transfer of CD45RBlowCD4+ T cells that contain a regulatoryT-cell subset expressing the CD25 IL-2 receptor did not cause in-flammation and suppressed the inflammatory response mediatedby the CD45RBhighCD4+ T cells when they were co-transferred Themechanism(s) for immune suppression by CD4+CD25+ T-regula-tory cells was further studied in H hepaticus-infected RAG-- micethat lack functional B and T cells In this model H hepaticus in-fection elicited T cell-mediated and T cell-independent intestinalinflammation both of which were inhibited by adoptively trans-ferred CD4+CD25+ regulatory T cells The T cell-independent pa-thology was accompanied by activation of the innate immunesystem that was also inhibited by CD4+CD25+ cells Suppression ofinnate immune pathology was dependent on T cell-derived IL-10and on the production of TGF-β (160)

Because these mice lack functional B and T cells the inflam-matory response to H hepaticus in RAG2-- micersquo is mediated bythe innate immune system RAG2-- mice naturally infected withH hepaticus developed severe colitis but IL-7-- RAG2-- mice colo-nized by the same flora did not develop spontaneous colitis sug-gesting that IL-7 plays a critical role in exacerbating a non-Tnon-B cell-mediated chronic inflammatory response Administra-tion of recombinant (r)IL-10 was able to prevent development ofcolitis in susceptible mice suggesting a pivotal role for macroph-ages (103) Helicobacter hepaticus-infected RAG2-- mice on the129SvEv background were reported to rapidly develop colitisand large intestinal carcinoma (Fig 4) (59) Adoptive transferwith CD4+CD45RBlowCD25+ regulatory T cells in these mice sig-nificantly inhibited H hepaticus-induced inflammation and coloncancer Regulatory cells lacking the anti-inflammatory cytokineIL-10 were unable to inhibit inflammation dysplasia or cancerindicating that IL-10 was required for the protective effects oflymphocytes in these experiments and that the regulatory T cellsexpressing IL-10 had an anti-inflammatory effect on the innateimmune system (60)

NF-kappaB is a transcription factor activated during the in-flammatory response and thus it was unexpected to discoverspontaneous colitis in mice lacking the p50 subunit and havingonly one allele of the p65 subunit of NF-kappaB (p50--p65+-) (57)These mice were rederived into a helicobacter-free health statusand the phenotype of spontaneous colitis decreased markedlyExperimental infection of NF-kappaB p50--p65+- mice with Hhepaticus caused severe colitis to develop within 6 weeks wasmilder in control genotypes such as p50--p65++ and p50++p65+-and was absent in wild-type mice suggesting that the p50 andp65 subunits of NF-kappaB have a role in inhibiting the develop-ment of colitis potentially through control of proinflammatoryIL-12 It was further documented that NF-kappaB activity is re-quired within the innate immune system to inhibit H hepaticus-induced colitis and that NF-kappaB regulatory activity ismediated at least in part through inhibition of the expression ofthe p40 subunit of IL-12 (246)

Figure 4 (A) Photomicrograph of a section of the transverse colon of a H hepaticus-infected RAG-- mouse at 14 months after infection Noticeseverely dysplastic glands with atypical epithelium infiltrating the submucosa and muscle and obliterating normal bowel structure (B) Extracellu-lar mucin was prominent in large pools partially lined by neoplastic epithelium HampE stain (A) bar = 250 microm (B) bar = 100 microm Figure used withpermission from Erdman and co-workers (60)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

Vol 54 No 2Comparative MedicineApril 2004

138

Mouse Models of Helicobacter-InducedGastritis

Helicobacter felis gastritis Isolation of H felis from the catstomach made it possible to model Helicobacter gastritis in miceafter it was documented that H felis would colonize germfreemice (147) and gnotobiotic rats (86) and induce gastritis BALBcmice infected with H felis for up to 22 months developed B-celllymphoid follicles in the gastric wall that were reminiscent ofMALT lymphoma of humans infected with H pylori (56) TheseBALBc mice did not develop gastritis until near the end of thestudy which was the first suggestion of strain difference in thehost response to H felis This was further described byMohammadi and co-workers (172) who observed that the patternand intensity of gastritis varied from minimal in BALBc to mod-erate in C3H to severe in C57BL6 mice The robust inflamma-tory response of C57BL6 mice to H felis was opportune forfurther model development of Helicobacter-induced gastritis be-cause the C57BL6 background is commonly used for producingtransgenic and knockout mice For example H felis infection inthe IL10-- mouse led to accelerated severe hyperplastic gastritiswith gastric epithelial cell dedifferentiation (9) a feature of Hpylori infection in humans that can progress to gastric atrophyand cancer (27) Many studies have followed that documented theroles of innate (130 131) and adaptive immune responses (171204) on impact of colonization levels and inflammatory responsesof mice to H felis Vaccine strategies to prevent infection with Hfelis (135) or therapeutically ameliorate gastritis (28) are of con-siderable interest for application to vaccine development in hu-mans The potential for sex differences in response to H felisinfection was reported in female C57BL6J mice infected with Hfelis for up to a year Infected females developed chronic gastricinflammation epithelial hyperplasia and oxyntic cell loss earlierthan did male mice with a trend over time for more severe le-sions to develop in female mice (29)

Infection with H felis has been used in mice to model gastriccancer Helicobacter pylori infection and adenomatous polyposiscoli (Apc) gene mutations have been linked to gastric cancer inhumans C57BL6 and Apc1638 heterozygous mice infected withH felis were studied for 75 months (78) The infected Apc1638mice had less epithelial proliferation and gastritis lower serumIgG responses and higher bacteria and urease scores than didinfected C57BL6 mice The Apc1638 truncating mutation led togastric dysplasia and polyposis of the antrum and pyloric junc-tion but H felis infection did not increase the risk of gastric neo-plasia Results of that study indicated that this Apc mutation isassociated with decreased immune inflammatory and gastrichyperplastic responses to H felis infection suggesting the possi-bility of a novel role for this tumor suppressor gene in the im-mune and local tissue responses to gastric bacterial infection Thep53 heterozygous mice infected with H felis for up to 15 monthsalso had lower risk of Helicobacter-associated cancer (94) Antralinflammation and submucosal invasive foci were observed inwild-type C57BL6 mice accompanied by invasion of adjacentsubmucosal blood vessels by glandular epithelia in a subset ofinfected C57BL6 mice None of these lesions were observed inp53+- mice infected or not and p53+- mice had significantlyhigher Helicobacter colonization consistent with an anti-inflam-matory Th2 response Proinflammatory Th1 responses were sig-nificantly higher in C57BL6 compared with p53+- mice Theseresults suggested that germline deletion of one p53 allele results

in a down-regulated Th1 response to gastric Helicobacter infec-tion possibly related to T-cell senescence which indirectly wouldbe protective against development of gastric cancer and other epi-thelial-derived neoplasms associated with chronic inflammationIn contrast infection with H felis in TSG-p53Big Blue mice(p53+-) resulted in enhanced gastritis and three-fold increase inmutations after 7 months compared with that in wild-type mice(133) Compared with wild-type mice TSG-p53Big Blue mice(p53+-) infected with H felis or H pylori for 6 months developedsevere gastritis that was associated with increased iNOS expres-sion and a four-fold and 17-fold higher rate respectively of mu-tation (247) After 12 months of infection gastric hyperplasia wasprominent but the mutagenic effects and iNOS expression de-creased in H pylori- and H felis-infected mice These data sug-gest a synergistic action between infection with H felis and p53deficiency in the accumulation of mutations in gastric tissue

Studies from our laboratories indicated that Th1-promotedgastric atrophy secondary to H felis infection in mice was amelio-rated by the Th2 host response to the murine helminthHeligmosomoides polygyrus (73) Those results suggest that thelow incidence of gastric cancer in African populations with a highincidence of H pylori infection is attributable in part to highprevalence of concurrent parasitic infection in Africans that tem-pers the host response to H pylori

Helicobacter pylori gastritis The ability to genetically engi-neer the host response in mice is a powerful advantage to studybasic mechanisms of H pylori gastritis and its sequelae Initialattempts to colonize mice with H pylori were unsuccessful untilthe Sydney strain of H pylori known as SS1 (cagA+ and vacA+)was isolated by dosing mice with a variety of fresh clinical iso-lates and evaluating long-term colonization (148) High levels ofcolonization were achieved in C57BL6 BALBc DBA2 andC3HHe mice and persistent colonization of mice led to chronicactive gastritis with progression to gastric atrophy The capabilityof SS1 to colonize mice and cause gastritis has had significant im-pact on H pylori research It should be noted that the functionalstatus of cagA+ in SS1 has been debated because several labora-tories have reported that SS1 does not induce IL-8 secretion fromhuman gastric epithelial cells in vitro (31) Thus although SS1 isprobably the best model strain of H pylori to use in mice to studycolonization and gastritis investigators interested in the cagPAIand in particular the significance of IL-8 as a proinflammatorymediator should use other H pylori strains and additionallymust use other species because mice do not produce IL-8

Mouse strains vary in their susceptibility to development ofgastric atrophy from Helicobacter-induced gastritis The geneticbasis for this has been examined for H felis models (239) as wellas H pylori models (238) Milder gastritis and lower predisposi-tion to atrophy was identified as a dominant trait associated withincreased production of IL-10 In a mouse model of H pylori in-fection a high-salt diet linked in humans to increased risk of gas-tric cancer was shown to exacerbate gastritis increase epithelialhyperplasia promote parietal cell loss and enhance H pylori colo-nization in C57BL6 mice (77) As for H felis there has been in-tense interest in developing vaccine strategies using H pylori inthe mouse (237) Therapeutic vaccines have been associated withtransient postimmunization gastritis (236)

Epidemiologic evidence in humans and from studies with micesuggests a link between a high-salt diet and H pylori infectionand the development of hypergastrinemia and preneoplastic gas-

