THE PATHOGENIC ENTERIC PROTOZOA:Giardia, Entamoeba, Cryptosporidium and

Cyclospora

World Class Parasites

VOLUME 8

Volumes in the World Class Parasites book series are writtenfor researchers, students and scholars who enjoy reading aboutexcellent research on problems of global significance. Eachvolume focuses on a parasite, or group of parasites, that has amajor impact on human health, or agricultural productivity, andagainst which we have no satisfactory defense. The volumesare intended to supplement more formal texts that covertaxonomy, life cycles, morphology, vector distribution,symptoms and treatment. They integrate vector, pathogen andhost biology and celebrate the diversity of approach thatcomprises modern parasitological research.

Series EditorsSamuel J. Black, University of Massachusetts, Amherst, MA,U.S.A.J. Richard Seed, University of North Carolina, Chapel Hill, NC,U.S.A.

THE PATHOGENIC ENTERIC PROTOZOA:Giardia, Entamoeba, Cryptosporidium and

Cyclospora

edited by

Charles R. Sterling

and

Rodney D. Adam

University of ArizonaTucson, Arizona

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Boston

TABLE OF CONTENTS

List of contributors vii

Preface

Section 1 – Epidemiology

1. Epidemiology and zoonotic potential of Giardia infections

R.C. Andrew Thompson

xi

1

2. Entamoeba histolytica and Entamoeba dispar, the non-identicaltwins

C. Graham Clark 15

3. Epidemiology and strain variation of Cryptosporidium

Rachel M. Chalmers and David P. Casemore 27

4. Cyclospora cayetanensis: An emergent and still perplexingcoccidian parasite

Charles R. Sterling and Ynes R. Ortega 43

Section 2 – Host parasite interactions

5. Antigenic variation of the VSP genes of Giardia lamblia

Rodney D. Adam and Theodore E. Nash 59

6. Pathogenesis and immunity to Entamoeba histolytica

Jessica L. Tarleton and William A Petri Jr 75

7. Innate and T cell-mediated immune responses incryptosporidiosis

Carol R. Wyatt and Vincent McDonald 91

Section 3 – Treatment and Control

8. Rationale approaches to treating Cryptosporidium,

Jan R. Mead and Pablo Okhuysen 103

9. Inactivation and removal of enteric protozoa in water

Frank W. Schaefer, III, Marilyn M. Marshall andJennifer L. Clancy 117

10. Monitoring of Giardia and Cryptosporidium in water inthe UK and US

Jennifer L. Clancy and Paul R. Hunter 129

Section 4 - Genomics

11. Entamoeba histolytica genome

James J. McCoy and Barbara J. Mann 141

12. Cryptosporidium parvum genomics: Impact on researchand control

Guan Zhu and Mitchell S. Abrahamsen

Index

153

165

Cyclospora, Giardia and Entamoeba

CONTRIBUTORS

Mitchell S. AbrahamsenAssociate ProfessorDepartment of Veterinary PathobiologyCollege of Veterinary MedicineUniversity of MinnesotaSt. Paul, MN 55108

Rodney D. AdamProfessorDept of Medicine and Microbiology/ImmunologyUniversity of Arizona College of MedicineTucson, AZ 85719

David P. CasemoreSenior Research FellowCentre for Research into Environment & HealthUniversity of WalesAberystwyth, SY23 2DB, UK

Rachel M ChalmersHead, Cryptosporidium Reference UnitNational Public Health Service Microbiology SwanseaSinglton HospitalSwansea SA2 8QA, UK

Jennifer L. ClancyPresidentClancy Environmental Consultants, Inc.PO Box 314St. Albans, VT 05478

C. Graham ClarkSenior Lecturer,Department of Infectious and Tropical DiseasesLondon School of Hygiene and Tropical MedicineKeppel Street,London, WC1E 7HT, UK

Paul HunterProfessor of Health ProtectionSchool of Medicine, Health Policy and PracticeUniversity of East AngliaNorwich NR4 7TJ, UK

Barbara J. MannAssociate ProfessorDepartments of Internal Medicine and MicrobiologyUniversity of VirginiaSchool of MedicineCharlottesville, VA 22908

Marilyn M. MarshallQuality Assurance OfficerUniversity of Arizona1203 N. MountainTucson, AZ 85721-0471

James J. McCoyResearch ScientistDepartment of Internal MedicineUniversity of VirginiaSchool of MedicineCharlottesville, VA 22908

Vincent McDonaldCentre for Adult and Paediatric Gastroenterology,Barts and the London School of MedicineQueen Mary CollegeUniversity of LondonTurner StLondon E1 2AD, UK

Jan R. MeadAssociate ProfessorAtlanta Veterans Affairs Medical Centerand Department of PediatricsEmory School of MedicineAtlanta, GA 30033

Theodore E. NashHead, Gastrointestinal Parasites SectionLaboratory of Parasitic DiseasesNational Institutes of Allergy and Infectious DiseasesNational Institutes of HealthBethesda, MD 20892

Pablo C. OkhuysenAssociate Professor of MedicineDivision of Infectious DiseasesProgram Director, University Clinical Research CenterThe University of Texas Health Sciences Center HoustonMedical School and School of Public HealthHouston, TX 77030

Ynes R. OrtegaAssistant ProfessorUniversity of GeorgiaCFS, Dept. Food Science and Technology 1109 Experiment St.Griffin, GA 30223

William A. Petri, Jr.Professor and ChiefDivision of Infectious Diseases and International HealthUniversity of VirginiaSchool of MedicineCharlottesville, VA 22908-1340

Frank W. Schaefer, IIIMicrobiologistNational Exposure Research LaboratoryU.S. Environmental Protection Agency26 West Martin Luther King DriveCincinnati, Ohio 45268-1320

Charles R. SterlingProfessorDepartment of Veterinary Science and MicrobiologyUniversity of Arizona1117 E. LowellTucson, AZ 85721

Jessica L. TarletonUndergraduate StudentUniversity of VirginiaCharlottesville, VA 22908

RC Andrew ThompsonProfessorWHO Collaborating Centre for the Molecular Epidemiology of ParasiticInfectionsSchool of Veterinary and Biomedical SciencesMurdoch UniversityMurdoch, Western Australia 6150

Carol R. WyattAssociate ProfessorDepartment of Diagnostic Medicine/PathobiologyCollege of Veterinary MedicineKansas State UniversityManhattan, KS 66506-5705

Guan ZhuAssistant ProfessorDepartment of Veterinary PathobiologyCollege of Veterinary MedicineTexas A&M UniversityCollege Station, TX 77843-4467

PREFACE

Giardia duodenalis (=G. lamblia), Entamoeba histolytica,Cryptosporidium parvum and Cyclospora cayetanensis are more than just amouthful for most who might encounter them. These protozoan parasiticagents contribute significantly to the staggering caseload of diarrheal diseasemorbidity encountered in developing world nations. Compounding the issueof their mere presence is the fact that standard ova and parasite examsfrequently do not detect these infections. Detectable stages may be shedintermittently or require specialized staining procedures. Added to this is theoften large number of asymptomatic carriers who serve as reservoirs forinfecting others. These parasites are also not strangers to more developednations, having responsibility for both small and large-scale diseaseoutbreaks. In such settings they may be even more difficult to detect simplybecause they are frequently overlooked in the grand scheme of diseasecausing possibilities.

They share common features; all are Protozoa, all possess trophicstages that inhabit the gastrointestinal tract, all have the ability to producedisease and in some instances death, and all produce environmentally stablecysts or oocysts, which ensure their transmissibility.

In other ways, these organisms are profoundly different. Giardia is aflagellate that inhabits the gut lumen in close association with enterocytes.Entamoeba is an amoeba that preferentially inhabits the mucosal region of thegut lumen, but which may, under certain circumstances, become invasive.Cryptosporidium and Cyclospora are obligate intracellular coccidians, eachtaking up a unique niche within their respective host enterocytes. Many otherdifferences have been observed in these organisms and have come to lightbecause of recent biological, molecular, and immunological studies. Thesedifferences likely contribute to unique mechanisms of disease production andhost responsiveness, many of which remain to be fully defined.

Giardia owns the distinction of having been described by the amateurDutch scientist Leeuwenhoek (1632-1723) who described many unicellularmicroorganisms from a variety of sources including Giardia from his ownstool samples. Giardia was long thought a strict commensal, but its frequentassociation with waterborne and day care center disease outbreaks, highprevalence in developing countries, especially among children, and relation totravel-associated diarrhea have all helped to change that picture. Despiteadvances in our knowledge of Giardia and giardiasis, this organism remainsone of the most poorly understood protozoan parasites. Why does it possesstwo nuclei and why does it display antigenic variability? What are theimmune mechanisms behind clearance and why do some individuals developchronic, long-lasting infections? Does this organism have true zoonotic

potential, and if so, what are the responsible genotypes and hosts. Are therestrain differences that influence pathogenicity? Finally, what is thephylogenetic relationship of this organism to other putatively basaleukaryotes?

Infection caused by Entamoeba histolytica severely compromises thelives of some 50 million individuals, largely from developing nations. Morethan 100,000 individuals will die annually from invasive amoebiasis. It is thethird leading cause of death among parasitic infections, being overshadowedonly by malaria and schistosomiasis. The ability to distinguish E. histolyticafrom the morphologically similar, but non-pathogenic E. dispar has assistedgreatly in defining the epidemiology of amebic disease since the latteraccounts for approximately 90% of all Entamoeba infections. The advent ofnew models of invasive amebic infection has provided important insights intothe pathophysiology of amoebiasis, but has also raised numerous importantquestions. What is the molecular basis for amebic invasion and the hostinflammatory response? Does the host response contribute to the diseaseprocess? What specific cytokines, chemokines or other inflammatorymediators participate in the invasion and extraintestinal phases of disease andhow are they modulated? Does ameba induced apoptosis play a role inamebic liver abscess progression? Finally, is there such a thing as protectiveimmunity to amoebiasis, and, if so, how is it mediated and can it be inducedartificially via vaccination?

Cryptosporidium parvum became recognized as a medicallyimportant parasite in humans following its discovery in AIDS patients andsubsequently in young children of developing nations. Further studies havedemonstrated its zoonotic potential as well as its ubiquitous presence innumerous animal species and the environment. It accounts for up to 20% ofdiarrheal episodes in children of developing countries and is a majorcontributor of diarrheal episodes in young farm animals worldwide. Thelargest documented waterborne parasitic disease outbreak in history isattributed to this organism and to date it remains refractory to all conventionaltherapies. It is also extremely resistant to disinfection. The uniqueintracellular but extracytoplasmic developmental location of this parasiteprompts numerous questions. Why has this parasite chosen this location forits development? Does this location somehow offer shelter fromantimicrobial therapy? How does Cryptosporidium obtain nutrients from itshost cell or the immediate environment? What immune effector mechanismsare operative against this organism at its intracellular and extracytoplasmiclocation? In addition, does the existence of human and zoonotic genotypeshave implications for organism virulence?

Cyclospora cayetanensis is the newcomer on the block. Its identityeluded the scientific community for almost a decade before its coccidian

nature was recognized. It is now seen as a disease-causing agent in AIDSpatients, children of developing nations and in immunocompetent individualswho are exposed to it. Several recent food borne disease outbreaks in theUnited States, arising from imported fruit, have heightened awareness of thisorganism’s existence. Despite what we have learned, this organism remainsan enigma. What are its principal transmission routes? Why does it appear tobe markedly seasonal? How does its apparently prolonged sporulation timerelate to the previous two questions? How is intestinal inflammation inducedin the apparent presence of very few organisms? Finally, are humans the onlysusceptible host?Past studies have enhanced our understanding of the biology, epidemiologyand host-parasite relationship of these complex organisms. This, in turn, hasled to the development of new strategies aimed at preventing, controlling andtreating infections caused by these protozoan parasites. Despite these efforts,however, the organisms that constitute the framework for this book remainproblematic. The numerous questions raised in this preface are addressed inchapters dealing with the respective organisms along with issues of a broadernature that encompass epidemiology, chemotherapy, biochemistry andgenomics. These chapters, written by acknowledged experts, are intended toprovide an overview of the current state of knowledge with respect to selecttopics, to stimulate thinking about the complex issues that face both parasiteand host in such a relationship, to present fresh and new approaches atdetection, treatment and control, and to make everyone aware that we haveyet to gain the upper hand against these ubiquitous denizens of ourgastrointestinal tract.

Charles R. SterlingRodney D. Adam

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EPIDEMIOLOGY AND ZOONOTIC POTENTIALOF GIARDIA INFECTIONS

RC Andrew ThompsonWHO Collaborating Centre For The Molecular Epidemiology Of Parasitic Infections andWestern Australian Biomedical Research Institute, Division Of Veterinary AndBiomedical Sciences, Murdoch University, Murdoch, Western Australia, 6150

ABSTRACTDetermining the source of infection is central to an understanding of theepidemiology of giardiasis. In this respect, the role of zoonotic transmissionhas been a matter of controversy for many years. This has been complicatedby the fact that the causative agent of giardiasis, Giardia duodenalis, is acommon parasite of people, domestic animals and wildlife. The developmentand application of molecular epidemiological tools has now made it possibleto directly genotype Giardia isolated from animals and environmentalsamples. These studies have shown that many species of mammals aresusceptible to infection with zoonotic and host-adapted genotypes of G.duodenalis and that they are often present in the same endemic foci. Recentstudies have also demonstrated that zoonotic transmission does occur innature. However, available data suggests that zoonotic transmission does notappear to play a major role in waterborne outbreaks of giardiasis. More studiesare required on the molecular epidemiology of Giardia infections in order tomore accurately determine the frequency of zoonotic transmission in localisedendemic foci and in outbreak situations.Key Words: Giardia; taxonomy; epidemiology; zoonoses; molecularepidemiology.

INTRODUCTIONMembers of the genus Giardia are ubiquitous, affecting the intestinal

tracts of numerous vertebrate species (Thompson et al.,1993). They areflagellated protozoans belonging to the Class Zoomastigophorea and OrderDiplomonadida. However, the phylogenetic affinities of Giardia have been amatter of controversy for many years. Giardia has a very simple intracellularorganization, with no mitochondria or peroxisomes and is thought to representan early branching eukaryote lineage that diverged before the acquisition ofmitochondria (Simpson et al., 2002). Giardia has therefore become a key

2 Thompson

organism in attempts to understand the evolution of eukaryotic cells. In thisrespect, recent research has revealed that Giardia has a primitive vesicularsecretory system that has been proposed as the archetype of the Golgisecretory apparatus in higher organisms (Marti et al., 2003a, b).

The protozoa that collectively comprise the genus Giardia haveintrigued biologists and clinicians for over 300 years, ever since Antony vanLeeuwenhoek first discovered the organism (Meyer, 1994). Despite its longhistory, our understanding of Giardia’s taxonomy, pathogenicity andrelationship with its hosts are still poorly understood. Giardia is not invasiveand lives and multiplies by asexual multiplication on the lumenal surface ofthe small intestine of its vertebrate host. Giardia has a very simple two-stagelife cycle. The organism produces environmentally resistant cysts which arevoided in the faeces and transmitted directly, or via water or food, to anotherhost with infection resulting from ingestion. Exposure first to an acidicenvironment in the stomach and then bile salts in the proximal small intestinestimulates release of trophozoites from the cyst which attach to and colonisethe mucosal surface. As trophozoites pass through the small intestine theyencyst and are passed in the faeces.

The pathogenesis of Giardia is not clearly understood and symptomswhich include persistent diarrhoea, abdominal pain and rapid weight loss, arehighly variable (Thompson et al., 1993) and may not be evident in asignificant proportion of infected individuals (Rodriguez-Hernandez et al.,1996). The risk factors for clinical giardiasis, particularly in humans, have yetto be resolved but clearly involve host and environmental factors, as well asthe ‘strain’ of the parasite.

Although species of Giardia inhabit the intestinal tracts of virtually allclasses of vertebrates, G. duodenalis (syn G. intestinalis; G. lamblia) is theonly species found in humans and most other mammals including dogs, catsand livestock (Thompson, 1998; Olson et al., 1995; Pavlaseck et al., 1995;Xiao and Herd, 1994; Xiao et al., 1994). G. duodenalis has a globaldistribution and is the most common intestinal parasite of humans indeveloped countries. In Asia, Africa and Latin America, about 200 millionpeople have symptomatic giardiasis with some 500,000 new cases reportedeach year (WHO, 1996). It is also a frequently encountered parasite ofdomestic animals and livestock.

Giardiasis is the most frequently diagnosed waterborne disease andalong with cryptosporidiosis, is the major public health concern of waterutilities in developing nations (Levine et al., 1990; Thurman et al., 1998). Therole of animals in water borne transmission has been difficult to determine.This is because, until recently, it has not been possible to ‘type’ isolates of theparasite obtained during outbreak situations as a means of determining thesource of contamination; i.e. whether the ‘strain’ of Giardia is of animal or

Thompson 3

human origin (see Thompson, 1998, 2000; Thompson et al., 1990; Erlandsen,1994; Thompson and Boreham, 1994). However, whether animals serve asthe original source of contamination or amplify the numbers of the originallycontaminating isolate, or both, remains to be determined (Bemrick andErlandsen, 1988; Thompson et al., 1990; Thompson, 1998). Similarly,although diagnosis of Giardia by traditional microscopic methods remains areliable indicator of infection, the detection of G. duodenalis by microscopy ormore sensitive techniques such as faecal ELISA are of limited epidemiologicalvalue, especially in terms of the source of infection, since they do not provideinformation on strain/genotype.

TAXONOMY AND HOST-SPECIFICITYFive species of Giardia are currently recognised (Table 1). This

represents a comprehensive taxonomic rationalisation proposed by Filice in1952 and since accepted by most authorities. The schemes proposed by Filicereflected a lack of morphological distinctness between most of the speciesdescribed earlier in Giardia and doubts over their assumed host specificity.When Filice proposed the G. duodenalis morphological grouping, he was wellaware that it was a temporary ‘holding’ place for a diverse group ofphenotypically variable yet morphologically uniform organisms. However, atthe time, the methodology was not available to reliably discriminate betweenthese variants or ‘strains’.

The recent application of PCR-based procedures which circumvent theneed for laboratory amplification using in vitro culture has enabled thecharacterization of previously inaccessible genotypes and thus the geneticcharacteristics of morphologically similar variants/strains (Van Keulen et al.,

4 Thompson

1998; Monis et al., 1998; Hopkins et al., 1997, 1999; Thompson et al. 1999).Using PCR-based procedures, in conjunction with analysis of a variety ofgenetic loci such as rDNA, elongation factor 1- alpha triose phosphateisomerase (tpi) and glutamate dehydrogenase (gdh), and with much larger datasets, it has been possible to elucidate the fundamental genetic divisions withinthe G. duodenalis morphological group (Table 2; Thompson et al.,1999;Monis and Thompson, 2003; Thompson 2003a).

Giardia isolates recovered from humans and many other mammalianspecies fall into one of the two major genotypic assemblages, A or B (Table2). Molecular analyses have shown that the genetic distance separating thesetwo assemblages exceeds that used to delineate other species of protozoa(Andrews et al., 1989; Mayerhofer et al., 1995; Monis et al., 1996). Molecularstudies have also demonstrated the existence of genetic subgroups within eachof these assemblages. Assemblage A consists of isolates that can be groupedinto two distinct clusters; AI consists of a mixture of closely-related animaland human isolates which are geographically widespread and most attentionregarding the zoonotic potential of Giardia has focused on this AI subgroup.In contrast, the second subgroup, A II consists entirely of human isolates.

Thompson 5

Assemblage B comprises a genetically more diverse group of predominantlyhuman isolates although some animal genotypes have been included (Moniset al., 1996,1998; Ey et al., 1997).

Some of these genetic divisions, or genotypic groupings, appear to beconfined to specific animal hosts. Giardia genotypes exhibiting a limited hostrange include those recovered from cats, dogs, rats, voles/muskrats andlivestock (Table 2). Unlike the uncertainty regarding the taxonomic status ofgenotypic assemblages A and B, there is probably sufficient data supportingthe restricted host range of these genotypes to warrant species designation(Monis and Thompson, 2003).