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

References1 Andersen L P 2001 New Helicobacter species in humans Dig

Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953

139

tric lesions Helicobacter infection in C57BL6 mice leads to alter-ations in gastrin and somatostatin concentrations as the gastritisprogresses to atrophy and is characterized by loss of parietal andchief cells and impairment of gastric acid secretion (43)Helicobacter felis infection in insulin-gastrin (INS-GAS) transgenicmice which develop hypergastrinemia accelerates developmentof gastric carcinoma (253) In the INS-GAS129 mouse modelgastric tissue from males responded more rapidly and aggres-sively to H pylori infection high-salt diet and the combinationcompared with tissue from females a finding that appears con-sistent with the greater incidence of gastric carcinoma in menOnly male INS-GAS mice infected with H pylori developed in situand intramucosal carcinoma (92) These results indicated that Hpylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia This same model ofINS-GAS mice infected with H pylori or an isogenic H pylori cagEmutant of the cagPAI developed atrophy intestinal metaplasiaand dysplasia by 6 weeks and carcinoma by 24 weeks (95) Inac-tivation of cagE delayed the progression to carcinoma but neo-plasia ultimately developed in all males infected with the Hpylori mutant These studies highlight the importance of usingboth sexes to investigate the pathogenesis of H pylori

lsquoHelicobacter suncusrsquo A gram-negative motile bacteriumwith bipolar sheathed flagella was isolated from the stomach ofhouse musk shrews (Suncus murinus) with chronic gastritis andwas found on gastric epithelial cells by use of electron microscopy(112) Sequence analysis of 16S rRNA suggested that this bacte-rium should be classified as a novel Helicobacter species with theproposed name lsquoHelicobacter suncusrsquo Gastritis from natural in-fection was reported and experimental challenge in shrews firstcleared of naturally acquired lsquoH suncusrsquo by use of antibiotics re-sulted in development of gastritis (110)

Natural Helicobacter Infections in RatsGastric spirals with morphology consistent with that of lsquoH

heilmanniirsquo were associated with mild gastritis in wild Rattusnorvegicus in Italy (108) Spiral organisms with morphology con-sistent with that of H trogontum were observed by use of micros-copy in bile from rats infected with the liver fluke Fasciolahepatica (69) Natural Helicobacter infections in research ratsappear to be common with prevalence of 19 on the basis of re-sults of testing rat cecal specimens submitted to a US veterinarydiagnostic laboratory and assayed for infection using genus- andspecies-specific PCR assays (153 198) In those two reports Hbilis was detected in 3 to 9 of rat cecal specimens 10 werepositive for H typhlonius 5 to 8 were positive for H hepaticusand H rodentium and unspeciated Helicobacter spp each weredetected in approximately 2 of the specimens Helicobacterrodentium was detected by use of PCR analysis of fecal speci-mens collected from 48 of 163 rats (29) housed in three colo-nies in Japan (113) Gross lesions were not observed andhistologic examination was not performed Except for H bilis in-fection of immunodeficient rats the potential for Helicobacterspp to cause disease in immunocompetent rats has not been re-ported to our knowledge

Helicobacter bilis Proliferative and ulcerative typhlocolitisand proctitis developed as a clinical problem in nude rats(CrNIH-rnu) (116) Helicobacter bilis was identified morphologi-cally in the colon crypts using modified Steinerrsquos silver stain was

recovered by culture and was further identified using PCRanalysis and amplicon sequencing Typhlocolitis was reproducedin H bilis-free nude rats that were injected intraperitoneallywith H bilis recovered from the same colony Helicobacter biliswas detected by use of PCR and RFLP analyses in 97 of 113 ratcecal specimens submitted to a veterinary diagnostic laboratory(198) To our knowledge the potential for H bilis to cause diseasein immunocompetent rats has not been reported

Helicobacter muridarum Spiral-shaped bacteria with dis-tinctive morphology were first isolated from the intestine of ratsand mice using a Campylobacter-selective medium andmicroaerobic incubation (190) and were later named Hmuridarum (149) To our knowledge pathology in rats attribut-able to H muridarum has not been reported

Helicobacter trogontum Helicobacter trogontum was iso-lated from the colon of Wistar and Holtzman rats (168) The or-ganism resembles H bilis with its rod shape entwined withperiplasmic fibers Like many large intestinal helicobacters Htrogontum is catalase and oxidase positive rapidly hydrolyzesurea and is susceptible to metronidazole and resistant to cepha-lothin and nalidixic acid Helicobacter trogontum is microaero-philic and grows at 37 and 42degC differentiating it from Hhepaticus and H muridarum which grow optimally at 37degC Onthe basis of 16S rRNA sequence analysis H trogontum is mostclosely related to H hepaticus Members of Helicobacter spflexispira 16S rDNA taxa 1 4 and 5 have been determined to bestrains of H trogontum (taxon 6) (121)

Experimental Helicobacter Infection ofRats

Rat models of Helicobacter gastritis In comparison withuse of mouse models use of rats for study of H pylori pathogen-esis has been limited Experimentally H felis and lsquoH heilmanniirsquocolonized the gastric antrum of rats in large numbers and in-duced mild gastritis (37) Wistar rats which were documented tobe free of naturally acquired gastric spirals were dosed with lsquoHsuisrsquo from swine and the stomach became colonized mild gastri-tis had developed when the rats were assessed two weeks later(167) Helicobacter felis was reported to colonize the gastric an-trum of germfree rats causing small foci of lymphocytes and eosi-nophils to form throughout the subglandular region of theantrum by two weeks after infection (86) At 8 weeks after infec-tion there were increased numbers of lymphocytes and eosino-phils in the subglandular areas and some focal aggregates oflymphocytes extending from the submucosa through the muscu-laris mucosa and lamina propria to the luminal surfaceHelicobacter felis was detected by use of the Warthin-Starrystain bacteriologic culture and a urease assay Cage-contact con-trol rats were not infected with lsquoH suisrsquo (167) or H felis (86) sup-porting the lack of fecal-oral spread of gastric helicobactersbetween rodents housed in the same cage

lsquoHelicobacter heilmanniirsquo infection was used to document thatHelicobacter infection can delay gastric emptying possibly re-lated to increased concentration of gastrin and suppressed con-centration of somatostatin (15) Short-term infection of rats withmouse-adapted H pylori has been used to indicate that H pyloriinfection can delay healing of iatrogenic gastric ulcers (151)Other studies in rats have involved examination of interaction be-tween H pylori lipopolysaccharide and administration of nonste-roidal anti-inflammatory drugs (228) and anti-ulcer drugs (191)

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

140

Natural Helicobacter Infections in GerbilsHelicobacter hepaticus In a survey of 50 gerbils housed in

four facilities in Japan shedding of H hepaticus in feces was de-tected by use of PCR analysis in 3 colonies but other Helicobacterspp were not detected (113) Gross lesions were not observed andhistologic examination was not performed

Helicobacter bilis Natural infection with H bilis in commer-cially available Mongolian gerbils was recently reported (11) In-fection was not associated with lesions or clinical problems exceptthat attempts to eradicate H bilis by use of antibiotics resulted inacute morbidity and mortality from overgrowth of Clostridiumdifficile

Experimental Helicobacter Infection ofGerbils

Gerbil models of Helicobacter gastritis The first success-ful models of gastric cancer and its precursor lesion intestinalmetaplasia attributable to chronic H pylori gastritis in a rodentmodel were reported using inbred Mongolian gerbils (MGSSea)(Meriones unguiculatus) infected either with clinical isolates of Hpylori or with strain ATCC 43504 (127 257) The gerbil model forgastric cancer has also combined H pylori infection with the car-cinogen N-methyl-N-nitro-N-nitrosoguanidine to establish thatchronic H pylori gastritis promoted incidence of chemically in-duced adenocarcinoma (245) Because risk factors for develop-ment of gastric adenocarcinoma in humans include high dietarysalt intake and H pylori infection gastric pathology resultingfrom H pylori infection and high dietary salt intake were exam-ined as independent variables in outbred Mongolian gerbils(Crl[MON]) infected with the Sydney strain of H pylori (10) Af-ter 37 weeks 5 of 5 infected gerbils had atrophic gastritis and in-testinal metaplasia Gastric atrophy and intestinal metaplasiawithout accompanying inflammation also were noted in 6 of 6uninfected gerbils that had consumed a 25 salt diet for 56weeks Thus outbred gerbils provide an excellent animal modelfor the study of several gastric cancer risk factors

Only a few H pylori strains have been documented to colonizeMongolian gerbils successfully A cagA+ strain 42GX isolatedfrom a Chinese patient colonized gerbils and induced severegastritis (252) The gastric mucosa of gerbils infected with thecagA+ H pylori SS1 strain for 36 weeks was analyzed for in-creased expression of pro- and anti-inflammatory cytokines (30)After 36 weeks female gerbils infected with H pylori had sig-nificantly increased expression of transcripts for IFN-γ and IL-12p40 but not TGF-β or IL-10 Significantly reduced IFN-γ andIL-12p40 responses were observed in infected male gerbils butepithelial proliferative and apoptotic responses were compa-rable between the sexes Thus female gerbil cytokine responsesto H pylori appear to have a stronger Th1 profile than do malesand the low expression of TGF-β or IL-10 may account for themore severe gastritis observed with H pylori infection in gerbilsthan in mice

The risk for gastric adenocarcinoma from H pylori infection inhumans may be related to increased gastric epithelial cell turn-over andor gastrin secretion Gerbils infected with wild-type Hpylori or isogenic mutants of cagA and vacA developed transientincreased rates of antral apoptosis followed by increased butsimilar rates of gastric epithelial cell turnover (186) Epithelialcell proliferation was significantly related to serum gastrin con-centration whereas antral apoptosis was inversely related to

acute inflammation and formation of lymphoid follicles Thus theresults from this gerbil model indicate that epithelial cell turn-over enhanced by H pylori infection may be mediated by gastrin-dependent mechanisms