CYCLES OF TRANSMISSIONAlthough the World Health Organization has considered Giardia to

have zoonotic potential for over twenty years, either through direct faecal-oralor waterborne routes of transmission, direct evidence has been lacking(Thompson, 1998, 2000). Clearly, the greatest zoonotic risk is from thosegenotypes of Giardia in genotypic assemblage A, particularly those in the AIsubgroup, and to a lesser extent genotypes in Assemblage B. In contrast, theanimal-specific genotypes appear to be host adapted, restricted to livestock,dogs, cats and rodents (Table 2). There is no epidemiological evidence tosuggest that they occur frequently in the human population and thus theirzoonotic risk appears minimal. However, from the point of view of zoonoticpotential the finding that similar genotypes are dispersed in different hosts isnot by itself conclusive evidence that zoonotic transmission is taking place.We therefore need to understand how the four major cycles of transmissionthat maintain the parasite in mammalian hosts involving transmission betweenhumans, livestock, dogs/cats or wildlife, may interact (Figure 1), anddetermine the frequency of transmission of zoonotic genotypes. A betterassessment for this will come from studies that examine the dynamics ofGiardia transmission between hosts living in the same locality or endemicfocus.

Human to human transmission of Giardia can occur indirectlythrough the accidental ingestion of cysts in contaminated water or food, ordirectly in environments where hygiene levels may be compromised, such asin day care centres or among the inhabitants of disadvantaged communities. Anumber of studies have been undertaken comparing the frequency ofoccurrence of Assemblage A and B genotypes in different populations ofpatients (Thompson, 2003b). Assemblage B appears to be more common thanAssemblage A, and interestingly the latter is more commonly associated withsymptomatic infections.

6 Thompson

In terms of livestock, cattle are most commonly infected and moststudies have concentrated on this species. Giardia is very common in bothbeef and dairy cattle throughout the world, and longitudinal studies haveconsistently demonstrated prevalence rates of 100% (O’Handley, 2002;O’Handley et al., 1999; Ralston et al., 2003; Xiao and Herd, 1994).Transmission occurs among infected calves as well as chronically infectedadults, but the frequency of transmission is particularly high amongst dairycalves (Xiao and Herd 1994; O'Handley et al., 1999; 2000). Recent studieshave demonstrated that calves in dairy and beef herds may harbour one of twogenotypes of G. duodenalis. Although the livestock genotype (Assemblage E)of Giardia appears to occur most frequently in cattle, studies in Canada andAustralia have shown that a small proportion of cattle in a herd may harbourgenotypes in Assemblage A, the most common genotypes affecting humans(O’Handley et al., 2000; Appelbee et al., 2003). However, the livestockgenotype may also occur to the exclusion of the zoonotic genotype(Thompson, 2003a).

Recent studies in Australia have found that G. duodenalis is the mostcommon enteric parasite of domestic dogs and cats (Bugg et al., 1999;McGlade et al., 2003), although it is rarely associated with clinical disease. It

Thompson 7

is also widely prevalent in dogs and cats in the USA and has been shown to becommon in pets in other countries (Thompson and Robertson, 2003).However, it has been suggested that prevalence rates of Giardia in companionanimals are often underestimated because of the low sensitivity ofconventional detection methods, the fact that the parasite may be present atsubclinical levels and the intermittent nature of cyst excretion (McGlade et al.,2003). Molecular epidemiological studies have shown that dogs may beinfected with their own, host adapted genotype of Giardia (C/D Table 2), aswell as with zoonotic genotypes (A/B Table 2).

Under natural, pristine conditions, what evidence there is availablesuggests that wildlife harbour their own genotypes/species of Giardia and notG. duodenalis. However, recent studies have confirmed that beavers in thewild can harbour infections with zoonotic genotypes of G. duodenalis(Appelbee et al., 2002).

ZOONOTIC TRANSMISSIONThe water connection

The consumption of unfiltered/untreated drinking water represents asignificant risk for giardiasis (Hoque et al., 2002; Jakubowski and Craun,2002). The majority of waterborne giardiasis outbreaks in humans haveoccurred in unfiltered surface or groundwater systems impacted by surfacerun off or sewage discharges (Jakubowski and Craun, 2002). Irrigation watersused for food crops that are traditionally consumed raw may also represent ahigh risk as a source of Giardia (Thurston et al., 2002). Environmentalcontamination of such water systems and supplies may result from human,agricultural and wildlife sources (Heitman et al., 2002).Wildlife

The occurrence of Giardia in wildlife, particularly of isolates that aremorphologically identical to G. duodenalis, has been the single mostimportant factor incriminating Giardia as a zoonotic agent. However, there islittle evidence to support the role of wildlife as a source of disease in humans,even though the role of wildlife has dominated debate on the zoonotictransmission of Giardia especially when water is the vehicle for suchtransmission. It was the association between infected animals such as beaversand waterborne outbreaks in people that led the WHO (1979) to categoriseGiardia as a zoonotic parasite. It is therefore surprising that so littleinformation is available on the genotypes of Giardia affecting wildlife, aswell as in people infected with Giardia as a result of a waterborne outbreak.

Although wildlife, particularly aquatic mammals, are commonlyinfected with Giardia there is little evidence to implicate such infections asthe original contaminating source in water borne outbreaks. It would appearthat such animals are more likely to have become infected from water

8 Thompson

contaminated with faecal material of human, or less likely, domestic animalorigin. Wildlife thus serve to amplify the numbers of the originallycontaminating isolate (Bemrick and Erlandsen, 1988; Monzingo & Hibbler,1987; Thompson, 1998; Thompson et al., 1990).

Some studies (eg. Isaac Renton et al., 1993) have geneticallycharacterised isolates associated with waterborne outbreaks, but the typingschemes used did not allow correlation with the currently recognisedassemblages. The one study that did genotype Giardia of beaver origin,confirmed previous suggestions that the source of Giardia infection inbeavers was likely to be of human origin (Dixon et al., 2002; Monzingo andHibbler, 1987; Rickard et al., 1999). In this study, 12 of 113 (10.6%) beaverfaecal samples from 6 of 14 different riverbank sites in southern Alberta,Canada, were positive for Giardia, and all those genotyped using the16S-rRNA gene belonged to the zoonotic genotype, Assemblage A (Appelbee etal., 2002).Cattle

Although the transmission process is complex and the risk is low,there is clearly a definite potential for microbial contamination of ground andsurface waters from livestock operations (Donham, 2000). Cattle aresusceptible to infection with zoonotic genotypes of Giardia and it has beenshown that calves infected with Giardia commonly shed from to cystsper gram of faeces (Xiao, 1994; O’Handley et al., 1999). Thus, even a fewcalves infected with genotypes in Assemblage A could pose a significantpublic health risk directly to handlers or indirectly as an important reservoirfor human waterborne outbreaks of giardiasis. This is of potential publichealth significance and may put producers, and other members of thecommunity, at risk. However, longitudinal studies in Australia suggest thatzoonotic genotypes may only be present transiently in cattle under conditionswhere the frequency of transmission with the livestock genotype is high andcompetition is thus likely to occur. The public health risk from cattle appearsto be minimal, at least based on studies in North America and Australia wheregenotyping has been undertaken and has shown that the livestock genotypeappears to predominate in cattle (O’Handley et al., 2000; Hoar et al., 2001).However, under certain circumstances, where Giardia infections may notpreviously have occurred or been common, an introduced genotype mayestablish and be perpetuated in the absence of competing genotypes. Forexample, a recent molecular epidemiological study showed that humansappear to have introduced Giardia into a remote national park in Uganda andare also thought to have been the source of zoonotic genotypes of Giardia in asmall number of cohabiting dairy cattle (Graczyk, et al., 2002).

Thompson 9

PetsAlthough the clinical significance of Giardia in dogs and cats appears

to be minimal, the public health significance of such infections in pets hasbeen the subject of much debate and is still a question of uncertainty forveterinarians. In domestic, urban environments of Australia, for example,zoonotic genotypes from Assemblage A and the ‘dog’ genotype, AssemblageD, are both equally common in dogs (Thompson et al., 1999). It is thereforeconsidered that two cycles of transmission probably operate in domestic urbanenvironments with the possibility of zoonotic transmission of Assemblage Agenotypes between pets and their owners. This was highlighted in the studyby Bugg et al. (1999) which found that dogs from multi-dog households weremore commonly infected with Giardia than dogs in single-dog households,emphasising the potential ease with which Giardia can be spread to in-contactanimals and therefore presumably humans (Bugg et al., 1999). In contrast, arecent survey of domestic dogs in Japan found all isolates to belong to thedog-specific genotype, Assemblage D (Abe et al., 2003).

Molecular epidemiological studies in localised endemic foci, wherethe frequency of transmission of zoonotic and non-zoonotic genotypes is high,will provide more useful information on the frequency of zoonotictransmission. For example, studies in Aboriginal communities in Australiahave shown that the dog genotype predominates in infected dogs (Hopkins etal., 1997). In contrast, in remote tea growing communities in Assamnortheast India, where Giardia occurs in both humans and their dogs, 20% ofdogs were found to be infected with Giardia, but they were all infected withzoonotic genotypes, mostly from Assemblage A (Traub et al., 2003). Thisdifference may reflect a closer association between individual dogs and theirowners in the tea growing communities, and the frequency with which dogsare able to eat human faeces in these communities (Traub et al., 2002). InAboriginal communities in Australia, such behaviour by dogs is less commonand the dogs tend to stay together in packs for much of the time. Inenvironments where the infection pressure is less, such as domestichouseholds in urban settings, dogs are just as likely to harbour zoonoticgenotypes of Giardia from Assemblage A as they are their own dog genotype(Assemblage D).

The study in Assam, India by Traub et al., (2003), has provided thefirst direct evidence of zoonotic transmission between dogs and humans, byfinding the same genotype of Giardia in people and dogs, not only in the samevillage, but also in the same household. Giardia isolates were characterised atthree different loci; the SSU-rDNA, elongation factor 1- alpha andtriose phosphate isomerase (tpi) gene. Evidence for zoonotic transmission wassupported by strong epidemiological data showing a highly significantassociation between the prevalence of Giardia in humans and the presence of

10 Thompson

a Giardia positive dog in the same household. A major finding of this studywas the importance of using multiple loci when inferring genotypes toGiardia in epidemiological investigations (Traub et al., 2003).

IN CONCLUSIONDomestic animals and wildlife appear to harbour their own host

adapted genotypes/species of Giardia although they are susceptible toinfection with zoonotic genotypes, principally from Assemblage A. However,what data that is available suggests that the occurrence of such zoonoticgenotypes is not common, and they are likely to be quickly diluted andexcluded by competitive interactions with host adapted genotypes. The publichealth risk of zoonotic genotypes in animals would appear to be through directtransmission. There is no convincing evidence that zoonotic transmissionimpacts significantly on the aetiology of waterborne outbreaks of giardiasis.Giardia of human origin appears to be the main source of water contaminationand as such may impact negatively on ecosystem health leading to infectionsin aquatic wildlife. Recent studies have demonstrated that filter-feedingmolluscs are useful indicators of the presence of waterborne pathogens.Genotypic characterisation was recently utilised in a study that isolatedGiardia cysts from clams in an estuary in North America (Graczyk, et al.,1999). All isolates were identified as belonging to genotype Assemblage A,highlighting contamination with faeces of mammalian origin, most probablyhuman, that contained G. duodenalis cysts of public health importance. Suchfilter-feeding molluscan shellfish can concentrate waterborne pathogens andthus in combination with appropriate genotyping procedures can serve asbiological indicators of contamination with Giardia cysts and can thus be usedfor sanitary assessment of water quality.

Further studies are needed on the molecular epidemiology of Giardiainfections in order to determine the frequency of zoonotic transmission inlocalised endemic foci and in outbreak situations, and to better understand theinteraction between the major cycles of Giardia transmission.

REFERENCESAbe, N., Kimata I., and Iseki M. 2003. Identification of genotypes of Giardia intestinalis

isolates from dogs in Japan by direct sequencing of the PCR amplified glutamatedehydrogenase gene. Journal of Veterinary Medicine and Science 61: 29-33.

Appelbee, A., Thorlakson, C., and Olson, M.E. 2002. Genotypic characterization of Giardiacysts isolated from wild beaver in southern Alberta, Canada. In: Olson, B.E., Olson, M.E.,Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford,UK, pp 299-300.

Appelbee, A.J., Frederick, L.M., Heitman, T.L. and Olson M.E. 2003. Prevalence andgenotyping of Giardia duodenalis from beef calves in Alberta, Canada. VeterinaryParasitology 112: 289-294.

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Andrews, R.H., Adams, and M. Boreham. P.F.L. et al. 1989. Giardia intestinalis:electrophoretic evidence for a species complex. International Journal for Parasitology19:183-190.

Bemrick, W.J. and Erlandsen, S.L. 1988. Giardiasis - is it really a zoonosis? ParasitologyToday 4:69-71.

Bugg, R.J., Robertson, I.D., Elliot, A.D. and Thompson, R.C.A. 1999. Gastrointestinalparasites of urban dogs in Perth, Western Australia. Veterinary Journal 57:295-301.

Dixon, B.R., Bussey, J., Parrington, L., Parenteau., Moore, R., Jacob, J., Parenteau, M.-P. andFournier, J. 2002. A preliminary estimate of the prevalence of Giardia sp. in Beavers inGatineau Park, Quebec, using flow cytometry. In: Olson, B.E., Olson, M.E., Wallis, P.M.(Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 71-79.

Donham KJ. 2000. The concentration of swine production. Effects on swine health,productivity, human health, and the environment. Veterinary Clinics of North. America. FoodAnimal. Practice. 16: 559-597.

Erlandsen, S.L. 1994. Biotic transmission - is giardiasis a zoonosis? in Giardia: fromMolecules to Disease, (eds R.C.A. Thompson, J.A. Reynoldson and A.J. Lymbery), CABInternational, Wallingford, pp. 83-97.

Ey, P.L., Mansouri, M., Kulda, J. et al. 1997. Analysis of Giardia from hoofed animals revealsArticodactyl-specific and potentially zoonotic genotypes. Journal of Eukaryotic Microbiology44:626-635.

Filice, F.P. 1952. Studies on the cytology and life history of a Giardia from the laboratory rat.University of California Publications in Zoology 57:53-146.

Graczyk, T.K., Thompson, R.C.A., Fayer, R., Adams, P., Morgan., U.M and Lewis, E.J. 1999.Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Baysubestuary, Rhode river. American Journal of Tropical Medicine and Hygiene 61:526-529.

Graczyk, T.K., Bosco-Nizeyi, J., Ssebide, B., Thompson, R.C.A., Read, C. and Cranfield, M R.2002. Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitatsof free-ranging human-habituated gorillas, Uganda. Journal of Parasitology 88: 905-909.

Heitman, T.L., Frederick, L.M., Viste, J.R., Guselle, N.J., Cooke, S.E., Roy, L., Morgan, U.M.,Thompson, R.C.A. and Olson, M.E. 2002. Prevalence of Giardia and Cryptosporidium andcharacterisation of Cryptosporidium spp. isolated from wildlife, human and agriculturalsources of the North Saskatchewan River basin in Alberta, Canada. Canadian Journal ofMicrobiology 48: 530-541.

Hoar, B.R., Atwill, E.R., Elmi, C. and Farver, T.B. 2001. An examination of risk factorsassociated with beef cattle shedding pathogens of potential zoonotic concern. Epidemiologyand Infection 127: 147-155.

Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A. and Thompson,R.C.A. 1997. Ribosomal RNA sequencing reveals differences between the genotypes ofGiardia isolates recovered from humans and dogs living in the same locality. Journal ofParasitology 83:44-51.

Hopkins, R.M., Constantine, C.C., Groth, D.M., Reynoldson, J.A. and Thompson, R.C.A.1999. DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer.Parasitology 118:531-539.

Hoque, M.E., Hope, V.T., Kjellstrom, T., Scragg, R., Lay-Yee, R., 2002. Risk of giardiasis inAucklanders: a case-control study. International Journal of Infectious Diseases. 6: 191

Isaac-Renton, J.L., Cordeiro, C., Sarafis, K. et al. 1993. Characterization of Giardiaduodenalis isolates from a waterborne outbreak. Journal of Infectious Diseases 167:431-40.

Jakubowski, W. and Graun, G.F. 2002. Update on the control of Giardia in water supplies. In:Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CABInternational, Wallingford, UK, pp 217-238.

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Levine, W.C., Stephenson, W.T. and Craun, G.F. 1990. Waterborne disease outbreaks, 1986-1988. Morbidity and Mortality Weekly Report 39:1-13.

Marti, M., Li, Y., Schraner, E.M., Wild, P., Kohler, P. and Hehl, A.B. 2003a. The secretoryapparatus of an ancient eukaryote: protein sorting to separate export pathways occurs beforeformation of transient golgi-like compartments. Molecular Biology of the Cell 14: 1433-1447.

Marti, M., Regos, A., Li, Y., Schraner, E.M., Wild, P., Muller, N., Knopf, L.G. and Hehl, A.B.2003b. An ancestral secretory apparatus in the protozoan parasite Giardia intestinalis.Journal of Biological Chemistry 278: 24837-24848.

Mayrhofer, G., Andrews, R.H., Ey, P.L. et al. 1995. Division of Giardia isolates from humansinto two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at27 loci and comparison with Giardia muris. Parasitology 111:11-17.

McGlade, T.R., Robertson, I.D., Elliott, A.D. and Thompson, R.C.A. 2003. High prevalence ofGiardia detected in cats by PCR. Veterinary Parasitology. 110: 197-205.

Meyer, E.A. 1994. Giardia as an organism. In Giardia: from Molecules to Disease, (eds R.C.A.Thompson, J.A. Reynoldson and A.J. Lymbery), CAB International, Wallingford, pp. 3-15.

Monis, P.T. and Thompson, R.C.A. 2003. Cryptosporidium and Giardia - zoonoses: fact orfiction? Infection, Genetics and evolution (in press).

Monis, P.T., Mayrhofer, G., Andrews, R.H. et al. 1996. Molecular genetic analysis of Giardiaintestinalis isolates at the glutamate dehydrogenase gene. Parasitology 112:1-12.

Monis, P.T., Andrews, R.H., Mayhofer, G. et al. 1998. Novel lineages of Giardia intestinalisidentified by genetic analysis of organisms isolated from dogs in Australia. Parasitology116:7-19.

Monzingo, D.L. Jr. and Hibler, C.P., 1987. Prevalence of Giardia sp. in a beaver colony andthe resulting environmental contamination. Journal of Wildlife Diseases. 23: 576-585.

O’Handley, R.M., 2002. Giardia in farm animals. In: Olson, B.E., Olson, M.E., Wallis, P.M.(Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 97-105.

O'Handley R, Cockwill C, McAllister TA, et al. 1999. Duration of naturally acquired giardiasisand cryptosporidiosis in dairy calves and their association with diarrhoea. Journal of theAmericam Veerinary Medical Association 214:391-396.

O'Handley RM, Olson ME, Fraser D, et al. 2000. Prevalence and genotypic characterisation ofGiardia in dairy calves from Western Australia and Western Canada. Veterinary Parasitology90:193-200.

Olson, M.E., McAllister, T.A., Deselliers, L. et al. 1995. Effects of giardiasis on production ina domestic ruminant (lamb) model. American Journal of Veterinary Research 56:1470-1474.

Pavlasak, I., Hess, L., Stehlik, I. et al. 1995. The first detection of Giardia spp. in horses in theCzech Republic. Veterinariya Meditsina (Praha), 40:81-86.

Ralston, B.J. McAllister T.A. and Olson M.E. 2003. Prevalence and infection pattern ofnaturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams.Veterinary Parasitology 114: 113-122.

Rickard, L.G., Siefker, C., Boyle, C.R., Gentz, E.J., 1999. The prevalence of Cryptosporidiumand Giardia spp. in fecal samples from free-ranging white-tailed deer (Odocoileusvirginianus) in the southeastern United States. Journal of Veterinary Diagnostic Investigation11:65-72.

Rodriguez-Hernandez J., Canut-Blasco A. and Martin-Sanchez A.M. 1996. Seasonalprevalence’s of Cryptosporidium and Giardia infections in children attending day carecentres in Salamanca (Spain) studied for a period of 15 months. European Journal ofEpidemiology 12:291-295.

Simpson, A.G., Roger, A.J., Silberman, J.D., Leipe, D.D., Edgcomb, V.P., Jermiin, L.S.,Patterson, D.J. and Sogin, M.L. 2002. Evolutionary history of "early-diverging" eukaryotes:

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the excavate taxon Carpediemonas is a close relative of Giardia. Molecular Biology andEvolution 19: 1782-91.

Thurman, R., Faulkner, B., Veal, D. et al. 1998. Water quality in rural Australia. Journal ofApplied Microbiology 84:627-632.

Thompson, R.C.A. 1998. Giardia infections. In: Zoonoses: Biology, Clinical Practice andPublic Health Control. (eds S.R. Palmer, E.J.L. Soulsby, and D.I.H. Simpson), OxfordUniversity Press, Oxford, pp. 545-561.

Thompson, R.C.A. 2000. Giardiasis as a re-emerging infectious disease and its zoonoticpotential. International Journal for Parasitology 30:1259-1267.

Thompson, R.C.A., 2002. Towards a better understanding of host specificity and thetransmission of Giardia: The impact of molecular epidemiology. In: Giardia: Olson, B.E.,Olson, M.E., Wallis, P.M. (Eds.), The cosmopolitan parasite. CAB International,Wallingford, UK, pp 55-69.

Thompson, R.C.A. 2003a. Molecular epidemiology of Giardia and Cryptosporidiuminfections. Journal of Parasitology, 89: S134-S140.

Thompson, R.C.A. 2003b The Zoonotic Significance and Molecular Epidemiology of Giardiaand Giardiasis Veterinary Parasitology (in press).

Thompson, R.C.A, and Boreham, P.F.L. 1994. Biotic and abiotic transmission, In: Giardia:from molecules to disease, (eds R.C.A. Thompson, J.A. Reynoldson and A.J. Lymbery),CAB International, Wallingford, pp. 131-136.

Thompson, R.C.A., Lymbery, A.J. and Meloni, B.P. 1990. Genetic variation in GiardiaKunstler, 1882: taxonomic and epidemiological significance. Protozoological Abstracts14:1-28.

Thompson, R.C.A., Reynoldson, J.A. and Mendis, A.H.W. 1993. Giardia and giardiasis.Advances in Parasitology 32:71-160.

Thompson, R.C.A., Hopkins, R.M. and Homan, W.L. 1999. Nomenclature and geneticgroupings of Giardia infecting mammals. Parasitology Today 16: 210-213.

Thurston-Enriquez, J.A., Watt, P., Dowd, S.E., Enriquez, R., Pepper, I.L. and Gerba, C.P.2002. Detection of protozoan parasites and microsporidia in irrigation waters used for cropproduction. Journal of Food Protection 65: 378-382.

Traub, R.J., Robertson, I.D., Irwin, P., Mencke, N. and Thompson, R.C.A., 2002. The role ofdogs in transmission of gastrointestinal parasites in a remote tea-growing community innortheast India. American Journal of Tropical Medicine and Hygiene 67: 539-45.

Traub, R.J., Monis, P., Robertson, I., Irwin, P., Mencke, N. and Thompson, R.C.A. 2003.Epidemiological and molecular evidence supports the zoonotic transmission of Giardiaamong humans and dogs living in the same community. Parasitology (in press).

Van Keulen, H., Feely, D.E., and Macechko, P.T. et al. 1998. The sequence of Giardia smallsubunit rRNA shows that voles and muskrats are parasitized by a unique species Giardiamicroti. Journal or Parasitology 84:294-300.

WHO, 1979. Parasitic Zoonoses. Report of a WHO Expert Committee with the participation ofFAO. Technical Report Series No. 637. World Health Organization, Geneva.

WHO, 1996. The World Health Report 1996. Fighting Disease Fostering Development. WorldHealth Organization, Geneva.

Xiao L. 1994. Giardia infection in farm animals. Parasitology Today 10:436-438.Xiao, L. and Herd, R.P. 1994. Infection pattern of Cryptosporidium and Giardia in calves.

Veterinary Parasitology 55:257-262.Xiao, L., Herd, R.P. and McClure, K.E. 1994. Periparturient rise in the excretion of Giardia

sp. cysts and Cryptosporidium parvum oocysts as a source of infection for lambs. Journal ofParasitology 80:55-59.

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ENTAMOEBA HISTOLYTICA AND ENTAMOEBADISPAR, THE NON-IDENTICAL TWINS

C. Graham ClarkDepartment of Infectious and Tropical Diseases, London School of Hygiene and TropicalMedicine

ABSTRACTOver the past 25 years a fundamental change has taken place in our

understanding of amebiasis, through the recognition of Entamoeba dispar as aspecies that is distinct from Entamoeba histolytica but is morphologicallyindistinguishable. This change in taxonomy has significant implications forthe diagnosis and treatment of infections, as well as for our ability to interpretthe earlier literature. The defining characteristic of the two species remainsthe ability of E. histolytica to cause invasive disease while E. dispar cannot,but the underlying genetic differences between the two that are responsible forthis remain to be defined. The ongoing comparative genome sequencing willhopefully shed light on the dichotomy.Key words: Entamoeba histolytica, Entamoeba dispar, amoebiasis,isoenzymes, monoclonal antibodies, DNA sequencing.

INTRODUCTIONIn 1875, Fedor Lösch described the first known case of disease caused

by an ameba (Lösch,1875, 1978). The remarkable diagrams he produced leaveno doubt that he was looking at what is now known as Entamoeba histolytica.The typical nucleus and ingested red blood cells are easily recognisable.Lösch went on to reproduce the disease by infecting dogs and, although hewas cautious in his interpretation of the results, it seems clear that he believedthat the amebae were responsible for causing the disease. Lösch referred tothe organisms as Amoeba coli, a descriptive rather than a taxonomic term.2003 was the 100th anniversary of the naming of Entamoeba histolytica byFritz Schaudinn (Schaudinn, 1903, 1978) and, while some aspects ofSchaudinn's description are very strange, the name of the organism hedescribed has been retained.

The naming of E. histolytica by Schaudinn was far from the end of thetaxonomic story, however. Over the next fifteen years or so a large number ofadditional species of enteric ameba were described, but their relationships to

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E. histolytica were often unclear. In 1919 Clifford Dobell (Dobell, 1919)reviewed the existing literature and concluded that there were only twospecies of Entamoeba infecting the human colon - E. histolytica producingcysts with 4 nuclei and E. coli producing cysts with 8 nuclei. In Dobell's viewat that time, E. histolytica was an obligate tissue parasite. He was later forcedto change this view when in 1925 E. histolytica was first grown in culture(Boeck and Drbohlav, 1925).

The gradual acceptance over the past 25 years that the organism knownas Entamoeba histolytica was in fact made up of two distinct speciesrepresents one of the most dramatic changes in human parasitology to takeplace during that time period. The story of this development starts in 1925when the eminent French parasitologist Emile Brumpt published apreliminary report (Brumpt, 1925) describing a new species that wasmorphologically indistinguishable from E. histolytica but was incapable ofcausing disease. He gave it the name Entamoeba dispar. His evidenceconsisted of observations on infected patients and on experimental infectionsof kittens. The latter were used as a very sensitive model for intestinalamebiasis at the time but showed no tissue invasion when infected with thenew organism.

Why was Brumpt's work not accepted by his contemporaries (Brumpt,1928)? There appear to have been two primary reasons. The first is thatmorphology was the accepted basis of all species descriptions at the time andin this case there were no differences. The second is that it had already beenestablished by the seminal work of Walker and Sellards (Walker and Sellards,1913) that not everyone infected with E. histolytica derived from a'convalescent carrier' would go on to develop disease, so Brumpt's patientswere not distinguishable from these asymptomatic experimental infections.Despite further experimentation by Brumpt (1926) and his studentTschedomir Simic (1931a, 1931b, 1935), their work was essentially ignoredfor the next 50 years.

The next evidence of two groups within E. histolytica did not emergeuntil 1972 when it was shown that amebae isolated from individuals withdisease had different lectin agglutination properties to those isolated fromasymptomatically infected individuals (Martínez-Palomo et al. 1973). Thebasis of this difference has recently been elucidated experimentally. Thesurface of E. histolytica is covered with a dense layer oflipophosphoproteoglycan, which is absent from E. dispar (Espinosa-Cantellano et al. 1998; Bhattacharya et al. 2000). In the initial publication thesurface properties were linked to differences in virulence among the strains inexperimental models but not, at this stage, to species differences.

In 1978, Sargeaunt and Williams published the first of a long series ofarticles on their isoenzyme studies in Entamoeba (Sargeaunt et al. 1978).

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Their examination of a large number of isolates in culture againdifferentiated two groups of organisms based on the migration of enzymes ingels. One of the groups was primarily isolated from symptomatic patientswhile the other was never isolated from individuals with invasive disease(Sargeaunt, 1987). The two types were named 'pathogenic' and'nonpathogenic' E. histolytica. Within 5 years Sargeaunt and Williams werealready raising the possibility in print that Brumpt had been right all along andthat the 'nonpathogenic' group they were detecting should be identified as E.dispar (Sargeaunt et al. 1982). The basis of this differentiation by isoenzymeshas now been shown to be due to sequence differences in the genes, at leastfor the most widely used enzyme, hexokinase (Ortner et al. 1997).

The main reason why Sargeaunt and Williams' proposal to resurrect thename E. dispar was not accepted more quickly is that, starting in 1986, anumber of reports were published that seemed to indicate that interconversionbetween the two forms could take place (Mirelman et al. 1986a, 1986b;Andrews et al. 1990; Mukherjee et al. 1993; Vargas and Orozco, 1993). Thisphenomenon was observed during attempts to grow the 'nonpathogenic' formunder axenic culture conditions. The results implied that the 'nonpathogenic'form was somehow activated, leading to a change in gene expression orprotein modification to give the 'pathogenic' isoenzyme phenotype and avirulent organism capable of causing disease. Not surprisingly this caused alot of controversy in the field of amebiasis research and generated a lot ofinvestigation into the observations.

During this same time period the first monoclonal antibodies and thefirst gene sequences were obtained from these organisms. Monoclonalantibodies often identified two groups of isolates that correlated with theirisoenzyme patterns (Strachan et al. 1988; Petri et al. 1990). Likewise, DNAfrom the two forms was also shown to be distinct (Garfinkel et al. 1989;Tannich et al. 1989). An extensive series of experiments attempting toreplicate the conversion phenomenon were unsuccessful (Clark et al. 1992).As the experimental data accumulated, it became more and more difficult toaccommodate the interconversion observations within known biologicalprocesses - PCR could not detect the presence of both gene sequence typeswithin the same organism for example. Finally, the first DNA-based typingsystem for Entamoeba isolates showed that the genotypes of 'converted'organisms matched those of laboratory reference strains implying that someform of cross-contamination was the most likely explanation for the observedchanges (Clark and Diamond, 1993a, 1993b). It has subsequently becomeclear that E. dispar cannot be made to grow axenically under the standardconditions used for E. histolytica (Clark, 1995; Kobayashi et al. 1998). Eventhe smallest number of E. histolytica cells will outgrow E. dispar under these

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conditions, leading to an apparent conversion of isoenzyme patterns (Clarkand Diamond, 1993b).

The evidence supporting the existence of two groups coupled with alikely explanation for the conversion phenomenon led to the redescription ofE. histolytica in 1993 to separate it from E. dispar (Diamond and Clark,1993). Although initially controversial, this change of nomenclature wasquickly accepted and in 1997 was given WHO approval (Anonymous, 1997).

IMPLICATIONS OF THE RECOGNITION OF E. DISPARAS A DISTINCT SPECIES

The splitting of E. histolytica and the acceptance of E. dispar as adifferent species is far from being simply an intellectual exercise in taxonomyand classification. It has real and significant implications for diagnosis andtreatment of infections as well as interpretation of published data. Indeed thechange has made the work of the diagnostic lab and the clinician much moredifficult.

Since Lösch's day, the primary method for identification of Entamoebainfections has been light microscopy. Under the microscope, the cysts andtrophozoites of E. histolytica and E. dispar appear identical irrespective of themethods of preparation and staining used. The only exception to this is incases of amebic colitis where trophozoites filled with red blood cells may beseen and these are indicative of an E. histolytica infection (González-Ruiz etal. 1993). In most samples, however, only cysts will be observed and thespecies involved will remain unidentifiable. When reading the literature frombefore 1980, and in many subsequent publications, we cannot in most casesidentify the species present when microscopy was the only method used fordiagnosis. Therefore interpretation of the data retrospectively is not possible.This will certainly be the case in population surveys where prevalence figuresare given.

We are now starting to obtain new prevalence data in which the speciesare separately identified. However, the data remain patchy and relatively fewcountries have been re-surveyed using species-specific technologies. To datemost studies have concentrated on defined and geographically restrictedpopulations. Broader studies have relied on less random sampling, studyinghospital patients, for example, who may not be representative of the wholepopulation. In general, E. dispar is found to be the more common species, in aratio of up to 10:1 in some areas. However this is not always the case.Recently E. histolytica was shown to be the more prevalent in a specificregion of Vietnam (Blessmann et al. 2002).

Retrospective interpretation of the literature may be possible in the caseof serological surveys. It appears from most studies that E. dispar infectiondoes not lead to seropositivity, at least when assayed using E. histolytica

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antigen. Those seropositive individuals infected with E. dispar are likely tohave been infected previously with E. histolytica (Gathiram and Jackson,1987). Interpretation of the infection rate relies on a clear picture of thepersistence of seropositivity and of infections in the absence of treatment. Atpresent the data appear to indicate that seropositivity persists for a year ormore after an infection is eradicated (Haque et al. 1999; Valenzuela et al.2001). The persistence of infection in the absence of treatment is less clear asdata from different studies do not agree. Children in Bangladesh appear toclear E. histolytica infections quickly but often become reinfected (Haque etal. 2002), while adult infections in Vietnam have a half-life of more than oneyear (Blessmann et al. 2003). If the latter proves to be the case in Mexico, theserological survey conducted there (which found 5.9% seropositivity) wouldindicate a much lower rate of new infection than previously suspected.

Microscopy continues to be the diagnostic method used in mostlaboratories around the world. Despite the commercial availability of specificdiagnostic tests that allow the differentiation of E. histolytica and E. disparusing ELISA or PCR, the cost of reagents and equipment remains beyond thereach of laboratories in most countries where the infection is prevalent.Recognising this, the WHO recommended the reporting of microscopy-baseddiagnosis of such infections as "E. histolytica /E. dispar" and suggested thatin the absence of proof or a strong suspicion that the organism being seen isE. histolytica the infected individuals should not be treated (Anonymous,1997). This latter recommendation is based on the relative prevalence data (E.dispar making up an estimated 90% of the cysts reported as E. histolytica /E.dispar) and on the observation that the vast majority of those infected with E.histolytica (in its redefined sense) never go on to develop invasive disease(Gathiram and Jackson, 1987; Haque et al. 1997, 2002; Blessmann et al.2003). Treatment of asymptomatic individuals is therefore likely to beunnecessary, and when the potential side effects of drug treatment and theexpense involved are considered it is difficult to justify this course of action.Ultimately, however, the decision on whether to treat a patient must be left upto the individual physician.

The prospects of a short-term solution to the diagnostic problem areslim for developing countries, as it would require a low technology,inexpensive method. At present the two commercially available diagnosticproducts do not match this description. The first to be marketed was based onthe existence of antigenic differences in the Galactose/N-Acetylgalactosamine-specific lectin found on the surface of both species of amebae(Haque et al. 1995, 2000) (Techlab, Inc., Blacksburg, VA, USA). This proteinis involved in both ameba attachment to the mucus layer of the colon and inbinding of bacteria for ingestion. During invasion, its properties lead the cellto bind host epithelial and other cells, which it then lyses. The diagnostic

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method is based on capturing the lectin from stool samples and detecting itspresence using species-specific monoclonal antibodies in an ELISA.Presumably the lectin is shed from the surface of the ameba or is derived fromlysed cells. It has not been reported on the surface of cysts to my knowledge.The method is relatively simple to perform and does not need complex andexpensive equipment, but the test itself is probably too expensive forwidespread use in many countries.

The second diagnostic method has not been used widely to date(Blessmann et al. 2002) and is even less accessible to developing countries,relying as it does on Real-Time PCR (Artus Biotech, Hamburg, Germany).Here both the reagents and the equipment are expensive. However, thesensitivity and specificity of the method appear to be even greater than for theELISA and the test may therefore find a niche in some N. American andEuropean diagnostic laboratories.

Despite the 'inconvenience' caused to diagnostic laboratories by theredescription of E. histolytica as a result of the acceptance of E. dispar'sexistence, the recognition that there are two species involved should leadultimately to a reduction in the unnecessary use of medication. The increasedawareness of E. dispar as will also, hopefully, lead to a reduction in theincorrect attribution of many intestinal problems to 'amebiasis' just because anameba resembling E. histolytica is present in the stool of an individual withgastrointestinal complaints.

HOW DO E. DISPAR AND E. HISTOLYTICA DIFFEROne of the problems with diagnosis of amebae is there are few

morphological characteristics to use. The lack of distinguishing morphologyis probably the primary reason it took so long for the existence of E. dispar tobe accepted. Nevertheless, when molecular characteristics are studied the twospecies become easily distinguishable. How different they are depends onwhat measure you use.

The differences initially identified and that led to the redescription of E.histolytica fell into three categories (Diamond and Clark, 1993). The first wasisoenzyme differences. The initial work of Sargeaunt and Williams, usingthree and then four enzymes, was later supplemented with additional enzymesby Blanc (Sargeaunt et al. 1982; Sargeaunt, 1987; Blanc, 1992). However theoverall picture remained the same - two clearly identifiable groups oforganisms persisted. Next came antigenic differences, where monoclonalantibodies identified two groups of organisms that correlated with theisoenzyme patterns obtained from the same isolates (Strachan et al. 1988).Shortly thereafter came DNA differences - from Southern blot analysis thenDNA sequencing (Tannich et al. 1989). These again identified two groups that

Clark

correlated with the isoenzyme patterns. It is in the field of DNA analysis thatmost of the subsequent differences have been detected.

No protein coding gene sequence has proven to be identical betweenthe two species. In fact the percentage sequence identity in coding regions oforthologous genes averages only 95% while in non-coding regions it drops to80% (Tannich et al. 1991; Willhoeft et al. 1999a). Variation within eachspecies has not been widely examined but appears to be significantly less than1% (Ghosh et al. 2000). This has allowed the design and testing of manydifferent PCR-based diagnostic methods in laboratories around the world. Noorganisms with characteristics intermediate between the two species havebeen identified. Thus, although E. histolytica and E. dispar are each other'sclosest relative within the genus Entamoeba (Silberman et al. 1999), they areclearly distinct and discrete species.

Qualitative analyses of their genomes are incomplete althoughcomparative genome sequencing is underway. Several significant differenceshave been found so far although the significance of most is as yet unclear. TheShort Interspersed Nuclear Element (SINE) known as IE or Ehapt2 isabundant in E. histolytica but rare or absent in E. dispar (Willhoeft et al.2002). The E. histolytica gene family encoding a surface protein known asAriel also appears to be absent in E. dispar (Willhoeft et al. 1999b). Thedifference that has generated the most excitement is the absence in E. disparof a functional gene homologous to the cysteine proteinase known as EhCP5in E. histolytica (Willhoeft et al. 1999a). The E. dispar chromosomal locushomologous to that in which EhCP5 is found has been sequenced, and adegenerate version of the gene was found that contained numerous mutationsand had no possibility of encoding a protein. The corresponding protein in E.histolytica is found on the surface of trophozoites (Jacobs et al. 1998) and istherefore suspected of playing a role in tissue invasion. It is anticipated thatadditional differences will be uncovered as genome sequencing progresses.

While it is still true to say that the two organisms appear identical underthe light microscope, using electron microscopy morphological differencescan be detected. The early cell surface difference detected by lectinagglutination was later identified as being due to the absence oflipophosphoproteoglycan and this can be visualised directly in transmissionelectron microscopy of the E. dispar cell surface. A thick surface coat seen onE. histolytica cells is missing from E. dispar leading to a difference in cellsurface charge (Espinosa-Cantellano et al. 1998). There have also beendifferences in the organisation of intramembrane particles reported (Pimentaet al. 2002). Another inter-specific difference involves the ingestion ofbacteria. While bacteria ingested by E. dispar are found individually invacuoles with membranes tightly delimiting the bacterium, in E. histolyticaseveral bacteria are found in the same phagocytic vacuole and with no close

21

22 Clark

apposition of the membrane (Pimenta et al. 2002). One note of cautionhowever; the structural differences reported are based on the study of onlyone isolate of E. histolytica in each paper and the same isolate of E. dispar inboth, so the possibility of these being strain differences rather than speciesdifferences cannot be excluded.

CONCLUSIONSIt is simplistic to say that the major difference between E. histolytica

and E. dispar is that one causes disease and the other does not. However,ultimately this will always be the characteristic that defines the two organismsfor most people. The genetic basis of this phenotypic difference remains to beestablished but it is clearly a major goal in current amebiasis research.