Natural Helicobacter Infections inHamsters

Helicobacter cinaedi Hamsters are an established reservoirhost for H cinaedi which also is the most commonly reportedenterohepatic helicobacter in immunosuppressed humans whofrequently have bacteremia or diarrhea as presenting signs (139)Septicemia and meningitis attributed to H cinaedi in a neonatewhose mother had contact with pet hamsters during pregnancywas reported (178) Helicobacter cinaedi DNA was identified in 8of 126 urease-negative (ie H pylori negative) human gastric bi-opsy specimens (187) Helicobacter cinaedi has also been isolatedfrom a rhesus monkey affected with chronic colitis and hepatitis(83) Helicobacter cinaedi produces a bacterial toxin that causesprogressive distention and death of Chinese hamster ovary(CHO) cells and HeLa cells and is termed cytolethal distendingtoxin (CDT) (242) The CDT causes cell cycle arrest at the G2Mphase and may be a mechanism for defense against immuneclearance This toxin has been identified in several enteropatho-genic bacteria including C jejuni C coli some Escherichia colistrains and Shigella spp as well as Haemophilus ducreyi andActinobacillus actinomycetemcomitans (20)

Homologues of the toxin B subunit from five species ofenterohepatic helicobacters including H hepaticus H bilis Hcanis and a novel Helicobacter sp isolated from mice and Hmarmotae from woodchucks have been identified and the bacteriallysates from four of these helicobacters have been documented tocause characteristic cytolethal distention of HeLa cells (20)

Helicobacter mesocricetorum A number of novelhelicobacters have been isolated from hamsters obtained from re-search colonies and commercial vendors Helicobactermesocricetorum was isolated from the feces of clinically normalSyrian hamsters (220) and appears to be most closely related toH rodentium

Helicobacter cholecystus This helicobacter was isolatedfrom the gallbladder of hamsters with cholangiofibrosis andcentrilobular pancreatitis (99) Sequence analysis of the 16SrRNA indicated close relationship to H pametensis isolated fromswine and birds

Helicobacter aurati This helicobacter was isolated from theinflamed stomach and cecum of adult Syrian hamsters (185) It isfusiform with multiple bipolar sheathed flagella and periplasmicfibers and is urease and γ-glutamyl transferase positive (Fig 5)Helicobacter aurati represents a distinct taxon and clusters withH muridarum H hepaticus and an unnamed Helicobacter iso-late (MIT 94-022) Chronic gastritis and intestinal metaplasiaassociated with naturally acquired colonization by H aurati andtwo other microaerobic species were reported in Syrian hamsters(184) from research and commercial facilities Helicobacteraurati a second novel urease-negative Helicobacter sp as well as asmaller urease-negative Campylobacter sp were cultured fromstomach specimens Helicobacter sp DNA was detected in a total of35 hamsters by use of Southern blot hybridization Campylobactersp DNA was detected in 15 of the 35 hamsters Argyrophilic bac-teria in gastric tissues had morphology consistent with that ofisolates recovered by culture The presence of Helicobacter but

141

not Campylobacter sp was correlated to gastritis severity andintestinal metaplasia Giardia spp were observed in the meta-plastic gastric pits of the most severe lesions These findings areconsistent with the development of intestinal metaplasia andgastric giardiasis in H pylori-infected humans

Experimental H pylori Infection ofGuinea Pigs

To our knowledge enterohepatic helicobacters have not beenreported in guinea pigs or rabbits but these species have notbeen extensively evaluated for Helicobacter infection (46) Guineapigs have been used in H pylori models of gastritis (197 218) be-cause of the ability of the widely used mouse-adapted SS1 strainof H pylori to colonize them (148) The vitamin C requirement ofguinea pigs is a valuable feature of the model because of the roleof vitamin C in protection against the formation of carcinogenicnitrosamines in gastric juice Experimental H pylori infection ofguinea pigs induced antral gastritis and gastric MALT in guineapigs (197 218) Dunkin-Hartley guinea pigs were infected withthe SS1 strain of H pylori and developed severe antral gastritisover 5 months (227) Natural Helicobacter infection was not de-tected in 5 control animals by use of culture and Helicobacter ge-nus-specific PCR analysis of tissues and feces The persistent

infection accompanied by severe gastritis and a prominent se-rum antibody response and the apparent absence of naturalHelicobacter infection makes the guinea pig a useful animalmodel for H pylori research

Orogastric vaccination of guinea pigs with H pylori sonicateand the adjuvant cholera toxin lowered the burden of infection(51) There have been differences in the response of guinea pigsand mice in response to vaccine strategies which highlights thatspecies differences need to be considered in the eventual applica-tion of a vaccine strategy to humans (137)

Interference with Research Attributableto Helicobacter Infections in Rodents

The discovery of H hepaticus was the result of an investigationinto an abnormally high incidence of hepatic tumors and hepati-tis in control AJCr mice that were part of a long-term carcino-genesis study (80 256) A review of the impact of H hepaticusinfection on 12 National Toxicology Program two-year carcino-genesis studies was published in 1998 (115) Male and femaleB6C3F1 mice from all 12 studies were naturally infected with Hhepaticus with associated hepatitis in many of the male mice re-ported from 9 of these studies The incidences of hepatitis andhepatocellular and hemangiosarcoma of the liver in H hepaticus-infected control male B6C3F1 mice were increased comparedwith those in uninfected control males Typical of the chronichepatitis attributable to H hepaticus the liver from infectedmales had increased hyperplasia and apoptosis Interestingly H-ras codon 61 CAA to AAA mutations were less common in liverneoplasms from infected males than those in historical and studycontrol specimens In inhalation toxicity and carcinogenicity stud-ies of cobalt sulfate male B6C3F1 mice had liver lesions consistentwith H hepaticus infection and increased incidence of hepatic he-mangiosarcoma Because of the confounding infection with Hhepaticus a conclusion could not be reached concerning an associa-tion between liver hemangiosarcoma and cobalt sulfate (16)

Helicobacter hepaticus contamination of nonfrozen transplant-able human tumors was infective to SCID mice via subcutaneousinjection of infected tissue (111) Contaminated but cryopreservedsamples were not infective To our knowledge this is the first re-port suggesting that H hepaticus has the ability to spread viabiomaterials and that freeze-thawing is able to reduce the num-bers of organisms to those that are insufficient for inadvertentinfection of immunodeficient mice Helicobacter bilis infectionwas documented to accelerate and H hepaticus infection to delaydevelopment of (otherwise) expected spontaneous colitis that de-velops with age in multiple drug resistance-deficient (mdr1a--)mice (159) The cause of spontaneous colitis has been attributedto a lack of P-glycoprotein that contributes to a presumed epithelialcell barrier defect in mdr1a-- mice Experimental H bilis infectioninduced diarrhea weight loss and typhlocolitis in mdr1a-- micewithin 6 to 17 weeks after inoculation and before the expectedonset of spontaneous typhlocolitis In contrast H hepaticus infec-tion delayed the onset of disease and spontaneous colitis was alsoless severe Similar effects were reported in IFN-γ-- mice natu-rally coinfected with H hepaticus and mouse hepatitis virus(MHV) Clinical disease that included pleuritis peritonitis hepa-titis pneumonia and meningitis developed Experimental infec-tion with H hepaticus alone did not induce clinical signs ofdisease but when mixed with MHV challenge H hepaticus infec-tion appeared to reduce the severity of MHV-induced lesions dur-

Figure 5 Electron micrograph of H aurati isolated from the stomachof hamsters Periplasmic fibers and multiple bipolar flagella are typi-cal features of the Flexispira taxa H aurati measures approximately06 by 48 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

142

ing the acute phase of infection and exacerbated hepatitis andmeningitis at a later time point (26)

Importantly subclinical effects of Helicobacter infection mayconfound research For example altered gene expression in cecaltissues prior to development of any features of pathology attrib-utable to H hepaticus infection was reported in AJCr mice (174)One month after infection and prior to any evidence of typhlitis25 genes were up-regulated and three were down-regulated in con-trast to up-regulation of 31 and down-regulation of two genes at the3-month time point when inflammation was becoming apparent Asubset of proinflammatory genes including IFN-γ IP-10 MIP 1αand serum amyloid A1 were among the up-regulated genes

Colony Management of Helicobacter-Free Animals

Many of the following principles have been applied to main-taining helicobacter-free cats and ferrets for controlled infectionstudies Based on management techniques similar to those ap-plied to rodents larger species such as swine or cats can be ob-tained by cesarean section and hand-raised in a barrier to avoidcontact with infected sowsqueens or fomites Helicobacter-free fer-rets have been obtained by antibiotic treatment of jills prior to andduring nursing followed by weaning kits in a barrier environment(5) Recrudescence of infection is a risk in the latter approach

Principles of Eradication of HelicobacterInfections in Mice

Before the impact of Helicobacter infection on the colony healthof research mice was appreciated the diagnostic laboratory at theMIT Division of Comparative Medicine frequently identifiedHelicobacter-infected mice from commercial and academicsources (210) Most commercial vendors can now supply micethat are free of H hepaticus and H bilis and by use of the sameproduction methods (ie cesarean section embryo transfer) itshould also be possible to supply mice that are free of otherHelicobacter spp such as H rodentium Natural infection withseveral murine Helicobacter spp remains common in conven-tional mouse colonies because of horizontal transmission throughfecal-oral contact (154 260) Because of the endemic nature of theinfection successful eradication strategies consist of restockingwith helicobacter-free mice close adherence to husbandry practicesproven to prevent introduction and dissemination of helicobactersand monitoring colony and sentinel mice to maintain thehelicobacter-free health status

Restocking with helicobacter-free mice Commercial ven-dors should be selected on the basis of the availability ofhelicobacter-free colonies and quality assurance practices that af-ford reasonable guarantee that the helicobacter-free health statuswill be maintained and adequately monitored A percentage ofnewly received animals should be tested by use of PCR analysis ofcecal scrapings or feces using genus-specific primer sets The vet-erinary staff should develop a working relationship with the ven-dor to emphasize the importance of helicobacter-free mice and toencourage rapid communication should a break in health status bedetected lsquoGift micersquo received from non-commercial sources can beassumed to be infected with one or more Helicobacter spp untilproven otherwise The best method for maintaining a helicobacter-free barrier is to purchase only helicobacter-free mice and ensurethat gift mice be re-derived into the facility via embryo transferas standard policy Although PCR screening is highly sensitive

and has reasonable specificity it requires skill to perform and issubject to false-negative results Thus reliance on diagnosticscreening of gift animals is subject to error