Our understanding of the genetic differences between E. histolytica andE. dispar is likely to change fundamentally within the next year as thecomparative genome analysis reaches fruition. The genome information byitself will not provide the complete picture, however, and it will need to befollowed by transcription and protein analyses as important differences mayprove to be quantitative rather than qualitative. Indeed, cell biological studieshint at this already as E. dispar is able to kill cells in culture (Espinosa-Cantellano et al. 1998), including neutrophils, almost as efficiently as E.histolytica.

From what we know at present, E. histolytica and E. dispar are rathersimilar organisms. They inhabit the same niche, eat the same food, aretransmitted in the same way, and are genetically closely related. Yet one iscapable of causing a serious disease that is often fatal if not treated, while theother is apparently benign. Understanding the reasons for this difference islikely to remain a challenge for several years to come.

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Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene84: 63-65.

Anonymous. 1997. WHO/PAHO/UNESCO report. A consultation with experts on amoebiasis.Epidemiological Bulletin PAHO 18: 13-14.

Bhattacharya, A., R. Arya, C.G. Clark and J.P. Ackers. 2000. Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar. Parasitology 120: 31-35.

Blanc, D.S. 1992. Determination of taxonomic status of pathogenic and nonpathogenicEntamoeba histolytica zymodemes using isoenzyme analysis. Journal of Protozoology 39:471-479.

Blessmann, J., H. Buß, P.A. Ton Nu, B.T. Dinh, Q.T. Viet Ngo, A. Le Van, M.D. Abd Alla,T.F.H.G. Jackson, J.I. Ravdin and E. Tannich. 2002a. Real-time PCR for detection anddifferentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. Journal ofClinical Microbiology 40: 4413-4417.

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Blessmann, J., P. Van Linh, P.A. Nu, H.D. Thi, B. Muller-Myhsok, H. Buß and E. Tannich.2002b. Epidemiology of amebiasis in a region of high incidence of amebic liver abscess incentral Vietnam. American Journal of Tropical Medicine and Hygiene 66: 578-583.

Blessmann, J., I.K.M. Ali, P.A. Ton Nu, B.T. Dinh, T.Q. Viet Ngo, A. Le Van, C.G. Clark andE. Tannich. 2003. Longitudinal study of intestinal Entamoeba histolytica infections inasymptomatic adult carriers. (submitted).

Boeck, W.C. and J. Drbohlav. 1925. The cultivation of Entamoeba histolytica. AmericanJournal of Hygiene. 5: 371-407.

Brumpt, E. 1925. Étude sommaire de l' "Entamoeba dispar" n. sp. Amibe à kystesquadrinucléés, parasite de l'homme. Bulletin de l'Academie de Médecine (Paris) 94: 943-952.

Brumpt, E. 1926. Individualité de l'Entamoeba dispar. Présentation de piéces. Bulletin de laSociété de Pathologié Exotique 19: 399-404.

Brumpt, E. 1928. Differentiation of human intestinal amoebae with four-nucleated cysts.Transactions of the Royal Society of Tropical Medicine and Hygiene 22: 101-114,Discussion pp. 115-124.

Clark, C.G. 1995. Axenic cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolitaGeiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879. Journal of EukaryoticMicrobiology 42: 590-593.

Clark, C.G., C.C. Cunnick and L.S. Diamond. 1992. Entamoeba histolytica : is conversion ofnonpathogenic amebae to the pathogenic form a real phenomenon? ExperimentalParasitology 74: 307-314.

Clark, C.G. and L.S. Diamond. 1993a. Entamoeba histolytica: a method for isolateidentification. Experimental Parasitology 77: 450-455.

Clark, C.G. and L.S. Diamond. 1993b. Entamoeba histolytica: an explanation for the reportedconversion of "nonpathogenic" amebae to the "pathogenic" form. Experimental Parasitology77: 456-460.

Diamond, L.S. and C.G. Clark. 1993. A redescription of Entamoeba histolytica Schaudinn,1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. Journalof Eukaryotic Microbiology 40: 340-344.

Dobell, C. 1919. The amoebae living in man. A zoological monograph. J. Bale, Sons, andDanielson, London., 155p.

Espinosa-Cantellano, M., A. González-Robles, B. Chávez, G. Castañon, C. Argüello, A.Lázaro-Haller and A. Martínez-Palomo. 1998. Entamoeba dispar : ultrastructure, surfaceproperties, and cytopathic effect. Journal of Eukaryotic Microbiology 45: 265-272.

Garfinkel, L.I., M. Giladi, M. Huber, C. Gitler, D. Mirelman, M. Revel and S. Rozenblatt.1989. DNA probes specific for Entamoeba histolytica possessing pathogenic andnonpathogenic zymodemes. Infection and Immunity 57: 926-931.

Gathiram, V. and T.F.H.G. Jackson. 1987. A longitudinal study of asymptomatic carriers ofpathogenic zymodemes of Entamoeba histolytica. South African Medical Journal 72: 669-672.

Ghosh, S., M. Frisardi, L. Ramirez-Avila, S. Descoteaux, K. Sturm-Ramirez, O.A. Newton-Sanchez, J.I. Santos-Preciado, C. Ganguly, A. Lohia, S. Reed and J. Samuelson. 2000.Molecular epidemiology of Entamoeba spp.: evidence of a bottleneck (Demographic sweep)and transcontinental spread of diploid parasites. Journal of Clinical Microbiology 38: 3815-3821.

González-Ruiz, A., M.A. Miles and D.C. Warhurst. 1993. Predictive value of diagnostic testsand prevalence of invasive Entamoeba histolytica infection. Journal of Infectious Diseases168: 513-514.

Guttiérez, G., A. Ludlow, G. Espinoza, S. Herrera, O. Muñoz, N. Rattoni and B. Sepúlveda.1992. Encuesta serológica nacional: II. Investigación de anticuerpos contra Entamoebahistolytica en la República Mexicana. Salud Publica de Mexico 34: 242-254.

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Haque, R., K. Kress, S. Wood, T.F.H.G. Jackson, D. Lyerly, T. Wilkins and W.A. Petri Jr.1993. Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based onmonoclonal antibodies to the galactose-specific adhesin. Journal of Infectious Diseases 167:247-249.

Haque, R., L.M. Neville, P. Hahn and W.A. Petri Jr. 1995. Rapid diagnosis of Entamoebainfection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. Journalof Clinical Microbiology 33: 2558-2561.

Haque, R., A.S.G. Faruque, P. Hahn, D.M. Lyerly and W.A. Petri Jr. 1997. Entamoebahistolytica and Entamoeba dispar infection in children in Bangladesh. Journal of InfectiousDiseases 175: 734-736.

Haque, R., I.K.M. Ali and W.A. Petri Jr. 1999. Prevalence and immune response to Entamoebahistolytica infection in preschool children in Bangladesh. American Journal of TropicalMedicine and Hygiene 60: 1031-1034.

Haque, H., N.U. Mollah, I.K.M. Ali, K. Alam, A. Eubanks, D. Lyerly and W.A. Petri Jr. 2000.Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoebahistolytica II antigen detection and antibody tests. Journal of Clinical Microbiology 38: 3235-3239.

Haque, R., P. Duggal, I.K.M. Ali, M.B. Hossain, D. Mondal, R.B. Sack, B.M. Farr, T.H. Beatyand W.A. Petri Jr. 2002. Innate and acquired resistance to amebiasis in Bangladeshi children.Journal of Infectious Diseases 186: 547-552.

Jacobs, T., I. Bruchhaus, T. Dandekar, E. Tannich and M. Leippe. 1998. Isolation andmolecular characterization of a surface-bound proteinase of Entamoeba histolytica.Molecular Microbiology 27: 269-276.

Kobayashi, S., E. Imai, H. Tachibana, T. Fujiwara and T. Takeuchi. 1998. Entamoeba dispar.cultivation with sterilized Crithidia fasciculata. Journal of Eukaryotic Microbiology 45: 3S-8S.

Pathologische Anatomie und Physiologie und für Klinische Medicin, von Rudolf Virchow65: 196-211.

Lösch, F.A. 1978. Massive development of amoebae in the large intestine (Translation). InTropical medicine and parasitology. Classical investigations, B.H. Kean, K.E. Mott and A.J.Russell (eds.). Cornell University Press, Ithaca, NY, p. 71-79.

Martínez-Palomo, A., A. González-Robles and M. De la Torre. 1973. Selective agglutination ofpathogenic strains of Entamoeba histolytica induced con A. Nature New Biology 245: 186-187.

Mirelman, D., R. Bracha, A. Chayen, A. Aust-Kettis and L.S. Diamond. 1986a. Entamoebahistolytica: Effect of growth conditions and bacterial associates on isoenzyme patterns andvirulence. Experimental Parasitology 62: 142-148.

Mirelman, D., R. Bracha, A. Wexler and A. Chayen. 1986b. Changes in isoenzyme patterns ofa cloned culture of nonpathogenic Entamoeba histolytica during axenization. Infection andImmunity 54: 827-832.

Mukherjee, R.M., K.C. Bhol, S. Mehra, T.K. Maitra and K.N. Jalan. 1993. Zymodemealteration of Entamoeba histolytica isolates under varying conditions. Transactions of theRoyal Society of Tropical Medicine and Hygiene 87: 490-491.

Ortner, S., C.G. Clark, M. Binder, O. Scheiner, G. Wiedermann and M. Duchêne. 1997.Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenicEntamoeba histolytica from nonpathogenic Entamoeba dispar. Molecular and BiochemicalParasitology 86: 85-94.

Petri, W.A., Jr., T.F.H.G. Jackson, V. Gathiram, K. Kress, L.D. Saffer, T.L. Snodgrass, M.D.Chapman, Z. Keren and D. Mirelman. 1990. Pathogenic and nonpathogenic strains of

Lösch, F. 1875. Massenhafte Entwicklung von Amöben im Dickdarm. Archiv für

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Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin. Infection and Immunity 58: 1802-1806.

Pimenta, P.F., L.S. Diamond and D. Mirelman. 2002. Entamoeba histolytica Schaudinn, 1903and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles. Journal of Eukaryotic Microbiology 49: 209-219.

Sargeaunt, P.G. 1987. The reliability of Entamoeba histolytica zymodemes in clinicaldiagnosis. Parasitology Today 3: 40-43.

Sargeaunt, P.G., J.E. Williams and J.D. Grene. 1978. The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Transactions of the RoyalSociety of Tropical Medicine and Hygiene 72: 519-521.

Sargeaunt, P.G., J.E. Williams, R. Bhojnani, J. Kumate and E. Jimenez. 1982. A review ofisoenzyme characterization of Entamoeba histolytica with particular reference to pathogenicand non-pathogenic stocks isolated in Mexico. Archivos de Investigación Médica (México)13 (suppl 3): 89-94.

Schaudinn, F. 1903. Untersuchungen über die Fortpflanzung einiger Rhizopoden. (VorläufigeMittheilung). Arbeiten der Kaiserlichen Gesundheitsamte 19: 547-576.

Schaudinn, F. 1978. On the development of some rhizopods. (Preliminary report) (Translation).In Tropical medicine and parasitology. Classical investigations, B.H. Kean, K.E. Mott andA.J. Russell (eds.). Cornell University Press, Ithaca, NY, p. 110-118.

Silberman, J.D., C.G. Clark, L.S. Diamond and M.L. Sogin. 1999. Phylogeny of the generaEntamoeba and Endolimax as deduced from small subunit ribosomal RNA gene sequenceanalysis. Molecular Biology and Evolution 16: 1740-1751.

Simic, T. 1931a. Étude expérimentale complémentaire de l’Entamoeba dispar Brumpt, deSkoplje, sur le chat. Annales de Parasitologie Humaine et Comparée 9: 497-502.

Simic, T. 1931b. Infection expérimentale de l'homme par Entamoeba dispar Brumpt. Annalesde Parasitologie Humaine et Comparée 9: 385-391.

Simic, T. 1935. Infection expérimentale du chat et du chien par Entamoeba dispar etEntamoeba dysenteriae. Réinfection et immunité croisée du chien. Annales de ParasitologieHumaine et Comparée 13: 345-350.

Strachan, W.D., W.M. Spice, P.L. Chiodini, A.H. Moody and J.P. Ackers. 1988.Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoebahistolytica. Lancet i: 561-563.

Tannich, E., R.D. Horstmann, J. Knobloch and H.H. Arnold. 1989. Genomic DNA differencesbetween pathogenic and nonpathogenic Entamoeba histolytica. Proceedings of the NationalAcadademy of Sciences USA 86: 5118-5122.

Tannich, E., H. Scholze, R. Nickel and R.D. Horstmann. 1991. Homologous cysteineproteinases of pathogenic and nonpathogenic Entamoeba histolytica. Journal of BiologicalChemistry 266: 4798-4803.

Valenzuela, O., F. Ramos, P. Morán, E. González, A. Valadez, A. Gómez, E.I. Melendro, M.Ramiro, O. Muñoz and C. Ximénez. 2001. Persistence of secretory antiamoebic antibodies inpatients with past invasive intestinal or hepatic amoebiasis. Parasitology Research 87: 849-852.

Vargas, M.A. and E. Orozco. 1993. Entamoeba histolytica: changes in the zymodeme of clonednonpathogenic trophozoites cultured under different conditions. Parasitology Research 79:353-356.

Walker, E.L. and A.W. Sellards. 1913. Experimental entamoebic dysentery. Philippine Journalof Science B Tropical Medicine 8: 253-331.

Willhoeft, U., L. Hamann and E. Tannich. 1999a. A DNA sequence corresponding to the geneencoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conservedbut highly degenerated in Entamoeba dispar. Infection and Immunity 67: 5925-5929.

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Willhoeft, U., H. Buß and E. Tannich. 1999b. DNA sequences corresponding to the ariel genefamily of Entamoeba histolytica are not present in E. dispar. Parasitology Research 85: 787-789.

Willhoeft, U., H. Buß and E. Tannich. 2002. The abundant polyadenylated transcript 2 DNAsequence of the pathogenic protozoan parasite Entamoeba histolytica represents anonautonomous non-long-terminal-repeat retrotransposon- like element which is absent in theclosely related nonpathogenic species Entamoeba dispar. Infection and Immunity 70: 6798-6804.

EPIDEMIOLOGY AND STRAIN VARIATION OFCRYPTOSPORIDIUM

R.M. Chalmers1 and D.P. Casemore2

1Head, PHLS Cryptosporidium Reference Unit, Swansea PHL, Singleton Hospital, SwanseaSA2 8QA, UK; 2Senior Research Fellow, Centre for Research into Environment & Health,University of Wales, Aberystwyth, SY23 2DB, UK.

ABSTRACTCryptosporidium parvum emerged in the 1970s as a common enteric

pathogen of young livestock and other animals and as an opportunistic andsometimes fatal infection in humans, primarily affecting theimmunocompromised. Since then it has become recognised as a worldwidecause of acute, self-limiting diarrhoeal disease in otherwise healthy humans. Itis a common cause of waterborne disease. The highest incidence is amongchildren under 5 years in developed countries, with a younger peak indeveloping countries. There are multiple sources and routes of infection,indicated initially by field epidemiology studies but subsequently confirmedby phenotypic and genotypic (molecular) methods. Such typing analyses haveshed new light on biology and epidemiology, providing a better understandingof the aetiology and public health control of cryptosporidiosis and also on theinvestigation of potential drug therapy. The last twenty years have thus been aperiod of exciting advance across many fields.Key words: Cryptosporidium, epidemiology, typing, public health,waterborne disease.

INTRODUCTIONHuman cryptosporidiosis – or its recognition - typifies the paradigm

of a disease whose time had come. Cryptosporidium parvum was discoveredin the early 1900s but was not described in humans until 1976 (Current, 1998;Fayer, 1997). It then emerged primarily as a cause of potentially fatal gastro-intestinal disease in immunocompromised patients, especially in the thennewly emerging condition, AIDS. It was seen initially, therefore, as a rareopportunistic infection. At the same time it was also identified increasinglywidely as an enteric pathogen in livestock animals. In the early 1980’sindependent studies in several parts of the world showed that it was in fact acommon cause of acute self-limiting gastro-enteritis in otherwise healthy

28 Chalmers and Casemore

people, especially children (Casemore, 1990; Cordell and Addiss, 1994;Current, 1998; Griffiths, 1998; Palmer & Biffin, 1990). Increasinglywidespread diagnosis and epidemiological investigation soon led to therecognition of waterborne disease as a significant public health problem(Meinhardt et al., 1996; Rose et al., 1997). It was assumed initially that allhuman infections were zoonotic and indeed infection from direct contact withlivestock is common (Casemore et al., 1997). This interpretation led,however, to some curious questions of biological plausibility, but thehypothesis that many infections were non-zoonotic (Casemore and Jackson,1984) remained unprovable until the emergence of suitable typing and tracingmethods.

SOURCES AND TRANSMISSIONThe epidemiology of cryptosporidiosis is complex, involving both

direct and indirect routes of transmission from animals to man and fromperson to person (Casemore et al., 1997). Cryptosporidium has been reportedworldwide and is common in man, in livestock animals and in wildlife;domestic pets were thought to be an uncommon source of infection. Zoonoticinfection by direct contact with mammalian livestock, especially lambs andcalves, is common, particularly in urban children visiting educational farms(Casemore, 1989). Indirect transmission, especially through water is alsocommon. Indeed, the widespread epidemic in the U.K. of foot and mouthdisease during 2001, and consequent control measures, led to a measurabledecline in incidence (estimated 35% overall) of cryptosporidiosis in thehuman population (Smerdon et al., 2003).

Direct faecal-oral transmission is common in children attendingplaygroups and daycare centers (Casemore, 1990; Cordell & Addiss, 1994),although the infection may be introduced, in the first instance, throughzoonotic contact (Casemore, 1989; Palmer & Biffin, 1990). Cryptosporidiumis a common cause of traveller’s diarrhoea, including that acquired duringvacation in the same country, probably due to increased and varied exposures.Hospital (nosocomial) transmission has been reported between patients andsometimes also to health care workers (Casemore et al., 1994). In HIV-positive patients increased risk is thought to be greatest from sexual high-riskbehaviours (Hunter and Nichols, 2002; Kim et al., 1998; Matos et al., 1998;Pedersen et al., 1996). There is no evidence of transmission across theplacenta. In livestock animals, oocyst excretion in dams is known to increaseduring the period around birth of offspring (Xiao et al., 1994). Foodborneinfection appears to be uncommon but has been associated with, for example,consumption of apple juice (Millard et al., 1995), unpasteurized milk,uncooked (non-fermented) sausages and salad (Casemore 1990; Palmer andBiffin, 1990).

Chalmers and Casemore 29

Water presents a major route of transmission, both drinking water andthrough recreational use (Meinhardt et al., 1996; Rose et al., 1997). Severaloutbreaks in the U.K. have involved around 500 laboratory confirmed cases,while an outbreak in Milwaukee in the U.S.A involved and estimated 403,000cases and cost the community millions of dollars. Outbreaks occur every yearassociated with potable public drinking supplies, including some associatedwith ground water sources previously believed to be safe. The incidence ofwaterborne infection may be amplified by secondary spread although theextent of this, and thus to some extent the size and duration of an outbreak,will reflect both the infecting strain and population immunity. (Frost et al.,2001; Meinhardt et al., 1996; Osewe et al., 1996). These factors may alsoinfluence the outcome of epidemiological studies (Harrison et al., 2003;Hunter and Quigley, 1998). It is difficult to assess the contribution of water tosporadic or endemic infection, although such transmission undoubtedlyoccurs. Concern over waterborne infection has led to the setting up of officialadvisory groups and issuing of advice in several countries (Harrison et al.,2003; Hunter 2000; Rose et al., 1997). Swimming pools are a significant riskfor transmission (Furtado et al., 1998; Meinhardt et al., 1996; Rose et al.,1997). Recent prospective population studies from Australia failed to confirmassociation between water consumption and endemic infection but did showthe importance of swimming pool use (Hellard et al. 2000; Robertson et al.,2002). Transmission associated with swimming pools result from faecalcontamination of the pool by users rather than of the mains water supply.Water quality parameter failures (e.g. raised turbidity) associated with unusuallevels of challenge to treatment and/or defects in treatment have been noted inmany outbreaks (Meinhardt et al., 1996; Rose et al., 1997). In view of thefrequency of heavy rainfall prior to many outbreaks it is interesting tospeculate on the potential effect of global climate change. Reports suggestthat this may lead to increased incidence in foodborne and waterborneinfections, and increased monitoring and control may need to be considered(Anon, 2002a; Rose et al., 2001). Private supplies may represent a particularrisk, especially for sporadic infection in visitors. These supplies generallyserve smaller numbers of consumers (Furtado et al. 1998; Meinhardt et al.,1996).