Embryo transfer Embryo transfer is the most efficientmethod of re-deriving mice into a helicobacter-free (as well asother bacterial viral and parasitic agents) health status It re-quires substantial investment in technical skills and requiressupport colonies (helicobacter-free recipient females and vasecto-mized males) that must be monitored closely for health statuschanges For large institutions that import substantial numbersof gift mice an embryo transfer program in a separate quaran-tine facility can be cost efficient and yields the greatest assuranceof maintaining helicobacter-free mice

Cross fostering Cesarean section with fostering ontohelicobacter-free dams is less ideal than embryo transfer but hasbeen successful (82 224) Neonatal mice appear to acquireHelicobacter infections soon after birth and thus immediatetransfer (within 24 h) of pups to clean mothers is imperative Thismethod is labor intensive and inherently subject to failure frominadvertent contamination Helicobacter hepaticus was isolatedfrom fetal viscera of 2 of 11 pups sampled late in gestation frominfected SCIDNCr females (152) suggesting that transplacentalinfection of H hepaticus is possible in immunodeficient miceCross-fostering or cesarean section to derive helicobacter-freemice should be reserved for immunocompetent strains of mice

Antibiotic therapy There have not been long-term large-scale studies that have indicated success in using antibiotics toeradicate helicobacters from a mouse colony Several oral antimi-crobial formulations have been evaluated for eradication of Hhepaticus (67 68) Amoxicillin- or tetracycline-based tripletherapy (amoxicillin or tetracycline in combination with metron-idazole and bismuth) given by oral gavage three times daily for 2weeks eradicated H hepaticus in 8- to 10-week-old AJCr mice(67) In a second study (68) AJCr male mice receivedamoxicillin-based triple therapy in drinking water or by oral gav-age or received tetracycline-based triple therapy in the drinkingwater Female DBA2J mice received amoxicillin-based tripletherapy in a specially formulated dietary wafer or by oral gavageor received enrofloxacin in drinking water All treatments weregiven for a 2-week period One month after treatment Hhepaticus was recovered from the liver cecum or colon of micereceiving amoxicillin- or tetracycline-based triple therapy indrinking water but not from those sites in mice receivingamoxicillin-based triple therapy by oral gavage Helicobacterhepaticus was not recovered from DBA2J mice receivingamoxicillin-based triple therapy in dietary wafers or by oral gav-age but was recovered from mice receiving enrofloxacin in waterAlthough these results indicate that amoxicillin-based tripletherapy administered in the diet or by oral gavage could be effec-tive in eradicating H hepaticus eradication of H hepaticus wasnot successful in a breeding colony of immunodeficient RAG1--

mice treated with amoxicillin-based triple combination in dietarywafers or in mice of various genetic backgrounds that had re-ceived medicated wafers 4 months after their manufacture Diar-rheic SCID mice infected with H rodentium and H bilisresponded clinically to medicated wafers but infection with bothhelicobacters persisted (215) Thus immunocompetence of themice and stability of antibiotic activity in wafers are necessaryconsiderations (68) Amoxicillin-based therapy also was effica-cious in preventing hepatitis and typhlitis in SCID mice infected

143

with H hepaticus but eradication data were not reported (205)Colony management Fecal-oral spread is the route of natu-

ral acquisition of infection with helicobacters in rodents and Hhepaticus (and others) can be transferred on soiled bedding (154260) Thus biocontainment necessary to prevent horizontaltransmission of H hepaticus within a mouse colony must includeprevention of fecal-oral contact Use of filter-topped caging trans-fer of mice by use of disinfected forceps and handling uninfectedmice before known infected mice or mice of unknown Helicobacterstatus provide a barrier to horizontal transmission (259)

Use of Sentinel Mice to Monitor forHelicobacter spp

Outbred Tac(SW)fBR sentinel mice have been used to monitorcolonies by use of PCR analysis and serologic testing for infectionacquired from colony mice shedding H hepaticus H rodentiumand H bilis (260) The experimental design incorporated an ongo-ing colony health surveillance program that required systematictransfer of dirty bedding from cages housing colony mice to thosehousing sentinel mice Screening colony mice by use of species-specific PCR analysis of cecal scrapings indicated that Hhepaticus and H rodentium were most prevalent Low incidenceof H bilis was noted Concurrence of Helicobacter infection de-tected by use of a PCR-based assay between colony mice and sen-tinels exposed through dirty bedding was 82 for H hepaticus88 for H rodentium and 94 for H bilis The anti-helicobacterserum IgG ELISA had high sensitivity but low specificity in de-tecting Helicobacter infection as early as one month after firstexposure to dirty bedding Low sensitivity may be related to thehigh antigenic challenge that sentinel mice experience when ex-posed long term to dirty bedding from a large number of colonymice Thus the high sensitivity of the serum ELISA predictsHelicobacter infection but the specificity of the assay was too lowfor reliable identification of Helicobacter species necessitatingthe use of culture and PCR analysis for confirmation

Transmission of H hepaticus infection to sentinel C57BL6Ncrmice was detected by use of PCR analysis as soon as 2 weeks af-ter exposure to dirty bedding twice a week (154) By 4 weeks afterexposure all sentinels were PCR positive and by 6 weeks all sen-tinels had seroconverted to H hepaticus as measured by use ofan ELISA Both of these protocols for sentinel exposure (154 260)relied on well-defined conditions consisting of controlled amountsand frequency of dirty bedding exposure as well as which cagesof colony mice were sampled As for other infective agents man-agement programs that depend on sentinel exposure to dirty bed-ding for reliable surveillance of the Helicobacter infection statusof rodent colonies must standardize their methods and poten-tially validate them using controlled studies Increased exposureto dirty bedding or testing an increased number of sentinel miceper time point may improve the sensitivity of surveillance pro-grams that depend on similar protocols

Helicobacter Infections in Non-RodentSpecies

Humans Helicobacter pylori was first identified in 1982 as aninfectious cause of chronic active gastritis and peptic ulcer Hpylori has been recognized as one of the most common human in-fectious diseases infecting 50 or more humans worldwide (see13 for review) Helicobacter pylori was classified by the WorldHealth Organization as a type-1 gastric carcinogen because of its

association with gastric adenocarcinoma and MALT lymphomain humans (128) Most H pylori-infected humans develop onlysubclinical gastritis but relevant disease with the risk of gastriccancer has an important impact on human health An enormousamount of investigation has examined the epidemiology treat-ment prevention and pathogenesis of this important bacterialinfection The literature base covering various aspects of H pyloriand associated disease syndromes has grown exponentially andis beyond the scope of this review Many of the features of H py-lori infection of humans are presented here in the context of us-ing animal models to study H pylori gastritis and gastric cancer

Far less is known about the enterohepatic helicobacters of hu-mans Helicobacter winghamensis was isolated from patientswith gastroenteritis including fever stomach malaise and diar-rhea (166) It is catalase urease alkaline phosphatase and ni-trate negative but oxidase and indoxyl acetate positive It growsat 37degC but not at 42degC and three isolates from two patients weresusceptible to nalidixic acid and cephalothin Sequence analysisof 16S rRNA indicates that the closest relative by phylogeneticanalysis is lsquoHelicobacter sp flexispirarsquo taxon 1 Helicobacterwinghamensis has one or two bipolar nonsheathed flagella how-ever periplasmic fibers a characteristic of the knownlsquoHelicobacter sp flexispirarsquo taxa are absent Other human iso-lates such as H cinaedi H fennelliae H canis H pullorum Hcanadensis and H bilis will be discussed in other sections

Natural Helicobacter Infections inNonhuman Primates

Helicobacter pylori Gastric and large intestinal Helicobacterinfections have been documented in Old and New World nonhu-man primates To our knowledge an association between clinicalsigns of disease and infection of nonhuman primates has notbeen reported Nonhuman primates commonly have subclinicalbut histologic evidence of chronic gastritis and are oftencoinfected with H pylori and other large spirals in the stomachsuch as lsquoH heilmanniirsquo (158) Gastric body and antral biopsyspecimens from 23 clinically normal young adult Chinese rhesusmacaques were evaluated for H pylori infection by use of PCRanalysis culture urease testing histologic evaluation and sero-logic testing (119) Results of PCR analysis were positive for 91of the animals 52 were culture positive urease tests were posi-tive in all animals and H pylori was evident histologically in48 of the macaques An ELISA using homologous rhesus H py-lori antigen and anti-monkey conjugate was superior in sensitiv-ity (90 of PCR-positive animals) to a commercial ELISAmarketed for humans

Because H pylori is typically acquired by humans early in lifenatural acquisition of H pylori infection has been examined in so-cially housed newborn rhesus macaques (229) By 3 months of age8 of 20 newborns were culture positive for H pylori with higher re-covery of H pylori from newborns from infected dams than fromuninfected dams By 1 year of age prevalence reached 90

Helicobacter cinaedi This helicobacter was implicated as acause of enteritis and bacteremia in immunocompromised hu-mans (1) but it also infects children and occasionally immuno-competent adults Helicobacter cinaedi was recently isolated fromthe colon liver and mesenteric lymph nodes of a 2-year-oldrhesus macaque with hepatitis and chronic diarrhea (83) Mild tomoderate biliary hyperplasia and hypertrophy with periportalinflammation and fibrosis were observed in the liver and diffuse

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

144

chronic typhlocolitis and glandular hyperplasia were noted in thelarge intestine Helicobacter cinaedi also has been recovered fromfeces of clinically normal rhesus macaques (516) indicating thatthese captive animals may be a reservoir for human infectionwith H cinaedi (63) or conversely like H pylori they may haveacquired the organism in the wild from humans Unfortunatelycolonic biopsy specimens were not taken to ascertain whethercolitis was present

Novel Helicobacter spp Novel Helicobacter spp have beenisolated from captive macaques with chronic moderate to severetyphlocolitis reactive lymphoid hyperplasia and multifocalmicroabscesses (84) Results of microbiological examination forSalmonella spp Shigella spp Clostridium difficile A and B tox-ins acid-fast bacteria herpes B virus simian T-lymphotrophicvirus simian retrovirus and simian immunodeficiency viruswere negative The helicobacters were recovered by culture of co-lonic biopsy specimens from 6 clinically affected and 2 clinicallynormal monkeys Nine strains were characterized biochemicallyand two separate biotypes were identified by use of 16S rRNAanalysis Type-1 strains from animals with subclinical colitis andtype-2 strains from animals with diarrhea and colitis differed incatalase activity ability to reduce nitrate to nitrite and suscepti-bility to cephalothin Complete 16S rRNA analysis of 5 strainsindicated that the organisms were newly recognized Controlledstudies are needed to determine whether Helicobacter spp con-tribute to chronic colitis in macaques Further microbiologicaland histologic analyses of clinical cases may prove useful in un-derstanding human IBD

Cotton-top tamarins (CTTs) can develop clinical signs of dis-ease that include weight loss chronic diarrhea and rectal bleed-ing with lesions similar to those of human ulcerative colitisFrom 25 to 40 of affected animals develop colon cancer within 2to 5 years A novel helicobacter with fusiform shape bipolar fla-gella and periplasmic fibers was isolated from feces collectedfrom a colony of CTTs with episodic colitis and colon cancer (207)It grew under microaerobic conditions at 37 and 42degC was ureasenegative and was positive for catalase and oxidase On the basisof other biochemical testing antibiotic susceptibilities and 16SrRNA gene sequence analysis the organism was classified as anovel helicobacter The cause of the syndrome in CTTs is unknownalthough epidemiologic studies in CTTs incriminate an infectiouscause (136) Identification of a novel helicobacter in affected ani-mals warrants further study to determine its potential relationshipwith chronic colitis and progression to adenocarcinoma

Experimental Helicobacter Infection ofNonhuman Primates

Helicobacter pylori Studies of H pylori infections in nonhu-man primates have been valuable because of the close phyloge-netic relationship of macaques to humans despite thedisadvantages of natural infection with H pylori andor lsquoHheilmanniirsquo (49 50) Strains of H pylori in humans as well as inrhesus macaques are heterogeneous and are believed to arisefrom complex adaptive responses of H pylori to its host environ-ment This was addressed in a study of rhesus macaques firsttreated to eliminate natural H pylori colonization then chal-lenged with a mixture of 7 human strains (48) The DNA finger-printing analysis of isolates recovered from serial samplesindicated that multiple strains would co-colonize the stomach formonths before one or more strains would predominate Prior

natural colonization was not protective against H pylori chal-lenge and subsequent infection

Microarray analysis of H pylori DNA recovered from experi-mentally challenged macaques indicated that babA one of a fam-ily of outer membrane proteins that mediates attachment of Hpylori to the Lewis B blood group antigen on gastric epitheliumwas deleted and in some cases replaced by babB anuncharacterized but related protein (230) In some isolates thebabA gene was present but was not expressed because of phasevariation in nucleotide repeats in the 5-coding region Strainslacking babA did not adhere to Lewis B which is expressed onmacaque gastric epithelium Absence of babA and duplication ofbabB were also seen in H pylori isolates derived from humanclinical samples suggesting that this gene conversion event isnot unique to experimentally infected rhesus monkeys These re-sults help support the hypothesis that H pylori regulates outermembrane protein expression in vivo by using antigenic varia-tion and phase variation potentially to facilitate adherence of Hpylori to the gastric epithelium and promote colonization

Rhesus macaques have also been used in therapeutic vaccinetrials Immunization with recombinant H pylori urease de-creased colonization following experimental infection of rhesusmacaques and was correlated with reduced gastritis (150)

Helicobacter cinaedi and H fennelliae The latterhelicobacter has no known animal reservoir but can be used toexperimentally infect nonhuman primates Experimental chal-lenge of 12- to 25-day-old pig-tailed macaques (Macacanemestrina) with H cinaedi or H fennelliae resulted in watery orloose feces without associated fever or fecal leukocytes within 3 to7 days after inoculation Results of culture of fecal specimens re-mained positive for three weeks after inoculation despite resolu-tion of the clinical illness All animals became bacteremic andsome had clinical signs of septicemia Acute inflammation wasobserved histologically in 1 of 5 rectal biopsy specimens and hy-perplasia of Peyerrsquos patches was noted in one additional animalthat was necropsied (64)

Natural Helicobacter Infections in FerretsHelicobacter mustelae Helicobacter mustelae was the first

non-human gastric Helicobacter isolate and shares many bio-chemical molecular and phenotypic characteristics with H py-lori (75) It is a gram-negative curved- to rod-shapedmicroaerophilic bacterium that like H pylori adheres to the gas-tric mucosa and is strongly oxidase catalase and urease positiveThe prevalence of H mustelae infection in adult domestic ferrets(Mustela putorius furo) approaches 100 and the organism hasbeen isolated from mink (241) Similar to that of H pylori infec-tion in humans persistent colonization of H mustelae is acquiredat an early age and is closely associated with development ofchronic active gastritis mucosal ulceration and experimentallywith promotion of carcinogenesis (96) The ferret is the only do-mestic animal with Helicobacter-induced gastritis associatedwith duodenal and gastric ulceration all sequelae of human Hpylori infection Both H pylori and H mustelae produce ureaseand increase blood gastrin concentration which alters mucosalpH and increases gastric acid production (189) The immune re-sponse of ferrets to H mustelae and of humans to H pylori is in-capable of eliminating active infection or preventing reinfectionafter antibiotic therapy

Gastric lymphoma resembling MALT lymphoma of humans

145

infected with H pylori was reported in four ferrets infected withH mustelae (58) Two ferrets developed low-grade small-cell lym-phoma and two ferrets developed high-grade large-cell lym-phoma at the lesser curvature of the pyloric antrum which is thesite of the most intense H mustelae-induced gastritis Premalig-nant gastric lesions associated with neoplasia were described byFox and co-workers (75) soon after the discovery of H mustelae asthe cause of chronic gastritis in ferrets Helicobacter mustelae-in-duced gastritis results in glandular atrophy of the proximal por-tion of the antrum which is associated with progression togastric adenocarcinoma in ferrets (76) and humans infected withH pylori (27) Thus the H mustelae-infected ferret can be used tomodel the progression of Helicobacter-associated chronic gastritisto the potential sequela of gastric cancer

Novel Helicobacter spp Hepatobiliary disease potentiallyrelated to Helicobacter infection was diagnosed in a single colonyof pet ferrets (104) Eight of 34 ferrets 5 to 8 years old andsampled over multiple years developed signs of chroniccholangiohepatitis There was histologic evidence of hepatocellu-lar hyperplasia progressing to carcinoma in two ferrets Bacteriawith morphology consistent with that of helicobacters were ob-served histologically in the liver of three ferrets including thetwo with carcinoma Sequence analysis of a 400-bp PCR productderived from fecal bacteria from one of the ferrets revealed 98and 97 similarity to H cholecystus and a novel strainHelicobacter sp 266-1 (90) respectively The prevalence of hepaticdisease in this single ferret colony suggests an infectious causethus the role of Helicobacter spp in ferret hepatitis and cancerwarrants further study Novel helicobacters have also been iso-lated from feces of wild mustelids California sea otters

Experimental Helicobacter-AssociatedGastritis in Ferrets

Experimental infection of the ferret with H mustelae is amodel of H pylori infection in humans (75 89) Helicobactermustelae infection in ferrets has been used to model potentialvirulence factors that influence colonization of the stomach andthe development of gastritis ulcers and gastric cancer Similar toH pylori in humans H mustelae adheres to ferret gastric tissuevia the formation of adhesion pedestals (179) Helicobactermustelae produces unique proteinacious surface ring structuresthat were investigated for their importance to gastric coloniza-tion Helicobacter mustelae-free ferrets were challenged withwild-type or surface ring isogenic mutants of H mustelae thenwere evaluated after 18 weeks Colonization and gastritis in fer-rets challenged with mutants was minimal compared with coloni-zation and more robust gastritis in animals infected withwild-type H mustelae This finding suggests that the novel sur-face proteins are important for H mustelae colonization of theferret stomach (183) As with H pylori flagellar motility of Hmustelae is an important virulence factor for colonization andpathogenesis of gastritis in ferrets Challenge of H mustelae-freeferrets with single or double isogenic mutant strains of the FlaAand FlaB flagellin proteins resulted in no colonization in doubleknockouts and low colonization in single mutants (2) Coloniza-tion with isogenic mutants of flagellar proteins slowly increasedover three months and the degree of gastritis was correlated withthe colonization burden of H mustelae Isogenic flagellum-nega-tive mutants of H mustelae were assessed for their ability to ad-here to primary ferret gastric epithelial cells Compared with

wild-type strains mutants deficient in production of FlaA FlaB orboth flagellar proteins did not manifest reduction in adherence toprimary ferret gastric epithelial cells (25) These findings suggestthat flagella do not play a direct role in promoting adherence of Hmustelae to gastric epithelial cells but genes regulating flagellarbiosynthesis may also regulate production of an adhesin

Helicobacter pylori and H mustelae may partially evade thehost immune response through expression of Lewis blood groupantigens that mimic those of the host a mechanism known asmolecular mimicry Cross-reactive antibodies to epitopes ex-pressed by gastric helicobacters and host gastric mucosa maycontribute to development of chronic atrophic gastritis throughdestruction of parietal cells Helicobacter mustelae expresses ablood group-like antigen A as part of its lipopolysaccharide cross-reactivity of serum from H mustelae-infected ferrets with ferretgastric tissue but not with duodenal or colonic mucosa has beendocumented (32 33) Absorption of the antiserum with ferret andrabbit gastric tissue removed cross-reactive antibodies (33) butpre-absorption with whole H mustelae cells or ferret erythrocytesfailed to reduce antibody binding to ferret gastric mucosa (32)Thus ferrets naturally infected with H mustelae generate anti-bodies that react with gastric mucosa but these antibodies do notappear to be generated through a mechanism involving molecu-lar mimicry (32)