EPIDEMIOLOGY – PERSON, TIME AND PLACEIn developing countries, infection is common in infants aged less than

1 year, while in developed countries infection is most common in childrenaged from 1 to 5 years, with a secondary peak in young, mainly urban, adults(Casemore 1990; Palmer & Biffin, 1990). A relative increase in incidence inadults is often seen in waterborne outbreaks (Meinhardt et al., 1996). Malesand females are generally affected with equal frequency although there is

30 Chalmers and Casemore

evidence from some studies in developing countries of a preponderance ofmale children, an observation common to a number of infectious diseases.Infection in children in developing countries may be associated withenteropathy and exacerbation of the effects of malnutrition, including immunedysfunction (Agnew et al., 1998; Clark, 1999; Checkley et al., 1998; Griffiths,1998).

There is evidence of seasonal peaks in several studies worldwide,particularly in spring and autumn, which do not necessarily both occur in anyone locality, nor recur year by year (Casemore, 1990). They coincidegenerally with lambing and calving, with other farming events such as muckspreading, with maximal rainfall, and with peak foreign travel.

Published reports show that the infection ranks about fourth in the listof pathogens detected in stools submitted to the laboratory. Among youngchildren in the U. K. cryptosporidiosis is more common than salmonellainfection, and during peak periods detection rates may exceed 20 per cent(Casemore 1990; Palmer & Biffin 1990). Cryptosporidiosis is generally oneof the most common causes of diarrhoea in AIDS patients and in some studiesprevalence exceeded 50 per cent (Pedersen et al., 1996; Clark, 1999; Hunterand Nichols, 2002;). Rates and/or severity of disease have declined recently inthe developed world, reflecting more effective anti-AIDS therapy. Infectionrates and severity are not generally increased for other immunocompromisedgroups unless profoundly compromised.

MOLECULAR STUDIES – STRAIN VARIATIONMolecular methods have answered many of the questions raised by

earlier field epidemiology since investigation of strain variation began in thelate 1980s and early 1990s. This included the observations that differentisolates have varying infective dose size and clinical responses (Fayer andUngar, 1986). Phenotyping tools tell us something about the characteristics anorganism expresses as a distinguishable trait. Those applied to C. parvuminclude protein analysis and antigenic diversity (Mead et al., 1988; McDonaldet al., 1991; McLauchlin et al., 1998; Nichols et al., 1991; Nina et al., 1992),and isoenzyme typing using various housekeeping enzymes (Ogunkolade etal., 1993). These showed that there were consistencies in the nature ofvariation detected, which had biological and/or epidemiological significance.Differences between species were reported and some differences within thespecies C. parvum, reflecting “animal types” and “human types” wereindicated (McDonald and Awad-el-Kariem, 1995). However, large numbersof oocysts were required from each source and since it is difficult to amplifyCryptosporidium oocyst numbers in the laboratory, only isolates being shed inlarge numbers by acutely infected animals or people could be studied:

Chalmers and Casemore 31

samples from sub-clinical infections and environmental samples simply didnot contain enough oocysts for the methods to be applied.

The application of the polymerase chain reaction (PCR), in which theamount of targeted genetic material is amplified from a theoretically very lowstarting point to produce huge amounts of replicated DNA, has providedresearchers with sufficient material to further investigate variation withinCryptosporidium (Morgan and Thompson, 1998). For this reason, almost allof the genetic methods applied to investigate strain variation within the genusrely on initial amplification of targeted gene loci by PCR. In addition, PCRdetection of Cryptosporidium in human and animal samples has been shownto be more sensitive and specific than traditional diagnostic microscopy(Webster, 1993; Morgan et al., 1998).

The main challenges to the application of PCR to Crypto-sporidiumare extracting DNA from the sporozoites within the robust oocysts andavoiding the effect of any inhibitors that might be present in the initialsample. Furthermore, the selection of the appropriate polymerase for aparticular biological sample type may be critical since polymerases can bedifferentially denatured, or inhibited by, for example, proteinases, phenols anddetergents present in the sample matrix. Over-coming inhibitors has beenachieved by the use of preliminary purification steps to remove sample matrixmaterial. Purification methods include floating oocysts from faecal matterusing two-phase systems (e.g. saturated salt solutions) or recovering oocystsfrom the sample matrix by immunomagnetic separation. Boiling the sampledestroys inhibitors, while DNA extraction methods and kits remove inhibitorysubstances (Boom et al., 1990; Elwin et al., 2001; Xiao et al., 2001a).

While direct comparison of nucleotide sequences is the ultimatemethod or gold standard for detecting DNA sequence variation, theidentification of consistent markers provides less complex tools forapplication to large numbers of samples required for epidemiologicalinvestigations. Such tools have been applied to identify species / genotypeswithin Cryptosporidium and include the investigation of randomly amplifiedpolymorphic DNA, restriction fragment length polymorphisms (RFLP)following locus-specific amplification by PCR, single-strand conformationpolymorphism analysis, and the application of real time PCR. PCR-RFLP hasbeen widely used and while it has limitations, it provides a reliable, specificand rapid species / genotype identification particularly if applied with qualitycontrol standards. The most widely targeted gene loci have been the smallsubunit ribosomal DNA (ssu rDNA or 18s) and the Cryptosporidium oocystwall protein (COWP) gene. Other target gene loci also includethrombospondin-related adhesive proteins (TRAP-C1 and TRAP-C2),dihydrofolate reductase-thymidylate synthase (dhfr-ts), ribonucleasereductase, internal transcribed rDNA spacers (ITS1 and ITS2), acetyl-CoA

32 Chalmers and Casemore

synthetase, beta tubulin and the 70kDa heat shock protein (hsp70) gene(Clark, 1999; Morgan et al., 1999a; Fayer et al., 2000).

Using molecular tools to investigate strain variation, it has beenshown that many of the differences within C. parvum actually represent twodifferent species: genotype 1, the “human” type, for which the name C.hominis has been proposed, and genotype 2, the “animal” or “cattle” type,which has retained the name C. parvum (Morgan-Ryan et al., 2002).

While it is evident that some primer pairs amplify all species withinthe genus (e.g. those for the ssu rRNA and COWP genes), others are morespecific (such as those for TRAP-C2 which are specific for C. parvum and C.hominis) (Elwin et al. 2001). However, others also amplify DNA from relatedprotozoan parasites, and some PCR-RFLP protocols differentiate species /genotypes more readily than others (Sulaiman et al., 1999). Primer pairs musttherefore be chosen according to the question being asked, as must therestriction enzymes applied since additional enzymes may be required todifferentiate all species. The importance of sequence confirmation of RFLPpatterns was illustrated by Chalmers and colleagues (2002a) who identified anovel RFLP pattern, very similar to C. hominis, in the COWP gene of isolatesfrom sheep, but sequence data clearly differentiated the isolate. Therefore,careful primer selection and PCR product analysis is required for detectionand characterization, particularly from environmental specimens where a widerange of cryptosporidia and other organisms may be present.

These broad methods have helped clarify the taxonomy within thegenus (although questions still exist above that level) and establish, withprinciples of classical parasitology, that there are 13 currently recognisedspecies (Fayer et al., 2000; Fayer et al., 2001; Alvarez-Pellitero and Sitjà-Bobadilla, 2002; Morgan-Ryan et al., 2002) (Table 1).

An increasing number of C. parvum genotypes have also beenidentified, some of which appear to be host-adapted since they have only beendetected in a limited range of host species. An example is the marsupialgenotype (Xiao et al., 1999) which has, so far, only been detected inmarsupials. Some of these genotypes may warrant species status. Declarationof species within the protozoa has traditionally relied upon classical criteriaencompassing morphological and ultrastructural data and life cyclecharacteristics including host range. Additional data are now available fromgenetic analyses, and these must be considered as complementary to theclassical criteria during assignation of species status.

Chalmers and Casemore 33

Whichever methods have been used to characterise isolates ofCryptosporidium at the species level have consistently told us that the vastmajority of human infection are caused by C. hominis and C. parvum. It hasbeen speculated that an intact immune system maintains host specificity sincefour other species have been detected in immunocompromised patients(Morgan et al., 1999b; Gatei et al., 2002). However, three of these specieshave now also been detected in clinical specimens from a small number ofimmunocompetent patients (Table 1), indicating that they are circulating inthe community and may pose an emerging public health risk, particularlysince little is known of their epidemiology, sources and transmission(Chalmers et al., 2002b). In Peru, these three other species were also found inboth diarrhoeic and non-diarrhoeic children who had no evidence of HIVinfection (Xiao et al., 2001b). Interestingly, in environmental studies ofsurface water and waste water samples in the USA, a cocktail ofCryptosporidium species was detected using IMS-PCR-RFLP, including C.

34 Chalmers and Casemore

parvum, C. hominis, C. felis, C. andersoni, C. muris, C. baileyi and a range ofC. parvum genotypes (Xiao et al., 2001b). Mixed infections have also beennoted in waterborne outbreaks (Patel et al., 1998). Molecular tools confirmthat people are exposed to a variety of potentially infective organisms fromenvironmental sources, as might be predicted (Meinhardt et al., 1996).

C. parvum has not only been detected in humans but also in a widerange of livestock and wild animals, while C. hominis appears to be largelyrestricted to humans, although there are published reports of natural infectionin a non-human primate and a dugong (Morgan et al., 2000) and experimentalinfections in pigs (Widmer et al., 2000), lambs (Giles et al., 2001) and calves(Akiyoshi et al., 2002). Despite this, C. hominis has a far more restricted hostrange than C. parvum and the detection of C. hominis in a sample indicates ahigh probability of a human source (Patel et al., 1998; Harrison et al., 2003).Enhanced surveillance and molecular epidemiology have further elucidatedthe epidemiology of human cryptosporidiosis and shown that regional andseasonal differences exist that may reflect differing exposures and behaviours(McLauchlin et al., 2000; Anon, 2002b). For example, regional differencesmay reflect urban/rural or human/zoonotic cycles of transmission. Seasonaldifferences may be linked to animal husbandry and reproduction, resulting ina spring increase in human C. parvum infections and to recent foreign travelwhich mainly occurs following the summer holidays resulting in an increasein C. hominis infections. This, however, is worthy of further investigation toidentify more precisely the risks during foreign travel. Furthermore, speciesvariations are observed when the data are analysed by countries visited(Anon, 2002b). Thus far, little is known of the epidemiology of non-C.parvum, non-C. hominis infections in humans, of which C. meleagridispredominates.

Analysis of outbreak samples has confirmed that urban trans-missionis not restricted to C. hominis but can occur with C. parvum. For example, inan outbreak associated with an indoor swimming pool in England, where thelikely source of contamination was human faecal material, C. parvum wasconfirmed in 34/41 cases (Anon, 2000). Outbreak investigations have alsobenefited from the identification of the species causing human illness in theform of the provision of advice regarding appropriate control measures. Forexample, an outbreak in Belfast, Northern Ireland, was epidemiologicallylinked to the drinking water supply and the source of contamination wasinitially assumed to be livestock since the source water arose in a rural areaand flowed through an aqueduct under agricultural land prior to distribution.However, PCR-RFLP of the COWP gene identified the human cases as C.hominis indicating human sanitation failure as the source of contaminationand infection (Glaberman et al., 2002). Inspection of the aqueduct showed apoint of ingress of domestic sewage and remedial action was taken.

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Recent studies have further identified separate risk factors for C.hominis and C. parvum. In a case control study of sporadic cases in Wales andthe North West of England, risk factors for cryptosporidiosis where C.hominis was detected were travel abroad, changing the nappies (diapers) ofchildren under five, and having contact with another infected person, whilefor C. parvum, the main risk factor was contact with a farmed animals(Hunter, 2003). This work has shown that there is epidemiological importanceto considering the two infections separately. Differing clinical pictures mayalso emerge, since studies in Peru have shown that oocysts were shed forlonger and in greater numbers in C. hominis infections than C. parvum (Xiaoet al., 2001b). Differences in pathogenesis between the two species have beenobserved in the pig model, in which C. parvum had a shorter pre-patent periodand resulted in more severe diarrhoea than C. hominis (Periera et al., 2002).These results are consistent with the hypothesis that C. hominis is moreadapted to the human host and C. parvum to animal hosts, and consistent withresults of infection of human cell lines (Hijjawi et al., 2001). This perhapsgives an explanation for the findings in an outbreak that involved watercontaminated from both human and agricultural sources, in which themajority of cases showed infection with C. hominis (Patel et al., 1998).Different isolates of C. parvum also vary in their infectivity for humans(Okhuysen et al., 1997), and such differences in strain infectivity, combinedwith differing levels of population immunity make it difficult to developmeaningful health-risk based standards for water.

These studies, combining clinical evidence, field epidemiology andgenotyping, show that, despite some of the limitations of PCR-RFLP as adiagnostic tool, a biologically plausible and consistent epidemiological picturehas emerged. This, importantly, provides a key to determining the sources ofinfection, routes of transmission and relevant interventions. However, it isclear from identifying separate species that further differences are presentwithin the strains circulating in host populations. It can be hypothesised thatthis is most likely within C. parvum for which both a human cycle and azoonotic cycle exists. Although a clonal population structure has beenpreviously suggested for Cryptosporidium (Gibbons et al., 1998), this isunlikely since the life cycle incorporates a sexual stage: the apparent absenceof recombinants of C. parvum and C. hominis supports their separate speciesstatus rather than clonal population expansion. The presence of recombinantswithin these species and a better understanding of population structures areworthy of further investigation and have implications for understanding theepidemiology and control of Cryptosporidium (Morgan et al., 1999a).

To further investigate intra-species variation, sub-typing or“fingerprinting” methods need to be applied. Such tools will also help betteridentify co-infections of mixed Cryptosporidium species or subtypes. A

36 Chalmers and Casemore

variety of approaches have been investigated to fingerprint Cryptosporidiumisolates, mostly based upon non-coding sequences that display a higher levelof polymorphism than ssu rDNA or genomic sequences. While introns andintergenic regions may be appropriate targets for some organisms, withinCryptosporidium these are short or rare. However, sequence repeats occurringas mini and microsatellite DNA are common in the Cryptosporidium genome,and have been used as genetic markers in other related protozoan parasitessuch as Plasmodium falciparum since microsatellite sequences show a higherlevel of polymorphism than coding sequences. Analysis of microsatellitemarkers in the U.K., Italy and Denmark, has shown variation within C.parvum and C. hominis, and the technique can be used to demonstrate linksbetween cases and sources of infection (Enemark et al., 2002; Mallon et al,2002; Caccio, 2003). Studies in Australia have used microsatellite-telomeremarkers to demonstrate the consistency of predominant strains of C. parvumin cattle on individual farms (Blasdall et al., 2002), and the technique haspotential for application to human epidemiological studies.

The discovery of dsRNAs in C. parvum and C. hominis isolates fromnatural, experimental and cell culture infections (Khramtsov et al., 1997) mayrepresent species-specific markers (Khramtsov et al., 2000). While Khramtsovand colleagues found that cDNA sequences of 306nt and 257nt of the ds-RNAdifferentiated between C. parvum and C. hominis (suggesting coevolutionwith the Cryptosporidium host cells), other workers sequencing a 173bpfragment identified wide variation but lack of specificity at the species leveland concluded that while the ds-RNA typing tool may offer utility as atracking tool for investigating the source of infection, it must be used inconjunction with other species / genotyping tools (Xiao et al., 2001c). Single-strand conformation polymorphism (SSCP) analysis and denaturingpolyacrylamide gel electrophoresis have been used to identify C. parvum andC. hominis and intra-species variation in human isolates, with SSCPappearing to offer superior intra-species variation (Gasser et al., in press).

DNA sequence analysis of the GP60 gene (Strong et al., 2000) hasalso provided useful epidemiological information, with respect to analysis ofstrains circulating in the community and to outbreaks, albeit currently inretrospective analysis (Sulaiman et al., 2002; Peng et al., 2002; Glaberman etal., 2002).

To provide practical tools for public health investigations andintervention strategies for control, methods identifying strain variation need tobe relatively rapid and simple in application. These will help, particularly atthe local level, elucidate routes of transmission, compare the relativeimportance of zoonotic and human transmission, track the spread of virulentstrains and establish the importance of parasite heterogeneity to the localcommunity. However, discriminatory methods with a multi-locus approach

Chalmers and Casemore 37

will provide both species confirmation and a better evaluation of parasitepopulation structure. In many other parasite groups, genomic variationindicates differences in virulence, host specificity and drug susceptibility(Thompson and Lymberry, 1996), which has potential importance for both theclinical management of infection and control and for predictive epidemiology,so why not in Cryptosporidium? Application of typing methods to infectingstrains may also help elucidate some of the problems of identifying specifictherapy. Other questions, including those of cross-immunity betweeninfecting isolates, as yet remain unanswered. However, the advances inidentification of strain variation have highlighted differing epidemiologicalpictures that can be interpreted and explored further for application to thecontrol of the spread of this fascinating parasite.

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Xiao, L., J. Limor, C. Bern, and A.A. Lal. 2001c. Tracking Cryptosporidium parvum bysequence analysis of small double-stranded RNA. Emerging Infectious Diseases 7:141-145.

CYCLOSPORA CAYETANENSIS: AN EMERGENTAND STILL PERPLEXING COCCIDIAN

PARASITE

Charles R. Sterling1 and Ynes R. Ortega2

1Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ857212Center for Food Safety, Dept. Food Science and Technology, 1109 Experiment St, Universityof Georgia, Griffin, GA 30223

ABSTRACT:Cyclospora infecting humans emerged as a pathogen in the late

1970s and has since been largely associated with disease of children inthe tropics, travelers and expatriates to developing countries, and theimmunocompromised. It has gained recent notoriety because of food-borne disease outbreaks that have sparked much interest in trying tobetter define the epidemiology of this most intriguing parasite. It isclear from studies performed on Cyclospora that much further researchis required to better understand certain facets of this parsite’s life cycle,interactions of the parasite with its host and interactions of the parasitewith the environment.Key words: Cyclosora, epidemiology, foodborne disease, detection.

BACKGROUNDAshford reported on the coccidian identity of what is now known as

Cyclospora cayetanensis in the late 1970s when he observed spherical oocystsmeasuring in diameter from 3 patients of Papua, New Guinea(Ashford, 1979). Ashford also reported on the delayed sporulation of isolatedoocysts and the eventual formation of 2 sporocysts, but was unable to clearlydelineate the eventual number of sporozoites per sporocyst. Thinking that 4might be present per sporocyst, he felt the oocysts could belong to anunnamed species of the genus Isospora, Toxoplasma,or Hammondia. Asmore reports of similar organisms from around the world appeared in theliterature, confusion as to its true taxonomic identity arose. Common namesthat appeared included Cyanobacterium-like body (a blue-green alga),Coccidian-like body and large Cryptosporidium, the latter deriving from itsacid-fast staining characteristics (Naranjo et al., 1989; Hart et al., 1990; Long

44 Sterling and Ortega

et al., 1990; Long et al., 1991; Shlim et al., 1991). Further sporulation studiesfinally demonstrated that isolated oocysts contained 2 sporocysts, each with 2sporozoites (Ortega et al., 1993). This clearly placed the organism within thegenus Cyclospora, with the species name cayetanensis finally being added in1994 (Ortega et al., 1994). C. cayetanensis has gained a measure of notorietyas an important pathogen of the immunocompromised, children of developingcountries, expatriates living in developing countries, travelers to developingcountries and more recently as the causative agent associated with severallarge foodborne outbreaks of disease in the United States, Canada andelsewhere (Herwaldt, 2000). While much has been learned about this parasiterecently, there remain substantial gaps in our knowledge. This chapterdescribes our current understanding of C. cayetanensis and attempts toidentify gaps in knowledge that should be filled so we may better understandthis emergent and still perplexing coccidian parasite.