Urease activity was observed to be essential for colonization ofthe ferret stomach by H mustelae (3) Ferrets that were specificpathogen free (SPF) for H mustelae were orally dosed with wild-type H mustelae or an isogenic urease-negative strain of Hmustelae then were evaluated for 25 weeks In those dosed withthe urease mutant H mustelae failed to colonize and the animalsdid not develop gastritis The urease from H pylori has been usedas an antigen combined with the mucosal adjuvant cholera toxinto therapeutically immunize ferrets naturally infected with Hmustelae (34) By six weeks after oral immunization H mustelaewas eradicated in 30 of all immunized ferrets and gastritis alsowas reduced

The H mustelae ferret model has been used to evaluate antibi-otic therapies for eradication of gastric helicobacters (5 161 180)Triple therapy consisting of amoxicillin (30 mgkg of bodyweight) metronidazole (20 mgkg) and bismuth subsalicylate(175 mgkg) administered orally twice daily for 21 to 28 dayscleared H mustelae in 71 (180) and 100 (5) of treated ferretsrespectively

Ranitidine bismuth citrate clarithromycin or a combination ofboth drugs was administered orally for 14 days to 7-month-oldferrets with H mustelae infection that was documented by gas-tric biopsy and culture (161) Ferrets were euthanized from 4 to43 weeks after therapy Dosages of clarithromycin and ranitidinebismuth citrate that suppressed growth of H mustelae were 125and 24 mgkg respectively given orally every 8 h Infection wasnot eradicated in ferrets treated with ranitidine bismuth citratealone but was eradicated in all 6 ferrets treated withclarithromycin and ranitidine bismuth citrate and in 4 of 6treated with clarithromycin alone A decrease in susceptibility toclarithromycin was detected for H mustelae isolates obtained af-ter treatment Mild or moderate antral gastritis was observedeven in ferrets from which infection was eradicated

Ferret kits acquire H mustelae from their jill near the time ofweaning via fecal-oral exposure (90) Therapeutic vaccination ofyoung ferret kits was attempted to determine whether this natu-

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

146

ral route of transmission could be interrupted (263) Weanlingkits were orally dosed with a whole-cell sonicate of H mustelaeand the adjuvant muramyl dipeptide All kits became colonizednaturally with H mustelae and most developed mild to severegastritis and duodenitis Unexpectedly kits that received soni-cate and adjuvant developed significantly greater gastritis andduodenitis that led to mucosal ulceration in some animals In ret-rospect the mucosal damage associated with this vaccinationregimen potentially enhances the value of the ferret model forstudying duodenal ulceration secondary to gastric Helicobacterinfection

Natural Helicobacter Infections in Catsand Dogs

Helicobacter felis lsquoH heilmanniirsquo (bizzozeronii) lsquoHrappinirsquo Because many of the helicobacters in the stomach ofanimals have been isolated only recently many earlier reportsdescribing these bacteria were based on morphologic criteriaThree morphologic forms of these organisms were reported byLockard and Boler in dogs (Fig 6) (157) All three of these mor-phologically distinct organisms are now known to behelicobacters on the basis of results of 16S rRNA analysisLockard type 1 is a bacterium fully entwined with periplasmic fi-bers and is now known as lsquoH rappinirsquo a member of the Flexispiragroup also isolated from aborted sheep fetuses (14) and from in-testines of normal mice (208) lsquoHelicobacter rappinirsquo has been iso-lated from feces of normal dogs and their owners affected byenteritis (202) and from a bacteremic human (232) Lockard bac-terium type 2 also has periplasmic fibers but they are moresparsely distributed on the organism and can appear singly or ingroups of two three or four This organism has been isolated inculture from the stomach of cats and dogs and has been namedH felis (182) The third morphologically distinct organism type 3is the bacteria most commonly seen in the animal stomach (dogscats non-human primates cheetahs swine) and occasionally inthe human stomach This bacterium though tightly spiraleddoes not have periplasmic fibers and has been given variousnamesmdashlsquoGastrospirillum hominisrsquo or lsquoH heilmanniirsquomdashand mostrecently has been cultured from dogs and has been named Hbizzozeronii (120) This bacterium measures 03 by 5 to 10 micromand has 10 to 20 sheathed flagella at both ends of the cell

Helicobacter pylori Although spiral bacteria were first ob-served to infect the stomach of dogs and cats more than 100 yearsago there has been renewed interest stimulated by the discoveryof H pylori in humans as well as the report of endemic H pyloriin one commercial colony of cats bred for research (118) Similarto human H pylori infection chronic H pylori infection in catsresults in chronic diffuse lymphofollicular atrophic gastritis withareas of mucosal dysplasia in the antrum and predominantly su-perficial gastritis in the body and cardia (61) Colonization pat-terns of H pylori in the naturally infected cat and the cytokineresponse such as up-regulation of IL-8 and IL-1β are similar tothose observations in infected people and nonhuman primates(222) Natural infection with H pylori has not been observed indogs except for one recent report of H pylori DNA (notably notrecovered by culture) in biopsy specimens from four pet dogs (17)Polymerase chain reaction amplification of 16S rDNA yielded a400-bp product that was subsequently cloned into E coli thenwas re-amplified by use of PCR analysis and 16S rDNA frag-ments were analyzed by use of RFLP analysis Two sequences

obtained from different dogs were identical with the correspond-ing sequences of H pylori strains Three sequences had moderatesimilarity to H pylori (966 to 980) and one sequence was simi-lar to H salomonis (973)

Helicobacter felis This helicobacter was first isolated fromcats and is a large spiral organism with periplasmic fibers (Fig 7)(182) Infection of domestic cats and dogs with gastrichelicobacters other than H pylori are not well characterized Incats H felis induces lymphoid follicular hyperplasia mild gastri-tis and seroconversion but is associated with normal gastricsecretory function (221) Helicobacter felis has been important todevelopment of H pylori gastritis models because it persistentlycolonizes mice and causes robust gastritis that recently was re-ported to progress to gastric cancer (253) The gastric mucosa ofhealthy pet cats is commonly colonized with an (until recently)uncultivatable lsquoH heilmanniirsquo (aka H bizzozeronii) that inducesmoderate or mild gastritis and has heterogeneity in its 16SrRNA sequence (176) To our knowledge the relationship be-tween the associated gastritis (inflammation atrophy of glandslymphofollicular hyperplasia) and common clinical syndromes inpets (vomiting anorexia) has not been established (175) Theprevalence of gastric Helicobacter spp in dogs and cats is highirrespective of clinical signs of disease and similar to that in hu-mans mode of transmission is unclear Urea breath testing andserologic plus urease testing histologic and cytologic examina-tions PCR analysis and culture of biopsy specimens are useful

Figure 6 Collage of the original photomicrographs of bacteria in thedog stomach as seen by electron microscopy taken from Lockard andBoler (157) Type 1A is lsquoH rappinirsquo-like Type 2A is H felis-like andType 3A is lsquoH heilmanniirsquo-like Arrows indicate periplasmic fibers in1A and 2A and suspected fibers in 3A Reprinted with permission ofComparative Medicine

147

diagnostically Therapeutic trials in dogs and cats have not beensuccessful in avoiding recrudescence or reinfection Gastrin secre-tion appears unaffected by Helicobacter infection (222) and a re-lationship of infection to gastrointestinal tract ulcers has notbeen reported

Miscellaneous helicobacters of cats and dogs Isolatedcase reports associating helicobacters with gastroenteritis inyoung cats and dogs have appeared in the literature Both Hcinaedi and lsquoH rappinirsquo along with C upsaliensis and twoAnaerobiospirillum spp were isolated from a puppy with bloodydiarrhea (170) Treatment with amoxicillin was successful sug-gesting a bacterial cause for the diarrhea A spiral-shaped bacillithat ultrastructurally resembled lsquoH rappinirsquo was found to heavilycolonize the stomach of a kitten with mucohemorrhagic enteritisit also was found in the small intestine and cecum of the kitten(140) lsquoHelicobacter rappinirsquo is a spiral-shaped helicobacterknown to colonize the intestine of dogs and mice The name isprovisional and is given to a taxonomic grouping of gram-nega-tive microaerophilic motile spindle-shaped microorganismswith spiral periplasmic fibers and bipolar tufts of sheathed fla-gella Sequencing of 16S rRNA has indicated that this formerFlexispira is a member of the genus Helicobacter but has notbeen formally named probably because lsquoH rappinirsquo strains repre-sent at least 10 Helicobacter taxa (40)

Helicobacter canis has been isolated from humans (70) and ex-otic Bengal cats (65) with diarrhea and from a puppy with signsof gastroenteritis in combination with histologic evidence ofWarthin-Starry-positive spiral to curved bacteria observed in alesion of necrotizing hepatitis (81) Thus H canis is associatedwith inflammation of the colon and potentially the liver of selecthosts particularly those individuals with immune dysregulationFurther studies are needed to determine the importance of Hcanis as a primary enterohepatic pathogen in dogs and cats and

the possible zoonotic threat to humansHelicobacter acinonychis (formerly H acinonyx) a species

closely related to H pylori was isolated from the stomach of chee-tahs (52) and from two Sumatran tigers with chronic gastritis(209) Helicobacter acinonychis was thought to contribute to anoutbreak of vomiting in a group of captive cheetahs (55) Histo-logic examination revealed chronic gastritis characterized by in-filtration of lymphocytes and numerous plasma cells epithelialerosions and mucosal hyperplasia Two groups of H acinonychisstrains were identified by use of randomly amplified polymorphicDNA fingerprinting analysis and gene sequencing one groupfrom cheetahs in a US zoo and two lions in a European circusand the other group from a tiger and a lion-tiger hybrid in thesame circus Results of PCR analysis and DNA sequencing indi-cated that each strain lacked the cag pathogenicity island andcontained a degenerate vacuolating cytotoxin (vacA) gene Micewere dosed with H acinonychis and H pylori and they becamepersistently colonized several variant strains causing a mixedinfection were recovered after 2 months (35) The ability of Hacinonychis to recombine with H pylori in vivo will allow furtherstudies of virulence mechanisms and genome evolution