TAXONOMY AND PHYLOGENYMorphological characteristics of fully sporulated oocysts have led to

C. cayetanensis being taxonomically placed in the subphylum Apicomplexa,subclass Coccidiasina, order Eucoccidiorida and family Eimeriidae. Thistaxonomic placement within the Eimeriidae was substantiated by molecularstudies of the 18SssrDNA that aligned C. cayetanensis closely with the genusEimeria (Relman et al., 1996). So much so, that some have posed thequestion of whether Cyclospora should be considered a mammalian Eimeriaspecies (Relman et al., 1996; Pieniazek and Herwaldt, 1997). Sequence dataobtained from Cyclospora isolated from Ethiopian monkeys and Tanzanianbaboons have demonstrated differences with C. cayetanensis from humansand differences in the organisms from the respective primate hosts fromwhich oocysts were isolated (Eberhard et al., 1999a; Lopez et al., 1999). Thishas led to the naming of three new Cyclospora species: C. colibi, C. papionisand C. cercopitheci. The C. papionis from Ethiopian monkeys is consideredlikely to be the same species of Cyclospora observed in Tanzanian baboonsbased on morphology and gene sequences. Phylogenetic trees for sequencedCyclospora and Eimeria species demonstrate a great deal of relatednessbetween the genera, with the Cyclospora species representing a distinctmonophyletic grouping (Olivier et al., 2001). Cyclospora species, therefore,appear to be more closely related to each other than to Eimeria. What ismissing from this equation is sequence data from the other named Cyclosporaspecies that constitute an interesting group of organisms reported fromreptiles, myriapods, insectivores and one murine host. It has been argued thatphenotypic-based traditional taxonomic schemes are complex andunsatisfactory and that molecular methods are arguably the best techniquesavailable for studying the relatedness among organisms. We would argue that

Sterling and Ortega 45

both methods have served us well so far in helping to recognize Cyclosporaand delineate the relatedness of species to each other and to the genusEimeria. As such, traditional taxonomy and molecular phylogeny both havetheir place in resolving Cyclospora’s true taxonomic placement.

BIOLOGY AND LIFE CYCLEC. cayetanensis is like the majority of eimerid coccidians in that the

end result of infection in its host is the production of an oocyst that undergoessporogony outside the host’s body. What sets this parasite apart from themajority of Eimeriidae is the apparent length of time it takes to completesporogony. Parasites such as Toxoplasma gondii and Eimeria tenella usuallycomplete sporogony within 1-5 days, the process being dependent on oxygenand temperature (Frenkel et al., 1970; Norton and Chard, 1983). Underfavorable laboratory conditions, C. cayetanensis completes sporogony in from8 – 14 days (Ortega et al., 1993; Smith et al., 1997) while a baboon isolatesporulated more rapidly (5 days) (Smith et al., 1997). Sporulation times andconditions that may affect them are not known for oocysts passed into theenvironment. Other cyclosporans, C. caryolytica and C. talpae, both ofmoles, have been reported to complete Sporulation in 4-5 and 12-14 daysrespectively (Ortega et al., 1998). Unfortunately, animal models for C.cayetanensis do not exist, so it is unknown whether oocysts that havecompleted Sporulation were infectious or not. There is an obvious need tostudy the parameters that affect Sporulation since this impacts on theepidemiology of disease transmission. Directly passed unsporulated oocystsare non-infectious and, therefore, direct person-to-person transmission isunlikely. One has to wonder then how contamination of raspberries occurredin association with the well-documented cases of foodborne disease outbreaksassociated with consumption of this fruit. Raspberries must be shipped underconditions that would not favor rapid Sporulation and must usually beconsumed within a few days after they are placed on the market. Theassumption being made is that fully sporulated oocysts must have somehowfound their way onto this delicate fruit (Herwaldt et al., 1997). Seasonalitypatterns of infection observed in many places suggest that oocysts maysurvive for extended periods in the environment. It is not known how long orunder what conditions this survival might transpire. An issue forconsideration is whether or not there are specific environmental cues thattrigger the final events of Sporulation that translate into oocyst infectivity.

Humans appear to be the only host for C. cayetanensis (Eberhard etal., 2000; Eberhard and Arrowood, 2002). Reports have appeared in theliterature of Cyclospora oocysts that might be the same as C. cayetanensisbeing described from a variety of host animals, but other than the recentreports of oocysts from primates, representing distinct species, they have not

46 Sterling and Ortega

been confirmed. In this regard, C. cayetanensis, like many Eimeriidae,probably displays host specificity.

Ingestion of sporulated and infectious oocysts leads to parasitecolonization of the jejunum by sporozoites (Ortega et al., 1997a). Theinfectious oocyst dose is not known, but as for Cryptosporidium and Giardia,is presumed to be low. Parasites take up residence within an intracellularlocation within a parasitophorous vacuole. Several studies have confirmedthe presence of distinctive intracellular asexual merozoite and sexualgametocyte stages, requisite forms for completion of the life cycle within asingle host (Bendall et al., 1993; Sun et al., 1996; Nhieu et al., 1996; Ortega etal., 1997a). The lack of in vitro models of cultivation and other experimentalhosts has clearly limited further studies on the biology and life cycle of thisparasite.

DISEASE POTENTIAL, IMMUNITY AND TREATMENTThe range of symptoms caused by infection with C. cayetanensis, as

with many intestinal protozoan pathogens, can be highly variable and dependon a variety of population and environmental factors. Individuals likely toexhibit symptoms of disease include young children of developing worldcommunities, naïve individuals visiting or living as expatriates in developingcountries, naïve individuals of developed countries exposed to imported foodsand the immunocompromised, particularly individuals with AIDS. Symptomsmay develop abruptly or gradually and may be of relatively short duration orlast an average of 7 weeks in immunocompetent individuals. This lattercontrasting situation was observed in noting symptoms of children livingwithin an endemic country, Peru, versus adult travelers and expatriates of aforeign country visiting or living within another endemic country, Nepal(Hoge et al., 1993; Madico et al., 1997). Interestingly, adult patients fromPeru who live in upper class communities display symptoms similar to adulttravelers and expatriates. It is quite probable that these individuals had noprior exposure to this organism because of their socioeconomic status. Inmany endemic settings where poor sanitary conditions prevail, the number ofasymptomatically infected individuals usually is higher than those displayingsymptoms. In addition, there are indications that in some settings, such as thepueblo jóvenes of Peru, infection early in life predisposes to some type ofimmunity since infections have not been detected in adults from the samesetting (Madico et al., 1997). Symptoms, when they do occur, are quitesimilar to those brought on by infection with Cryptosporidium. Waterydiarrhea, mild to severe nausea, anorexia and abdominal cramping are thechief complaints (Shlim et al., 1991; Wurtz; 1994; Herwaldt, 2000). Adultpatients may experience weight loss of 5-10% and diarrhea alternating withconstipation has been commonly reported (Ortega et al., 1998).

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C. cayetanensis has been recognized with increasing frequency frompatients with AIDS (Long et al., 1990; Pape et al., 1994); Sifuentes-Osornio etal., 1995). This is particularly true in individuals living within or who havetraveled to developing countries. The symptoms are identical to those seen inimmunocompetent individuals, but may be prolonged. The prolonged courseof infection experienced in these patients is likely the result of continuedreplication of first generation meront stages in the absence of effectiveintestinal immunity. A lower than expected prevalence of C. cayetanensisinfection in AIDS patients is observed in some developing countries, such asPeru. This may be due to the prophylactic use of TMP-SMX against possiblePneumocystis carinii infection (Ortega et al., 1998). The rather highprevalence rate reported in adult AIDS patients of Haiti may be due to aninfrequent use of TMP-SMX prophylaxis in that country (Pape et al., 1994).

Immunity to C. cayetanensis has not been extensively studied. IgMand IgG antibodies are detected in response to infection and these antibodiesrecognized a wide range of parasite antigens, many of which are shared byCryptosporidium (Ortega, et al., 1998). An interesting feature of thisinfection that has been noted is the profound inflammation seen in histologicsections taken from intestinal biopsies and the paucity of parasitedevelopmental stages encountered (Ortega et al., 1997a). This raises thequestions as to whether the parasite might stimulate inflammation bymodulating certain pro-inflammatory cytokine responses. Otherwise, thesimilarities noted by host response, or lack thereof, to C. cayetanensis andCryptosporidium infections in immunocompetent and AIDS patients mightindicate that immune responses operate similarly towards these parasites.

Early studies aimed at elucidating the identity of Cyclospora alsonoted a high degree of infection resistance to conventional antimicrobialtherapy. In an initial report from Peru, subjective treatment withtrimethoprim-sulfamethoxazole (TMP-SMX) resulted in symptom cessationin one adult and four children after 4 days (mean) of treatment (Madico et al.,1993). The effectiveness of this treatment was confirmed in double-blindrandomized placebo-controlled trials involving expatriates in Nepal andchildren in Peru (Hoge et al., 1995; Madico et al., 1997). TMP, 160mg, andSMX, 800mg, bid for 7-10 days remains the recommended drug treatment forthis infection. In one randomized-controlled trial in patients infectedwith C. cayetanensis, ciprofloxacin, although not as effective, was deemed tobe an acceptable drug for patients who could not tolerate TMP-SMX (Verdieret al., 2000).

48 Sterling and Ortega

DISTRIBUTION, SEASONALITY AND OUTBREAKSITUATIONS

Infections with C. cayetanensis have been reported from around theworld, with most occurring in developing countries of the tropics and sub-tropics (Sterling and Ortega, 1999). Where surveys have been conducted intemperate zoned developed countries, infection rates have been exceedinglylow (<0.5%) and have largely been attributed to international travel orconsumption of imported food products. Indigenous transmission within theU.S., with no apparent foodborne association, has been suggested in a numberof reports. In these instances, the suggested vehicle of transmission was waterfrom 4 different sources: tap, lake, run-off and well (Hale et al., 1994; Wurtz,1994; Huang et al., 1995; Oii et al., 1995). In another report from the U.S.,infection was linked to gardening and working with soil (Koumans et al.,1996). In no instance, however, was there any direct evidence that infectionoccurred via these contact routes. Oocysts of Cyclospora have been isolatedfrom water sources of Nepal, Peru and Guatemala, where infections in thegeneral population are more common, suggesting that water plays an importrole in transmission (Rabold et al., 1994; Sturbaum et al., 1998; Bern et al.,1999; Sherchand et al., 1999; Sherchand and Cross, 2001).

An interesting feature associated with C. cayetanensis infections fromendemic areas is the rather marked seasonality. In Nepal infections are mostcommon prior to and during the warm monsoon months, but decrease beforethe rains end (Shlim et al., 1991). In Guatemala they follow a somewhatsimilar pattern, however the temperature is more moderate (Bern et al., 1999;Bern et al., 2000). In Haiti, the infections were recorded as most commonduring the drier and cooler months (Eberhard et al., 1999b), while in Lima,Peru, which is very dry throughout the year, infections usually occur duringthe warmer months (Ortega et al., 1993; Madico et al., 1997). Finally, inIndonesia, cases commonly appear during the cooler wet months (Fryauff etal., 1999). Presumably, factors such as moisture and temperature affectoocyst sporulation and survival. If so, in what way and how does one accountfor the variations in rather marked seasonality observed in these diverse areasof the world? Could factors such as the amount of sunlight or UV radiationover extended periods of time affect sporulation or oocyst survival? Theseare important questions since they would likely affect transmission patterns ofthis parasite.

Water and food have been identified as two sources of infection inassociation with disease outbreaks caused by C. cayetanensis. Zoonotic andperson-to-person transmission is unlikely since suspected animal reservoirs ofthis parasite have not been confirmed as carrying this parasite and sinceoocyst sporulation requires times of a week or longer under favorableconditions.

Sterling and Ortega 49

The first outbreak of cyclosporiasis in the US occurred in 1990among residents of a physicians’ dormitory in Chicago and it washypothesized that water from rooftop reservoirs was the contaminating source(Huang et al., 1995). The reservoirs received water from a municipal source,were covered with canvas and located in an unprotected building roof area. Inaddition, there had been a recent pump failure, resulting in stagnating water.People who became ill during this outbreak had attended a catered party, buthad also consumed tap water in the dormitory. Because informationregarding the food eaten was not obtained, it was not known if there mighthave been an association between a given food source and the attack ratesobserved. There was, on the other hand, a high correlation between attackrates and the consumption of tap water coming from the reservoirs.

A definite association of disease with water consumption was made inNepal in 1994 among British expatriates associated with a military facility(Rabold et al., 1994). Twelve of 14 persons became ill and oocysts wereidentified in the stools from 6 of 8. Oocysts were identified in grab samplesfrom a sealed tank receiving a mixture of river and municipal water that waschlorinated. Although data are lacking, oocysts of Cyclospora, like those ofCryptosporidium, are thought to be highly chlorine resistant. Consumption ofuntreated water had earlier been identified as a risk factor in association withillness seen at outpatient clinics serving expatriates in Nepal (Hoge et al.,1993). Organisms resembling Cyclospora were identified from the untreatedtap water of a home of one of the study patients. C. cayetanensis oocystshave also been isolated from wastewater of sewage lagoons near an area ofendemic disease in Lima, Peru (Sturbaum et al., 1998). While not implicatedin direct waterborne transmission in this instance, water from these lagoonswas used to irrigate certain food crops.

The association of C. cayetanensis with foodborne diseasetransmission was first suggested in 1995 by illness in an airline pilot who hadconsumed food prepared in a Haitian kitchen and brought on board theairplane (Connor and Shlim, 1995). Foodborne association was againimplicated in 1995 in small outbreaks in the United States (Koumans et al.,1998; Herwaldt, 2000). Larger outbreaks, occurring in 1996 and 1997 in boththe United States and Canada contributed to this organism’s notoriety andcalled attention to the fact that imported foodstuffs facilitated in thedistribution of this parasite (Herwaldt et al., 1997, 1999). Investigationsassociated with initial outbreaks in 1995 and 1996 implicated a variety ofsources, including raspberries and strawberries, as possibly being theresponsible agents of disease transference. Interestingly, the implication thatstrawberries might have been responsible caused considerable consternationand economic loss among US growers through early 1996 becausestrawberries were being pulled from the shelves of supermarkets in several

50 Sterling and Ortega

states (anon, 1996). They were subsequently dissociated from the outbreakswhen ultimately it was shown that raspberries were more significantlyassociated with illness by multivariate analysis of the data from the Floridaoutbreak of 1995 and outbreaks of early 1996 (Koumans et al., 1998). Inaddition, it was shown that strawberries were just as likely to be distributed toparts of the country where disease was not occurring as they were to parts ofcountry where disease outbreaks occurred.

The outbreaks of 1996 involving C. cayetanensis were instrumental toour understanding of foodborne transmission and the source of contamination.A total of 1,465 cases of cyclosporiasis were reported to the CDC and almosthalf were event associated (Herwaldt et al., 1997). Cases were reported from20 states, the District of Columbia and two Canadian provinces. In 55 eventrelated clusters, raspberries were definitely served at 50 and possibly at 4more. Traceback data, which is often complicated by complex distributionand handling of food sources, pointed to Guatemala and a number ofGuatemalan farms as the source of infected fruit. Because of the manner bywhich raspberries are grown and shipped, it was hypothesized that the berrieshad been sprayed with insecticides or fungicides prepared using oocystcontaminated water (Herwaldt et al., 1997). While not proven, this raises thequestion as to the potential effect of such agents on oocyst viability? Theoutbreaks of 1996 and 1997 led the US Food and Drug Administration torestrict the importation of raspberries during 1998. Canada did not follow thiscourse of action and experienced yet more outbreak clusters (Herwaldt, 2000).

Other imported fruits and vegetables have also been linked tofoodborne outbreaks of cyclosporiasis in the United States. Blackberries fromGuatemala, mesclun lettuce from Peru and basil from Mexico were suspectedof being associated with outbreaks from Georgia, Ontario, Florida, the D.C.area and Missouri from 1997-2000 (Herwaldt, 2000). It was noted that thevegetables involved could have come from the United States, and in the caseof basil, the outbreak in the D.C. area could have been due to contaminationfrom food-handlers since several were ill at the time of the outbreak. In theMissouri outbreak involving basil, oocysts were actually detected by bothmicroscopy and PCR in frozen leftovers (chicken pasta salad) from one of theparties. The first foodborne outbreak of cyclosporiasis from central Europewas reported in 2002 (Doller et al., 2002). In this instance, an attack rate of85% was noted in attendees of 4 independent luncheon parties. The suspectedsource of contamination was one of several types of lettuce grown in eitherthe south of France or in Italy.

One of the big questions relating to foodborne transmission is howand in what manner did the suspect food become contaminated? Was it use ofcontaminated water or human feces used as fertilizer that contained fullysporulated oocysts that served as the primary source of contamination? Given

Sterling and Ortega 51

the large quantities of consumable vegetables and fruits that are imported intothe United States, are there measures that can be taken to prevent furtherfoodborne disease outbreaks of cyclosporiasis? Despite attempted controlmeasures aimed at improving hygiene, sanitation and water quality, outbreakslinked to imported Guatemalan raspberries continued after the 1997 season.Studies using gamma irradiated Toxoplasma and Eimeria have demonstratedthat dose levels of 0.5 and 1.0 kGy, respectively, are effective in killingoocysts of these species seeded onto fruit as judged by animal infectivitystudies (Dubey et al., 1998; Lee and Lee, 2001). These studies also point tothe utility of these organisms as models for studies with Cyclospora sinceanimal models for the latter do not exist.

DETECTIONOocysts of C. cayetanensis have been detected from fecal,

environmental and food samples by microscopy (Eberhard et al., 1997;Visvesvara et al., 1997; Sturbaum et al., 1998; Ortega et al., 1997b; Lopez etal., 2001). This has not been without challenges, however, since special stainsor filters must be used to enhance their detection. Detection in feces isusually accomplished by means of employing a modified acid-fast stain thatmust be requested, as for detection of Cryptosporidium. A microscopeequipped with an ocular micrometer should be used since acid-fastCyclospora oocysts measure whereas those of Cryptosporidiummeasure Ultraviolet fluorescence microscopy is useful in confirmingthe presence of Cyclospora oocysts since they will autofluoresce green usinga 450-490 nm dichroic filter and blue using a 365nm dichroic filter (Berlin etal., 1998; Ortega et al., 1998). This latter method has been used to identifyCyclospora oocysts from food and water, but is deemed extremely laborintensive and insensitive to likely have much utility. Unfortunately,monoclonal antibody based fluorescent detection or immunomagneticseparation techniques have not been developed to enhance detection of thisparasite from fecal, environmental or food sources.

Environmental and food samples present challenges of oocystisolation and identification not likely to be encountered in fecal samples. Thepresence of plant and animal debris and a multitude of chemicals andprotozoan organisms that may be genetically quite similar in such samples arelikely to lead to disappointing diagnostic outcomes when applied to thedetection of Cyclospora. These problems dictate that various samplecollection, concentration, purification, differential diagnostic and oftenviability detection steps have to be applied to samples to come up with adesired diagnosis. The steps to be employed in achieving a diagnosticoutcome are also often confounded due to the presence of small oocystnumbers in such matrices. Initially, recovery methods for Cyclospora oocyst

52 Sterling and Ortega

detection derived from those used to detect Cryptosporidium.Acknowledging that these techniques are cumbersome and that antibody-based reagents are unavailable for Cyclospora, investigators have turned tomolecular-based approaches to address this deficiency. The use of suchtechniques, however, still requires use of specific recovery and concentrationsteps before they can be applied. It is beyond the scope of this chapter toevaluate all of these and the reader is referred to the excellent review ofShields and Olson (2003).

Use of the polymerase chain reaction (PCR) following the use ofspecific recovery and concentration steps has the potential to be moresensitive and specific when applied to C. cayetanensis detection fromvirtually any matrix. Initial primers developed for the PCR reaction showedcross reactivity with Eimeria (Relman et al., 1996). Further primermodifications and the addition of restriction fragment length polymorphism(RFLP) analysis permitted a more definitive diagnosis of C. cayetanensis(Jinneman et al., 1996; Jinneman et al., 1998; Sturbaum et al., 1998). It isacknowledged, however, that the primers used would not permit C.cayetanensis to be distinguished from other Cyclospora species. Analysis ofthe internal transcribed spacer ITS-1 region has been used to distinguishhuman and primate species of Cyclospora and has demonstrated that thisregion is highly variable within and between samples (Adam et al., 2000;Olivier et al, 2001). In the majority of samples tested this variability did notcorrelate with geographic origin of the samples and, therefore, may not be asuitable marker for molecular epidemiology studies. Despite the observedvariability, however, conserved species-specific ITS-1 sequences showedconsistent and remarkable diversity among Cyclospora spp. ITS-1 sequencesargue for polyparasitism and simultaneous transmission of multiple strainsrather than multiple and different copies in one organism. This may explainthe presence of a single ITS-1 sequence in epidemiologically associatedisolates (Adam et al., 2000). Both conditions seem to occur inCryptosporidium. Two calf-propagated Cryptosporidium contained twodifferent types of ssrDNA sequences whereas a human isolate contained onlyone (Le Blancq et al., 1997, Widmer et al., 1999). Examining more outbreakassociated isolates and propagating the organism in animal models couldresolve the issue of polyparasitism, or multiple copies in one organism.

An oligoligation assay, used to distinguish PCR products, has beendeveloped to differentiate Cyclospora from Eimeria (Jinneman et al., 1999).This technique may have utility in differentiating Cyclospora species oncetheir hyper-variable regions have been determined. A quantitative real-timePCR assay using both a species-specific primer set and a dual fluorescent-labeled C. cayetanensis hybridization probe has recently been developed todetect this parasite (Varma et al., 2003). DNA from as few as 1 oocyst per

Sterling and Ortega 53

reaction volume could be detected. It was acknowledged that a morerobust testing of the DNA extraction method would be required to ensuresuitability for a wide variety of environmental or clinical samples and thatfurther improvements in DNA extraction would enhance the overall efficiencyof the assay.

The viability of recovered C. cayetanensis oocysts has not beenextensively studied. Oocysts can be induced to sporulate and followingtreatment with bile salts, taurocholate and mechanical pressure can beexcysted (Ortega et al., 1993). At present it is unknown if excystedsporozoites can infect cell cultures or cause actual infections in humans.Excystation, and therefore viability, may not be related to infectivity. A noveltechnique, electrorotation, that is able to detect changes in the physiochemicaland morphologic properties of an oocyst, has also been used to indicate anoocyst’s viability (Dalton el al., 2001). Oocysts rotate dependent upon theirconductivity and permittivity in an electric field and the resultingelectrorotation spectra measurements determine whether the oocyst is viableor not.

CONCLUSIONSOutbreaks of cyclosporiasis, particularly those resulting from

consumption of imported foods, have brought C. cayetanensis our attention.Numerous questions relative to the life cycle, biology, immuneresponsiveness, epidemiology and detection of this organism have been posedin the foregoing chapter and require addressing if we are to better understandand control this emergent and still perplexing coccidian parasite.

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Hale, D., W. Aldeen, and K. Carroll. 1994. Diarrhea associated with cyanobacteria-like bodiesin an imunocoompetent host. An unusual epidemiological source. Journal of the AmericanMedical Association. 271:144-145.

Hart, A.S., M.T. Ridinger, R. Soundarajan, C.S. Peters, A.L. Swiatlo, and E. Kocka. 1990.Novel organisms associates with chronic diarrhea in AIDS. Lancet. 335:169-170.

Herwaldt, B.L. 2000. Cyclospora cayetanensis: A review, focusing on the outbreaks ofcyclosporiasis in the 1990s. Clinical Infectious Diseases. 31:1040-1057.

Herwaldt, B.L., M-L. Ackers, and the Cyclospora Working Group. 1997. An outbreak in 1996of cyclosporiasis associated with imported raspberries. New England Journal of Medicine.336:1548-1556.

Herwaldt, B.L., M.J. Beach, and the Cyclospora Working Group. 1999. The return ofCyclospora in 1997: another outbreak of cyclosporiasis in North America associated withimported raspberries. Annals of Internal Medicine. 130:210-220.

Hoge, C.W., D.R. Shlim, M. Ghimire, J.G. Rabold, P. Pandey, A. Walch, R. Rajah, P. Gaudio,and P. Echeverria. 1995. Placebo-controlled trial of co-trimoxazole for Cyclospora infectionsamong travelers and foreign residents in Nepal. Lancet. 345:691-693.

Hoge, C.W., D.R. Shlim, R. Rajah, J. Triplett, M. Shear, J.G. Rabold, and P. Echeverria. 1993.Epidemiology of diarrhoeal illness associated with coccidian-like organism among travelersand foreign residents in Nepal. Lancet. 341:1175-1179.

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Huang, P., J.T. Weber, D.M. Sosin, P.M. Griffin, E.G. Long, J.J. Murphy, F. Kocka, C. Peters,and C. Kallick. 1995. The first reported outbreak of diarrheal illness associated withCyclospora in the United States. Annals of Internal Medicine. 123:409-414.

Jinneman, K.C., J.H. Wetherington, A.M. Adams, J.M. Johnson, B.J. Tenge, N-L. Dang, andW.E. Hill. 1996. Differentiation of Cyclospora sp. and Eimeria sp. By using the polymerasechain reaction amplification products and restriction fragment length polymorphisms. Foodand Drug Administration Laboratory Information Bulletin LIB No. 4044.

Jinneman, K.C., J.H. Wetherington, W.E. Hill, A.M. Adams, J.M. Hohnson, F.J. Tenge, N-L.Dang, R.L. Manger, and M.M. Wekell. 1998. Template preparation for PCR and RFLP ofamplification products for the detection and identification of Cyclospora sp. and Eimeria spp.oocysts directly from raspberries. Journal of Food Protection. 61:1497-1503.

Jinneman, K.C. J.H. Wetherington, W.E. Hill, C.J. Omiescinski, A.M. Adams, J.M. Johnson,B.J. Tenge, N-L. Dang, and M.M. Wekell. 1999. An oligonucleotide-ligation assay for thedifferentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplificationproducts. Journal of Food Protection. 62:682-685.

Koumans, E.H.A., D.J. Katz, J.M. Malecki, S. Kumar, S.P. Wahlquist, M.J. Arrowood, A.W.Hightower, and B.L. Herwaldt. 1998. An outbreak of cyclosporiasis in Florida in 1995: aharbinger of multistate outbreaks in 1996 and 1997. American Journal of Tropical Medicineand Hygiene. 59:235-242.

Koumans, E.H., D. Katz, J. Malecki, S. Wahlquist, S. Kumar, A. Hightower, et al. 1996. Novelparasite and mode of transmission: Cyclospora infection-Florida. Annual EpidemicIntelligence service Conference 45:60.

Le Blancq, S.M., N.V. Khramtsov, F. Aamani, S.J. Upton, and T.W. Wu. 1997. ribosomalRNA gene organization in Cryptosporidium parvum. Molecular and BiochemicalParasitology. 90:463-478.

Lee, M.B., and E.H. Lee. 2001. Coccidial contamination of raspberries: mock contaminationwith Eimeria acervulina as a model for decontamination treatment studies. Journal of FoodProtection. 64:1854-1857.

Long, E.G., A. Ebrahimzadeh, E.H. White, B. Swisher, and C.S. Callaway. 1990. Algaassociated with diarrhea in patients with acquired immunodeficiency syndrome and intravelers. Journal of Clinical Microbiology. 28:1101-1104.

Long, E.G., E.H. White, W.W. Carmichael, R.R. Quinlisk, B.L. Swisheer, H. Daugharty, andM.T. Cohen. 1991. Morphologic and staining characteristics of a Cyanobacterium-likeorganism associated with diarrhea. Journal of Infectious Diseases. 164:199-202.

Lopez, A.S., D.R. Dodson, M.J. Arrowood, P.A. Orlandi Jr., A.J. Da Silva, J.W. Bier, S.D.Hanauer, R.L. Kuster, S. Oltman, M.S. Baldwin, K.Y. Won, E.M. Nace, M.L. Eberhard, andB.L. Herwaldt. 2001. Outbreak of cyclosporiasis associated with basil in Missouri in 1999.Clinical Infectious Diseases. 32:1010-1017.

Lopez, F.A., J. Manglicmot, T.M. Schmidt, C. Yeh, H.V. Smith, and D.A. Relman. 1999.Molecular characterization of Cyclospora-like organisms from baboons. Journal ofInfectious Diseases. 179:670-676.

Madico, G., R.H. Gilman, E. Miranda, L. Cabrera, and C.R. Sterling, 1993. Treatment ofCyclospora infections with co-trimoxazole. Lancet. 342:122-123.

Madico, G., J. McDonald, R.H. Gilman, L. Cabrera, and C.R. Sterling. 1997. Epidemiologyand treatment of Cyclospora cayetanensis infection in Peruvian children. Clinical InfectiousDiseases. 24:977-981.

Narango, J., C. Sterling, R. Gilman, E. Miranda, F. Diaz, M. Cho, and A. Benel. 1989.Cryptosporidium muris-like objects from fecal samples of Peruvians (abstract 324, In:Program and abstracts of the Annual Meeting of the American Society of TropicalMedicine and Hygiene (Honolulu), 10-14 December, 1989.

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Nhieu J.T., R. Nin, J. Fleury-Feith, M.T. Chaumette, A. Schaeffer, and S. Bretagne. 1996.Identification of intracellular stages of Cyclospora species by light microscopy of thicksections using hematoxylin. Human Pathology. 27:1107-1109.

Norton, C.C., and M.J. Chard. 1983. The oocyst sporulation time of Eimeria species from thefowl. Parasitology 86:193-198.

Oii, W.W., S.K. Zimmerman, and C.A. Needham. 1995. Cyclopora species as agastrointestinal pathogen in immunocompetent hosts. Journal of Clinical Microbiology.33:1267-1269.

Olivier, C., S. van de Pas, P.W. Lepp, K. Yoder, and D.A. Relman. 2001. Sequence variabilityin the first internal transcribed spacer region within and among Cyclospora species isconsistent with polyparasitism. International Journal for Parasitology. 31:1475-1487.

Ortega, Y.R., R.H. Gilman, and C.R. Sterling. 1994. A new coccidian parasite (Apicomlexa:Eimeriidae) from humans. Journal of Parasitology. 80:625-629.

Ortega, Y.R., R. Nagle, R.H. Gilman, J. Watanabe, J. Miyagui, H. Quispe, P. Kanagusuku, C.Rojas, and C.R. Sterling. 1997a. Pathologic and clinical findings in patients withcyclosporiasis and a description of intracellular parasite life-cycle stages. The Journal ofInfectious Diseases. 176:1584-1589.

Ortega, Y.R., C.R. Roxas, R.H. Gilman, N.J. Miller, L. Cabrera, C. Taquiri, and C.R. Sterling.1977b. Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetablescollected in markets of an endemic region in Peru. American Journal of Tropical Medicineand Hygiene. 57:683-686.

Ortega, Y.R. C.R. Sterling, and R.H. Gilman. 1998. Cyclospora cayetanensis. Advances inParasitology. 40:399-418.

Ortega, Y.R., C.R. Sterling, R.H. Gilman, V.A. Cama, and F. Diaz. 1993. Cyclospora species: anew protozoan pathogen of humans. New England Journal of Medicine. 328:1308-1312.

Pape, J.W., R.-L. Verdier, M. Boncy, J. Boncy, and W.D. Johnson. 1994. Cyclospora infectionin adults infected with HIV. Clinical manifestations, treatment and prophylaxis. Annals ofInternal Medicine. 121:654-657.

Pieniazek, N.J., and B.L. Herwaldt. 1997. Reevaluating the molecular taxonomy: is the humanassociated Cyclospora a mammalian Eimeria species? Emerging Infectious Diseases. 3:381-383.

Rabold, J.G., C.W. Hoge, D.R. Shlim, C. Kefford, R. Rajah, and P. Echevenia. 1994.Cyclospora outbreak associated with chlorinated drinking water. Lancet. 344:1360-1361.

Relman, D.A., T.S. Schmidt, A. Gajadhar, M. Sogin, J. Cross, K. Yoder, O. Sethabutr, and P.Echeverria. 1996. Molecular phylogenetic analysis of Cyclospora, the human intestinalpathogen, suggests that it is closely related to Eimeria species. Journal of InfectiousDiseases. 173:440-445.

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Sherchand. J.B., J.H. Cross, M. Jimba, S. Sherchand, and M.P. Shrestha. 1999. Study ofCyclospora cayetanensis in health care facilities, sewage water and green leafy vegetables inNepal. Southeast Asian Journal of Tropical Medicine and Public Health. 30:58-63.

Shields, J.M., and B.H. Olson. 2003. Cyclospora cayetanensis: a review of an emergingparasitic coccidian. International Journal for Parasitology. 33:371-391.

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Sifuentes-Osornio, J., G. Porras-Cortes, R.P. Bendall, F. Morales-Villarreal, G. Reyes-Teran,and G.M. Ruiz-Palacios. 1995. Cyclospora cayetanensis infection in patients with andwithout AIDS: biliary disease as another clinical manifestation. Clinical Infectious Diseases.21:1092-1097.

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ANTIGENIC VARIATION OF THE VSP GENESOF GIARDIA LAMBLIA

Rodney D. Adam1 and Theodore E. Nash2

1Dept of Medicine and Microbiology/Immunology, University of Arizona College of Medicine,Tucson, AZ, USA2Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD, USA

ABSTRACTGiardia lamblia, a protozoan parasite inhabiting the small intestine, is a

common infection worldwide that frequently results in chronic diarrhea,malabsorption and upper gastrointestinal symptoms. Giardia undergoessurface antigenic variation in humans and animal model infections, aphenomenon that may account for both chronicity of infections and therelatively broad mammalian host specificity with genotypically identicalorganisms found in humans, cats, beavers, and other mammals. The variant-specific surface proteins (VSPs) are an unusual family of related cysteine-richproteins, from 50 kD to over 200 kD in size, that coat the surface of thetrophozoite. Only one VSP of the estimated 150 or more vsp genes isexpressed on an individual at any specific time. However, the repertoires ofvsp genes may differ depending on the genetic group. VSPs switchspontaneously every 6-12 generations although some Giardia also switchduring encystation/excystation. All VSPs have a high cysteine content ofabout 12 % cysteines that are mostly present as CXXC motifs as well as ahighly conserved C-terminus, a surface Zn finger motif, a GGCY motif andother common features. The biological function(s) of VSPs are uncertain butthey undergo both immune and biological selection. The molecularmechanism(s) involved in antigenic variation are unknown but because thereis the absence of gene movement or gene mutations and only one of 4practically identical alleles (Giardia are tetraploid) is expressed; epigeneticprocesses are likely involved. Further studies of the mechanism of antigenicvariation and the biological role of the VSPs promise to contribute to ourunderstanding of G. lamblia and its pathogenesis.

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DISCOVERY AND DOCUMENTATION OF ANTIGENICVARIATION

Antigenic variation of major surface proteins or glycoproteins hasemerged as a major virulence factor in the protozoan pathogens, as well as inbacteria and viruses. In Giardia, the possibility of antigenic variation was firstsuggested by the marked difference in surface antigens (Nash and Keister,1985) among isolates that were quite similar genetically (Nash et al., 1985).These surface antigens are secreted into culture medium in large quantitiesand are the dominant molecules found when trophozoites are surface labeled.They were initially called excretory-secretory products (Nash et al., 1983;Nash and Keister, 1985), and are now called variant-specific surface proteins(VSP) (Mowatt et al., 1991).

Further evaluation of the surface antigens was possible when amonoclonal antibody (MAb 6E7) was produced for a major 170 kD surfaceantigen of the WB isolate (Nash and Aggarwal, 1986). “Mutants” weresubsequently identified that had lost reactivity to MAb 6E7. Subsequently, theWB isolate, which had already been doubly cloned in soft agar, was againcloned twice by limiting dilution. The resulting cloned organisms (WBA6)expressed a 170 kD surface antigen (initially called CRP170 and subsequentlyVSPA6) that was reactive with MAb6E7. MAb6E7, which is cytotoxic fororganisms expressing VSPA6 (Nash and Aggarwal, 1986) was used to selectfor organisms that were resistant to the antibody and no longer expressedVSPA6. These organisms were cloned and characterized. One of these clonedlines expressed a 64 kD surface antigen (CRP64, VSP1267), while anotherexpressed a 68 kD surface antigen (CRP68, VSP1269). The process ofantigenic variation was then carried out one step farther when a MAb forVSP1267 (MAb5C1) was produced and used to select variants that expressedneither VSPA6 nor VSP1267). This variation occurs spontaneously at a rateof approximately once every six to 13 generations (Nash et al., 1990a).Presumably, any given population of trophozoites is dominated by whateverVSP type is favored by the current growth conditions.

Giardia lamblia isolates that commonly infect humans fall into two majorgenotypes (Adam, 2001). The first (Genotype A; Nash Groups 1 and 2,Mayrhofer Assemblage A) includes the WB isolate in which antigenicvariation was first documented. Antigenic variation has also been documentedin the other major genotype (Genotype B; Nash Group 3, MayrhoferAssemblage B), of which the GS isolate is the most studied member. The vspgene repertoires of these two genotypes are very different in terms ofantigenicity and sequence similarity. Nonetheless, the general features of theVSPs and vsp genes are the same for both genotypes.

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CHARACTERISTICS OF THE VSPsThe VSPs are encoded by a repertoire of genes estimated at 150 in

number (Nash et al., 1990a) and perhaps even larger. All of the VSPs are richin cysteines, which make up approximately 12% of the amino acid contentand are frequently found in a CXXC motif (Adam et al., 1988). Earlier workhad suggested the presence of free thiol groups on the surfaces of Giardiatrophozoites (Gillin et al., 1984), so it was of interest to determine whether thecysteines of the VSPs were present with disulfide bonds or free thiol groups.In the case of TSA417, it was found that all or nearly all the cysteines werepresent as intrachain disulfide bonds, both in intact trophozoites and in thesolubilized protein (Aley and Gillin, 1993). There was no evidence forinterchain disulfide bonds. In nonreducing conditions, TSA417 was partiallyresistant to trypsin, but reduced protein was fully susceptible, suggesting thatthe disulfide-bonded cysteines played a role in protection from proteases.These findings have also been verified for another VSP (VSP4A1)(Papanastasiou et al., 1997a).

The N-termini of the VSPs are signaling peptides approximately 14(Lujan et al., 1995b) to 17 (Aley and Gillin, 1993) amino acids in length. Theremainder of the N-terminal regions of the VSPs are quite variable and arelikely responsible for most of the antigenic variability of VSPs. Of thoseMabs for which the recognized epitopes have been determined, they reactwith the N-terminal region or a repeat (in repeat-containing VSPs such asVSPA6).

The approximately 38 amino acids comprising the C terminus are highlyconserved, with greater than 90% identity among VSPs, and end in anabsolutely conserved CRGKA (Mowatt et al., 1991). Approximately 36 AAof the C terminus is cleaved from the secreted protein (Papanastasiou et al.,1996). Therefore, much of this conserved region appears to be involved inmembrane anchoring, while the CRGKA is a cytoplasmic tail.

VSPs have a highly conserved GGCY motif of unknown function whichis generally found toward the C-terminal end, but not in the conserved portion(Nash et al., 1995). VSPs have a number of common motifs including a Znfinger binding motif that consists of a combination of LIM and RING fingerZn finger motifs (Nash and Mowatt, 1993). It is the only known Zn fingermotif on the surface of any organism. In other organisms, LIM and Ringfinger proteins have diverse functions. Although initially described as DNAbinding proteins, most are associated with protein-protein interaction. ManyRING finger proteins in higher eukaryotes play a role in ubiquination(Joazeiro and Weissman, 2000). RING proteins self assemble and are able tocataylze or inhibit particular reactions depending on the protein (Kentsis et al.,2002). The potential role of the RING motif in Giardia is unclear but it ispossible that VSP bind to one another on the surface or alternatively there are

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RING-like interactions between VSP and gut. Although the biological role ofthis motif in VSPs is not known, similar motifs in higher mammalian cellsplay a role in DNA binding and protein-protein interactions. RING fingerproteins are capable of interacting between themselves and self-assemblysuggesting that VSPs interact between each other on the surface or interactwith particular proteins on the surface of the small intestine.

In vitro, Zn finger motifs (Nash and Mowatt, 1993; Zhang et al., 1993)are capable of binding Zn and other cations (Lujan et al., 1995b).Surprisingly, Zn binding is not limited to the Zn finger, but is dependent onthe presence of free thiol groups (Nash and Mowatt, 1993; Papanastasiou etal., 1997a). The dependence on free thiol groups for binding of zinc topurified VSP suggests that binding is mediated by the cysteines. There arenumerous CXXC groups whose spacing suggests they can bind metals.

The lack of binding specificity raises questions about biological role ofzinc binding for VSP function, but it should be emphasized that the metalsnaturally bound by zinc fingers have uncommonly been determined in othersystems. In the case of the VSPs, it may be that Zn is not the only cation thatis present naturally. In fact, differences in cation binding by different VSPscould contribute to their biological diversity. It is also possible that as VSPare shed in rather enormous amounts, smaller peptides are formed that bindmetals and contribute to Zn deficiency.