During a survey study of gastric helicobacters in dogs Hsalomonis sp nov a canine gastric isolate related to H felis andH bizzozeronii was identified (132) Helicobacter salomonis wasdistinguished from H felis and H bizzozeronii by its morphology(slight spiral 5 to 7 microm long by 08 to 12 microm wide) and unusualslow wavelike motility Phylogenetic analysis by use of 16S rDNAsequence comparison revealed H salomonis to be a distinctHelicobacter species Reports of associated clinical disease havenot been published

Novel Helicobacter spp Cats have been colonized withmixed infections of enteric Helicobacter and Campylobacter spp(211) Feces from commercially bred cats contained isolates iden-tified as Campylobacter-like organisms on the basis of biochemi-cal and phenotypic characteristics Use of PCR analysis withprimers specific for Helicobacter and Campylobacter spp at thegenus level identified mixed infections Use of species-specificprimers identified coinfection with C helveticus C upsaliensisand C jejuni Five RFLP patterns were obtained by digestion ofthe 12-kb PCR products of the 16S rRNA isolated from 19Helicobacter isolates The RFLP and sequence analyses verifiedthat two isolates clustered with lsquoFlexispirarsquo taxon 8 one clusteredwith H bilis one with H canis and the remaining appearedclosely related to a novel helicobacter H marmoatae isolatedfrom a woodchuck (93) These results illustrate the potential un-certainty in identifying isolates solely on the basis of chemicaland phenotypic characteristics The clinical relevance of entericHelicobacter and Campylobacter coinfection in cats is unknownImportantly there is the potential for zoonotic transmission Sys-tematic evaluation of dogs for enterohepatic helicobacters has notbeen reported to our knowledge

Experimental H pylori and H felisInfection of Cats and Dogs

Similar to that in dogs experimental infection of the cat withH pylori results in more severe pathologic changes than does in-fection with H felis Cats free of known gastric helicobacters in-cluding H pylori with normal gastric mucosae were susceptibleto colonization by the same H pylori strain which was negativefor the cagPAI (cytotoxin gene-associated pathogenicity island)

Figure 7 Morphology of H felis as imaged by electron microscopy (Aand C) and phase contrast microscopy (B) demonstrating spiral mor-phology and periplasmic fibers Reproduced with permission from DrAdrian Lee Bar = 02 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

148

that was isolated from naturally infected cats from a commercialvendor (72 118) All cats were persistently colonized over a studyperiod of 7 months and developed multifocal gastritis consistingof lymphoid aggregates plus multiple large lymphoid nodulesthat were most noticeable in the antrum Organisms with mor-phology consistent with that of H pylori were distributed in glan-dular crypts of the antrum and two of four cats had high IgGtiter to H pylori The H pylori recovered at the end of the studywas confirmed to be the same as the inoculating strain by use ofRFLP analysis specific for the flaA gene (72) Similar resultswere obtained after experimental infection of cats with a humancagA+ strain of H pylori (188) Results of experimental studieshave indicated that H pylori can be recovered from salivary se-cretions gastric juice and gastric tissue by use of culturing andthe organism has been detected in dental plaque and feces of in-fected cats by use of PCR analysis (91) Gnotobiotic beagle pupswere orally challenged with H pylori at 7 days of age and werepersistently colonized for 30 days but at a lower density thanthat typically seen in humans Small (lt 1 mm) lymphoid follicleswere grossly visible in the stomach and histologic lesions con-sisted of focal to diffuse lymphoplasmacytic infiltrates with fol-licle formation and mild to moderate infiltration of neutrophilsand eosinophils in the gastric lamina propria A second group ofdogs with exposure contact only also became colonized with theH pylori during a 30-day study period (195)

Natural Helicobacter Infections in SwinelsquoHelicobacter suisrsquo Domestic swine were described as a pri-

mary host of lsquoH heilmanniirsquo (203) as are cats dogs (176) andrarely humans (240) lsquoHelicobacter heilmanniirsquo in pigs was re-named lsquoH suisrsquo on the basis of analysis of samples obtained frompig stomach using 16S rRNA sequencing results of which iden-tified the isolates as a novel species (23 38) The potentialzoonotic connection between gastric helicobacters of pigs andfarmers was highlighted by the observation of high prevalence inhumans of lsquoH heilmanniirsquo-associated gastritis in a small pre-dominantly rural area (240) The pig stomach is also colonizedwith Campylobacter spp which has complicated the interpreta-tion of studies Characterization of large gastric spirals that ap-pear to infect most slaughter pigs has been hampered by theinability to successfully recover these organisms by use of cultur-ing (169) Thus genus- and species-specific PCR assays in combina-tion with RFLP and sequence analyses are the most sensitive andreliable diagnostic methods for use in swine studies (121 203)

lsquoHelicobacter suisrsquo has been linked to a clinical condition infarm pigs ulceration of the pars esophagea (194) which histori-cally has been attributed to a complex interaction of dietary par-ticle size gastric fluidity dietary carbohydrate content andpresence of certain species of commensal gastric organisms ca-pable of fermenting dietary carbohydrates Unlike that in hu-mans the relevance of the role of helicobacters in development ofporcine gastric ulcers is undefined The causal relationship be-tween lsquoH suisrsquo and gastritis or ulceration in the pig remains to beclarified This organism colonizes mainly in the antral and bodymucosa whereas ulceration develops in the esophagus-cardiajunction

Helicobacter pametensis This helicobacter has been isolatedfrom swine feces but birds are thought to be the primary host

lsquoHelicobacter sp flexispirarsquo taxa Potentially 10 distinctmembers of the lsquoFlexispira rappinirsquo taxa have been described us-

ing 16S rRNA sequencing (40) and include fusiform-shaped bac-teria with helical periplasmic fibrils and bipolar tufts of sheathedflagella Three lsquoFlexispira rappinirsquo members that have been for-mally renamed as Helicobacter spp include H bilis Htrogontum and H aurati Evaluation of gastric organisms fromFinnish farm pigs that were characterized by use of phenotypicbiochemical and molecular techniques revealed close homologyto H flexispira taxa 4 and 5 and H trogontum (taxon 6) but aless-close relationship to other members including taxa 1ndash3 and8 H bilis and H aurati (121)

Experimental Infection of Swine withHelicobacter pylori

Germ-free and gnotobiotic pigs (54) SPF farm pigs (192) andminiature pigs (141) have been used to study the pathogenesis ofH pylori-induced gastritis Gnotobiotic piglets have been used todocument that the humoral response to H pylori is initiatedwithin the gastric compartment and matures over time to an IgA-dominated mucosal and a systemic IgG-dominated humoral im-mune response (142) Germ-free neonatal piglets infected withpiglet-adapted H pylori strain 26695 were used to document thatneither the cagPAI nor the ability to induce IL-8 in vitro is essen-tial for colonization or neutrophilic inflammation in vivo (54)

Natural Helicobacter Infections in SheeplsquoHelicobacter rappinirsquo Vertical transmission of a

helicobacter was first proposed when lsquoH rappinirsquo was suspectedto have crossed the placenta and caused hepatic necrosis in fe-tuses during natural infection in presumably immunocompetentsheep The same isolate given as an experimental challenge topregnant guinea pigs resulted in liver necrosis in the fetuses (14)lsquoHelicobacter rappinirsquo was reported to cause recurrent bacteremiain an adult human dialysis patient which suggests that lsquoH rap-pinirsquo is a pathogen in immunocompromised patients (232) Thepotential for zoonotic transmission of lsquoH rappinirsquo between hu-mans and dogs has been reviewed (71)

Helicobacter pylori A higher incidence of H pylori infectionin a group of sheep herders compared with that in the neighbor-ing population suggested a zoonotic connection with sheep (or atleast the occupation) but this remains unresolved (181)

Natural Helicobacter Infections in CattlelsquoHelicobacter bovisrsquo lsquoHelicobacter bovisrsquo is the proposed

name for a helicobacter detected in the abomasum of cattle inEurope (39) Although culture of lsquoH bovisrsquo has not been success-ful helicobacter-like organisms were detected in abomasal biopsyspecimens from adult cattle by use of electron microscopy andbiochemical and immunohistochemical methods Bacterial 16SrDNA was amplified by use of PCR analysis and was sequencedPhylogenetic analysis placed the organism corresponding to thereference sequence R2XA within the genus Helicobacter A diag-nostic PCR assay was designed that differentiated all of the bo-vine 16S rDNA sequences from Helicobacter and Wolinellaspecies Helicobacter bilis appears to be the most related speciesbut sequence similarity is low (928) indicating that lsquoH bovisrsquo isa novel Helicobacter species In a separate survey of slaughtercattle lsquoH bovisrsquo was detected in 85 of the animals (114) In thesame survey the abomasum of goats was tested and the resultsindicated failure to detect Helicobacter infection

149

Natural Helicobacter Infections in BirdsHelicobacter pullorum This helicobacter was isolated from

the liver duodenum and cecum of chickens and from humanswith gastroenteritis (233) Supported by relative DNA homologyand similar protein electrophoretic patterns 16S rRNA sequenceanalysis identified 7 strains as belonging to a single Helicobacterspecies Helicobacter pullorum is biochemically differentiatedfrom other helicobacters and has typical ultrastructure exceptfor a non-sheathed flagellum as seen by use of electron micros-copy Similar to H fennelliae in immunocompromised humans orH cinaedi in a range of different hosts H pullorum represents anon-gastric urease-negative Helicobacter species that colonizesthe large intestine Because H pullorum has been isolated in cul-ture from feces of diarrheic humans and has been isolated frompoultry and poultry products it may cause an associated zoonosis(12) A species-specific PCR assay for H pullorum is availableand will further facilitate identification of the helicobacter andclarification of its epidemiology A putative virulence factor in Hpullorum is CDT (265) Isolates of H pullorum from birds andhumans have produced CDT activity in cell culture and are PCRpositive for the CDT genes

Helicobacter canadensis This helicobacter has been impli-cated in a human bacteremic patient and was cultured from aseries of 11 patients with diarrhea in Canada (74 243)Helicobacter canadensis was previously characterized as Hpullorum by determination of its biochemical profile and by useof fatty acid and RFLP analyses In a reassessment of this classi-fication (74) four isolates differed biochemically from Hpullorum by their inability to hydrolyze indoxyl acetate and theirresistance to nalidixic acid Using complete 16S rRNA and RFLPanalyses these four strains clustered near H pullorum but hada sequence difference of 2 thereby representing a novelhelicobacter renamed as H canadensis Other investigators re-examined the taxonomic position of Campylobacter-like isolatesrecovered from barnacle geese (Branta leucopsis) and Canadageese (Branta candensis) (251) From a collection of 21 isolates 7strains were analyzed by use of extensive phenotypic testing andfour strains were characterized by use of 16S rRNA sequenceanalysis The results identified the bird isolates as H canadensisthus geese are an animal reservoir for this potentially zoonotichelicobacter

Helicobacter pametensis An organism in fecal isolates fromwild birds and domestic swine was identified as a novelhelicobacter with the suggested name of H pametensis Thishelicobacter was differentiated from other known Helicobacterspecies by use of full 16S rRNA sequencing and on the basis ofunique biochemical and morphologic features (42) The potentialfor H pametensis to cause disease is unknown Two additionalputative Helicobacter species from birds were phenotypicallysimilar to H pametensis but had a different biochemical profileincluding being positive for urease and hydrolysis of indoxyl ac-etate and resistant to cephalothin Because only a limited num-ber of isolates were characterized they were not formally namedbut are referred to as Helicobacter sp lsquoBird-Brsquo and Helicobactersp lsquoBird-Crsquo

Natural Helicobacter Infections inWoodchucks

Helicobacter marmotae The association between wood-chuck hepatitis virus (WHV) infection of Marmota monax and

the high incidence of hepatocellular carcinoma in woodchucks isa model for hepatitis B virus-associated hepatocellular carcinomaof humans Liver specimens from WHV-infected woodchucks 50of which had viral-associated hepatic tumors and liver speci-mens from uninfected woodchucks without tumors werescreened for Helicobacter infection by use of culture and PCRanalysis with Helicobacter genus- and species-specific primers(93) Under microaerobic culture one liver specimen from 10 tu-mor-positive woodchucks yielded a urease- catalase- and oxi-dase-positive bacterium typical of an enterohepatic helicobacter(Fig 8) Nine of 10 tumor-bearing liver specimens and 5 of 10 tu-mor-free specimens were PCR positive yielding an expected1200-bp product which was further verified to represent a novelHelicobacter sp by use of Southern blot hybridization and se-quence analysis Using 16S rRNA species-specific primers de-signed for the novel isolate two additional liver specimens fromthe nontumor group had PCR-positive amplicons confirmed byuse of Southern blot hybridization By use of 16S rRNA analysisand on the basis of biochemical and phenotypic characteristicsthe organism was classified as a novel Helicobacter sp and wasnamed H marmotae (93) Four additional isolates from cat feceswere found to be identical to the woodchuck isolate by use of bio-chemical phenotypic and 16S rRNA analyses Controlled chal-lenge studies are required to ascertain whether H marmotaepromotes hepadnavirus-associated tumors in woodchucks orcauses enterohepatic disease in cats

Natural Helicobacter Infections inDolphins and Whales

Helicobacter cetorum For decades gastric ulcers have beenreported in cetaceans with clinical signs of disease including

Figure 8 Morphology as imaged by electron microscopy of H marmotaeisolated from woodchucks and cats H marmotae measures approxi-mately 02 by 15-5 microm

Natural and experimental Helicobacter infections

Vol 54 No 2Comparative MedicineApril 2004

150

inappetance lethargy and anorexia Ulcers were previously asso-ciated with parasitic infections or had undefined causes FiveHelicobacter isolates have been obtained from dolphins (122) andwhales (125) both wild and captive animals in habitat rangingfrom Boston to Hawaii These isolates are identical and novel asevidenced by unique RFLP patterns from digestion of a 12-kbPCR product using Helicobacter genus-specific primers and fur-ther sequencing of 16S rRNA Positive culture results have beenobtained for fecal and biopsy specimens from the gastric mucosain animals with clinical signs consistent with gastric diseaseThis novel helicobacter now formally named H cetorum poten-tially causes gastritis and ulcers in marine mammals Positivefecal culture results indicate potential fecal-oral transmissionComparison of minimally invasive techniques for detecting theprevalence H cetorum in wild dolphins was undertaken as partof a long-term health study of the animals off the coast of Florida(123) Using serum samples and fecal specimens results of sero-logic testing culture and PCR analysis using genus- and Hcetorum-specific primers and Southern blot hybridization indi-cated at least 50 prevalence of H cetorum infection in thisgroup of wild dolphins

Novel helicobacters isolated from pinnipeds Novelhelicobacters have been isolated from harp seals and Californiasea lions Two novel helicobacters were isolated in culture fromthe gastric mucosa of four harp seals (Phoca groenlandica)stranded on the Massachusetts coastline (124) Both isolateswere catalase and oxidase positive One isolate was fusiformshaped with Flexispira morphology and was urease positive andthe second was spiral shaped and was urease negative Mild dif-fuse lymphoplasmacytic gastritis in the superficial mucosa of thepyloric antrum was associated with slender spiral and fusiform-shaped bacteria detected by use of the Warthin-Starry stain Onthe basis of results of 16S rRNA analysis the urease-negative iso-late clustered with H canis and the urease-positive isolate clus-tered with other isolates from sea lions and sea otters Thesefindings suggest that Helicobacter spp play a role in gastrointes-tinal tract diseases of pinnipeds

ConclusionThe genus Helicobacter has rapidly expanded due to the isola-

tion of new species from a wide range of animals Many domesticand wild animal species have not been systematically evaluatedfor Helicobacter infections therefore more novel isolates can beanticipated with future efforts The genus now includes 26 for-mally named species as well as numerous other novelhelicobacters currently being characterized Continued study ofthe genus Helicobacter is yielding new information for varied dis-ciplines within biology and medicine The zoonotic potential ofHelicobacter spp has been established but is likely under re-ported Diagnostic evaluations of human and veterinary patientswill increasingly include Helicobacter-associated disease in thedifferential list of potential diagnoses Advanced molecular tech-niques have given investigators the ability to study these fastidi-ous organisms when culture has been difficult or appearedunachievable The advances of evaluating host and bacterial geneexpression using microarray technology is in its infancy and willbe very applicable to the study of interactions betweenHelicobacter spp and their hosts Genomic studies of Helicobacterspp as pathogens and of their hosts will no doubt shed light oncritical genes and associated molecular events that could be tar-

gets for preventing or treating infectious diseases in the futureAs examples new insights are emerging concerning thedysregulated host response to enteric flora thought to be the ba-sis for IBD of humans Particularly noteworthy mouse models ofHelicobacter-associated disease are strengthening the link be-tween chronic infections and the associated tissue damage fromchronic inflammation and the progression of dysplastic lesions tocancer in the liver and gastrointestinal tract As more is learnedabout the helicobacters that naturally infect the species that bio-medical research historically has and will continue to rely on aspredictable models of human disease management of helicobacter-free research colonies will continue to gain in importance

AcknowledgmentsSupported in part by the ACLAM Foundation (MTW JGF) the Henry

and Lois Foster Grant (MTW JGF) RO1 CA 67529 CA 87958 AI37750AI50952 P01 CA 26731 and RR07036 from the National Institutes ofHealth (JGF) and grants from Astra Glaxo Merck and Pfizer Labora-tories (JGF) The authors also gratefully acknowledge the indispens-able contributions of colleagues in our laboratories as well as those ofour collaborators

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Dis 19112-1152 Andrutis K A J G Fox D B Schauer R P Marini X Li L

Yan C Josenhans and S Suerbaum 1997 Infection of the fer-ret stomach by isogenic flagellar mutant strains of Helicobactermustelae Infect Immun 651962-1966

3 Andrutis K A J G Fox D B Schauer R P Marini J CMurphy L Yan and J V Solnick 1995 Inability of an isogenicurease-negative mutant stain of Helicobacter mustelae to colonizethe ferret stomach Infect Immun 633722-3725

4 Aras R A J Kang A I Tschumi Y Harasaki and M JBlaser 2003 Extensive repetitive DNA facilitates prokaryoticgenome plasticity Proc Natl Acad Sci U S A 10013579-13584

5 Batchelder M J G Fox A Hayward L Yan B Shames JC Murphy and L Palley 1996 Natural and experimentalHelicobacter mustelae reinfection following successful antimicro-bial eradication in ferrets Helicobacter 134-42

6 Beckwith C S C L Franklin R R Hook Jr C L Besch-Williford and L K Riley 1997 Fecal PCR assay for diagnosis ofHelicobacter infection in laboratory rodents J Clin Microbiol351620-1623

7 Beckwith C S D J McGee H L Mobley and L K Riley2001 Cloning expression and catalytic activity of Helicobacterhepaticus urease Infect Immun 695914-5920

8 Berg D J N Davidson R Kuhn W Muller S Menon GHolland L Thompson-Snipes M W Leach and D Rennick1996 Enterocolitis and colon cancer in interleukin-10-deficientmice are associated with aberrant cytokine production and CD4(+)TH1- like responses J Clin Invest 981010-1020

9 Berg D J N A Lynch R G Lynch and D M Lauricella1998 Rapid development of severe hyperplastic gastritis with gas-tric epithelial dedifferentiation in Helicobacter felis-infected IL-10(--) mice Am J Pathol 1521377-1386

10 Bergin I L B J Sheppard and J G Fox 2003 Helicobacterpylori infection and high dietary salt independently induce atro-phic gastritis and intestinal metaplasia in commercially availableoutbred Mongolian gerbils Dig Dis Sci 48475-485

11 Bergin I L N S Taylor and J G Fox 2000 Antibiotic-associ-ated Clostridum difficile colitis in Mongolian gerbils (Merionesunguiculatus) treated with amoxicillin metronidazolebismuthwafers Contemp Topics Lab Anim Sci 3953