PROTEIN TRAFFICKING AND POST-TRANSLATIONMODIFICATION OF THE VSPs

The VSPs are found in the ER and lysosome-like vacuoles, as well asdiffusely coating the surface of the trophozoite (Pimenta et al., 1991;McCaffery et al., 1994)(Fig. 1). Even though the vegetative trophozoites do

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not have Golgi that can be identified by EM, they do exhibit complextrafficking of proteins (McCaffery et al., 1994), as well as Brefeldin A (BFA)-inhibited protein transport, suggesting that functional Golgi may exist (Lujanet al., 1995a). In fact, VSP trafficking was also inhibited by BFA, suggestingthe possibility that the VSPs are transported and processed by Golgi or aGolgi-like organelle (Lujan et al., 1995a). This possibility is supported by thefinding that VSPs using their own signal peptide are transported through theGolgi in COS cells and become surface-localized (Nash, Conrad, Kulakova,unpublished). The full transmembrane portion of the peptide is required forproper transport of VSPs to the surface (Nash and Kulakova, unpublished).Addition of a peripheral vacuole (PV) localization signal diverts the VSP tothe PV suggesting that the default pathway of membrane bound molecules isto the surface unless other signals are present (Touz et al., 2003). Mutation ofthe Zn finger results in the VSP being stuck in the ER. When the highlyconserved GGCYmotif is deleted, the VSP cannot be detected, suggestingeither loss of antigenicity or early destruction (Nash and Kulkova,unpublished results).

Glycosylation has been proposed for one VSP (Papanastasiou et al.,1997b), but has not been found in two other VSPs (Nash et al., 1983; Lujan etal., 1995b; Marti et al., 2002). There is also evidence for a palmitoylation sitewhich is probably in the conserved C-terminus that is cleaved upon secretionfrom the membrane (Papanastasiou et al., 1997b; Hiltpold et al., 2000;Papanastasiou et al., 1996)

FEATURES OF THE VSP GENESThe vsp genes demonstrate a number of important similarities, but have

an equally important list of differences. The first vsp gene to be described(vspA6 or CRP170) consists of a 99 bp 5’ region followed by about 21 to 23copies of a 195 bp tandem repeat, then 1338 bp at the 3’ end (Adam et al.,1992; Yang and Adam, 1994). The 3’ 120-130 bp are highly conserved for allvsp genes, in keeping with the amino acid conservation in that region. Theputative polyadenylation signal (AGTPuAAPy) found in all Giardia genes isimmediately preceded by “PuCTPyAGPuT”, which may begin in the stopcodon.

Giardia is polyploid (most likely tetraploid), so there are approximatelyfour alleles of each gene. One of the interesting characteristics of the Giardiagenome is the remarkably small degree of allelic heterozygosity; this holdstrue for the vsp genes as well. The only example of allelic sequenceheterozygosity documented to date for a vsp gene is the observation that theexpressed allele of the vspA6 gene (only one of the four alleles is expressed;see below) has eight nucleotide substitutions in comparison to the otheralleles. The other difference between the expressed and other alleles is that the

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expressed allele has approximately 22 copies of the repeat while thenonexpressed alleles have 8 or 9 copies. It is likely that the repeats encode animmunologically important region, since the cytotoxic monoclonal antibody(MAb6E7) reacts with the repeat region (Mowatt et al., 1994).

Some other vsp genes also contain tandem repeats. The vspC5 genecontains a 66 bp 5’region, approximately 26 copies of a 105 bp repeat in theexpressed allele, and a 135 bp 3’ region comprised primarily of the conservedregion. Like the vspA6 gene, the expressed allele has more copies of therepeat than do the nonexpressed alleles (20 or 21), but in contrast, there are nosequence differences among the alleles. CRP136 has 23 copies of a 120 bprepeat (Chen et al., 1995) and CRP65 has four copies of a 228 bp repeat(Upcroft et al., 1997). However, the presence of tandem repeats is notuniversal and in fact may represent the exception rather than the rule. Whenrepeats are found, they begin near the N terminus and are highlyimmunogenic, but their biologic functions are not known.

There are a number of examples of vsp genes that are highly similarthroughout the entire reading frame, suggesting that the vsp gene repertoirehas been expanded by duplication and divergence. Perhaps the mostremarkable example is the vsp1267 gene, which consists of two identicalcopies in a tail-to-tail arrangement approximately three kb apart. Anotherexample of gene duplication is the vspG3M-B gene (Mowatt et al., 1994), alsocalled vspA6-S1 (Yang and Adam, 1995b), which is highly similar to thevspA6 gene, except that it contains between one and two copies of the 195 bprepeat and is located on a different chromosome. Interestingly, much of thegene has been sequenced from the WB isolate (probably from Afghanistan)(Yang and Adam, 1995b) and the G3M isolate (Mowatt et al., 1994) (fromPeru) and the sequences were identical. This observation suggests that thedivergence is not occurring rapidly. In other cases, vsp genes are highlysimilar in the 5’ region followed by substantial divergence, suggesting that therepertoire has been expanded by recombination (Yang and Adam, 1995a).Two WB-derived genes, vsp9B10A and B encode proteins that react with thesame MAb. Yet, the genes have enough sequence differences to allow readydistinction (Nash et al., 2001; Carranza et al., 2002). These observationsindicate that similar vsp genes may encode VSPs with cross-reactive epitopes.From the GS isolate, the H7 and H7-1 genes (Nash et al., 1995) as well asanother member of the H7 gene family (Nash, unpublished) provide anexample from the GS isolate, indicating that this expansion of the repertoirealso occurs in the genotype B (Group 3) organisms.

Unlike the African trypanosomes, telomeric sites are not required for vspgene expression. The genomic locations of two vsp genes (vspA6 and vspC5)have been determined in isolates expressing these genes and in isolates whichhave lost expression (Yang and Adam, 1994; Yang and Adam, 1995a). In

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both cases, they were in a single chromosome-internal site which did not varyin the gain or loss of expression. A telomeric location has been documentedfor some vsp genes (Upcroft et al., 1997; Arkhipova and Morrison, 2001), butthe expression competency for these genes has not yet been determined. Thus,the vsp genes are dispersed throughout the genome, being found inchromosome-internal sites as well as telomeric sites and are found on most orall of the chromosomes.

CONTRO OF EXPRESSION OF THE VSPsLack of DNA sequence alterations or rearrangements associated with

antigenic variation - No examples have yet been documented in which aDNA sequence alteration or DNA rearrangement has been associated withgain or loss of expression of a vsp gene. A number of vsp genes have beensequenced from variants in which the gene is expressed and one in which it isnot expressed; to date, no sequence alterations or rearrangements have beencorrelated with gain or loss of expression.

In the initial description of antigenic variation (Adam et al., 1988), therewas significant variation in the Southern blots of DNA from several clonedlines which either expressed or did not express vspA6 (CRP170). However,there was no clear correlation between the pattern and whether the vspA6gene was expressed in that particular cloned line. In retrospect, the variationwas due do variation of repeat copy number of a 195 bp repeat found in thevspA6 gene.

Allele-specific expression - As tetraploid organisms, G. lambliatrophozoites are expected to have four copies of each allele of a vsp gene. Inthe cases of the vsp genes that do not contain tandem repeats, there have beenno examples of allelic sequence heterozygosity, so the alleles cannot bedistinguished from each other. However, in the case of the vspA6 gene, whichcontains a 195 bp repeat, the alleles can be distinguished from each otherbecause of differing repeat copy numbers (8, 9, or approximately 23).Therefore, the alleles can be distinguished by Southern blotting andtranscripts can be distinguished by Northern blotting, leading to theobservation that only the allele containing 23 repeats is expressed (Yang andAdam, 1994). Interestingly, there are eight nucleotide (five amino acid)substitutions in the open reading frame of the expressed allele compared tothe nonexpressed alleles, but no detected changes in the upstream ordownstream noncoding regions. vspC5 contains a 105 bp repeat (Yang et al.,1994). The alleles have 26, 21, or 20 copies of the repeat, and again, the allelewith the largest number of repeats is expressed. The allele-specific expressionof these vsp genes suggests an epigenetic form of control for their expression.More direct evidence for lack of gene movement has been provided by theintegration of a tagged vsp into the genome. Southern blots indicate the lack

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of gene movement during gain or loss of expression (Nash and Kulakova,unpublished).

Loss of expressed allele - In one case of antigenic variation, theexpressed vspA6 allele was lost from the variant that had lost vspA6expression (Adam et al., 1992). Whether this resulted from subchromosomaldeletion or loss of an entire chromosome has not yet been determined. Itshould also be noted that the exact relationship between loss of the expressedvsp allele and loss of expression has not been determined. It may be acommon or uncommon etiology of loss of expression, or may simply be anepiphenomenon.

Antigenic variation with encystation and excystation - When exposedto conditions favoring the formation of cysts, trophozoites rapidlydifferentiate into cysts. During the process of encystation using trophozoitesexpressing the VSP, TSA417, the VSP disappears from the cell surface(McCaffery et al., 1994). During excystation of these encysted cells, TSA417is then found in the peripheral vacuoles, suggesting that they might beendocytosed (Svärd et al., 1998). Within 90 minutes after cells are induced toexcyst, TSA417 transcripts disappear and are replaced by other vsptranscripts, of which there is one predominant vsp and others found in lesseramounts. This suggests a population shift induced by the process ofencystation and excystation, in contrast to the selection of an alternative vspfollowed by preferential replication as occurs during vegetative growth. Themechanism by which this process occurs is not known, but does not appear toinvolve gross DNA rearrangements. In another study using a WB lineexpressing VSP1267 (WB1267), the proportion of trophozoites expressingVSP1267 decreased from 99% to 94.2% during encystation, while theproportion expressing a different VSP (VSP9B10B) increased from 0 to38.1%. The numbers add up to greater than 100% because some of thetrophozoites expressed more than one VSP while switching. Interestingly,VSP switching during encystation and excystation does not occur with vspH7of the GS isolate in vitro (Svärd et al., 1998), in mice (Gottstein and Nash,1991), or in humans (Nash et al., 1990b).

ANTIGENIC VARIATION IN VITRO AND IN VIVO:CLUES TO THE BIOLOGICAL ROLE OF ANTIGENICVARIATION

Since surface antigen variation was initially identified as an in vitrophenomenon of Giardia, a subsequent determination of the biologic role ofantigenic variation has been of special interest. Individual trophozoitesstudied during in vitro growth express only one VSP on their surface at anytime except during switching when two VSPs can be transiently detected.

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Rates of switching are both VSP and isolate dependent. Selection oftrophozoites following random expression of VSPs appears to be the majorway trophozoites expressing certain VSPs predominate.

The course of in vivo infection and VSP expression over time isimperfectly documented and depends upon a number of factors including thehost species and maturity of their immune system, type of Giardia isolate andparticular VSP expressed. Most experimental studies have employed a cultureof the GS isolate expressing VSPH7 as the predominant VSP. In humansVSPH7 is expressed until day 17 post-infection when switching to other VSPsbegins (Nash et al., 1990b). The course of infection after 21 days has not beenstudied. In the adult mouse the infection peaks around day 6-10, thendecreases so that by day 21 there are barely detectable numbers of organisms.VSP switching occurs at the time humoral responses are detected during thesecond week of infection (Byrd et al., 1994). The pattern of infection inneonatal mice is similar to that of adult mice, where switching and decreasednumbers are evident by day 14; thereafter small numbers of organisms arelikely maintained (Gottstein et al., 1990). The study of antigenic variation ingerbils employed different isolates and clones and therefore cannot be directlycompared (Aggarwal and Nash, 1988). None of these experimental modelsshow the expected waves of intestinal parasites expressing one and thenanother VSP that has been characteristic of some blood borne parasitesundergoing antigenic variation. Both immunological and non immunologicalprocesses have been identified that act as selection factors in vivo. Therefore,the remainder of the discussion will center on the selection of spontaneouslyoccurring variants.

The role of adaptive immunity - Adaptive immunity plays an importantrole in VSP selection. Specific anti-VSP antibodies are easily detected as aresult of infection and clearly have growth inhibitory and/or parasiticidaleffects on organisms expressing VSPs specific to these antibodies (Nash et al.,1990b; Gottstein et al., 1990; Stager et al., 1997b; Stager et al., 1997a; Stagerand Muller, 1997; Stager et al., 1998; Nash and Aggarwal, 1986; Hemphill etal., 1996). Some VSP specific monoclonal antibodies (Mabs) causecomplement independent killing of trophozoites in vitro (Nash and Aggarwal,1986), and depending on the concentration, almost all VSP specificpolyclonal or Mab antibodies cause agglutination and growth inhibition. Anumber of model systems have suggested a role of surface-reactive intestinalIgA antibodies, including anti-VSP antibodies, in controlling infections(Heyworth and Vergara, 1994; Langford et al., 2002; Gottstein et al., 1993;Stager and Muller, 1997; Stager et al., 1998).

Following experimental infections of immunocompetent mice withGS/H7, humoral responses to the VSPH7 develop at about the same time thatthe number of trophozoites in the intestine declines in mice (Byrd et al.,

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1994). Similarly, experimental infections in humans demonstrated VSPswitching at the same time as an antibody response to the original VSP wasmounted (Nash et al., 1990b).

Experimental infections using B cell deficient mice confirm theimportance of humoral antibodies in VSP selection in vivo (Singer et al.,2001; Stager and Muller, 1997; Langford et al., 2002). B cell deficient miceinfected with GS/VSPH7 are able to control infection, but the organisms donot undergo antigenic variation (Singer et al., 2001), implying that in thismodel control of infection is T cell mediated but VSP expression is controlledby antibodies or biological selection (see below). In contrast, another study ofB cell deficient adult mice concluded that humoral responses were essential tocontrol infections (Langford et al., 2002). Despite the different conclusions ofthese studies, they confirm that humoral responses to the VSPs occur and thatthese humoral responses can negatively select particular VSPs.

Although the humoral immune response has generally been consideredthe most important means of controlling Giardia infections, there isincreasing evidence of the importance of the T cell response in Giardia murisinfections (Stevens et al., 1978; Roberts-Thomson and Mitchell, 1978) and inmice infected by G. lamblia (Singer and Nash, 2000; Gottstein and Nash,1991). The relative importance of humoral and cell mediated immunity in theresolution of human infections is not known.

The role of biological selection - There is an increasing body of evidencesuggesting that non-immunological mechanisms also play an important roleVSP selection (biological selection). The effects of proteases on the VSPexpression and survival of Giardia in vitro was the first indication of theimportance of biological selection (Nash et al., 1991). When trophozoiteswere grown in high concentrations of either trypsin or chymotrypsin, cloneswere either protease-resistant and survived, or protease-sensitive and died,being replaced by protease-resistant trophozoites that subsequently regrewand repopulated the culture. These organisms expressed a new VSP resistantto the same concentration of protease that killed the earlier population. Theexistence of protease sensitive and protease resistant VSPs underscoressequence diversity of the VSPs which results in varied biologicalcharacteristics.

Biological selection has been demonstrated in vivo during the course ofexperimental infections. Human volunteers were inoculated with trophozoitesof the GS isolate expressing low numbers of many different VSPs, includingVSPH7 and another VSP recognized by MAb3F6 (Nash et al., 1987; Nash etal., 1990b). Within the first two weeks; before the adaptive immune systemcould play a role, VSP expression changed so that most now expressedVSPH7 and essentially none expressed the VSP recognized by MAb3F6.Similarly, gerbils inoculated with clones of the WB isolate expressing

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primarily one VSP, began expressing many VSPs within a few days, beforethe adaptive immune responses could possibly play a role. The change of VSPexpression before an adaptive immune response indicates that biologicalselection had occurred.

The clearest example of biological selection in vivo was seen inexperimentally infected SCID mice and irradiated immunodeficient gerbils(Singer et al., 2001). In the absence of an adaptive immune system,differences in VSP expression after inoculation of clones expressing one VSPcannot be due to acquired immune responses. Clones of trophozoites, eachexpressing a different VSP, were inoculated into SCID mice. In contrast tonormal mice who are able to suppress the level of infection to barelydetectable numbers by 2-3 weeks post inoculation, these animals maintainhigh numbers of trophozoites in the small intestine. Preferences for certainVSPs were noted. While certain VSP expressing clones were maintained andhighly expressed in the intestines during the course of the infection, otherVSPs were not maintained, but were replaced by other VSP expressingtrophozoites. Parallel experiments using the same clones were performed ingerbils that were immunosuppressed by irradiation, and VSP preferences werealso found, but were not identical to those of mice. These results suggest thatone of the roles for diversity of VSP expression may be to broaden the hostrange of the parasite.

Therefore the presence of specific VSPs in a host is determined bymultiple factors. The expressed VSP must be suitable to the particularintestinal environment and must not be eliminated by the host immuneresponse. It is likely that the number of VSPs that fulfill these criteria for eachhost is much smaller than the entire VSP repertoire.

The precise biological roles of VSPs are not known. The dramaticprotease resistance of certain VSPs suggests that they may protect the parasitefrom the harsh intestinal environment. However, the mechanism by which theparticular structure and unusual motifs are beneficial to the parasite is unclear.Although antigenic variation per se is commonly said to be a mechanismdesigned to escape the host’s immune response, this is not their only rolesince biological selection also occurs. When we understand the reason for thelarge repertoire of vsp genes and the important differences and similarities ofthese genes, we will likely have a much better understanding of therelationship between Giardia and its hosts.

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SUMMARY

Table 1:

Features of antigenic variation in Giardia lamblia

1.2.3.4.5.

6.7.

8.9.

High frequency of change (every 6 to 13 generations)Large repertoire of vsp genes (estimated at 150)Most or all vsp genes appear to have full open reading framesSequence alterations have not been associated with antigenic variationDNA rearrangements do not appear to be associated with antigenic

variationExpression is allele-specificVsp gene repertoires differ among different genotypes, but are highly

similar within genotypesTelomeric location is not requiredSwitching during encystations/excystation has been documented but is not

universal

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Antigenic variation of a cysteine-rich protein in Giardia lamblia. Journal of ExpimentalMedicine 167: 109-118.

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Aggarwal,A., and T.E. Nash. 1988. Antigenic variation of Giardia lamblia in vivo. Infectionand Immunity 56: 1420-1423.

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Gottstein,B., and T.E. Nash. 1991. Antigenic variation in Giardia lamblia: Infection ofcongenitally athymic nude and scid mice. Parasite Immunology 13: 649-659.

Hemphill,A., S. Stager, B. Gottstein, and N. Muller. 1996. Electron microscopical investigationof surface alterations on Giardia lamblia trophozoites after exposure to a cytotoxicmonoclonal antibody. Parasitology Research 82: 206-210.

Heyworth,M.F., and J.A. Vergara. 1994. Giardia muris trophozoite antigenic targets for mouseintestinal IgA antibody. Journal of Infectious Diseases 169: 395-398.

Hiltpold,A., M. Frey, and P. Köhler. 2000. Glycosylation and Palmitoylation are CommonModifications of Giardia Variant Surface Proteins. Molecular and Biochemical Parasitology109: 61-65.

Joazeiro,C.A., and A.M. Weissman. 2000. RING finger proteins: mediators of ubiquitin ligaseactivity. Cell 102: 549-552.

Kentsis,A., R.E. Gordon, and K.L. Borden. 2002. Self-assembly properties of a model RINGdomain. Proceedings of the National Academy of Sciences U. S. A 99: 667-672.

Langford,T.D., M.P. Housley, M. Boes, J. Chen, M.F. Kagnoff, F.D. Gillin, and L. Eckmann.2002. Central importance of immunoglobulin A in host defense against Giardia spp.Infectection and Immunity 70: 11-18.

Lujan,H.D., A. Marotta, M.R. Mowatt, N. Sciaky, J. Lippincott-Schwartz, and T.E. Nash.1995a. Developmental induction of Golgi structure and function in the primitive eukaryoteGiardia lamblia. Journal of Biological Chemistry 270: 4612-4618.

Lujan,H.D., M.R. Mowatt, J.J. Wu, Y. Lu, A. Lees, M.R. Chance, and T.E. Nash. 1995b.Purification of a variant-specific surface protein of Giardia lamblia and characterization of itsmetal-binding properties. Journal of Biological Chemistry 270: 13807-13813.

Marti,M., Y. Li, P. Kohler, and A.B. Hehl. 2002. Conformationally correct expression ofmembrane-anchored Toxoplasma gondii SAG1 in the primitive protozoan Giardiaduodenalis. Infection and Immunity 70: 1014-1016.

McCaffery,J.M., G.M. Faubert, and F.D. Gillin. 1994. Giardia lamblia: Traffic of a trophozoitevariant surface protein and a major cyst wall epitope during growth, encystation, andantigenic switching. Experimental Parasitology 79: 236-249.

Mowatt,M.R., A. Aggarwal, and T.E. Nash. 1991. Carboxy-terminal sequence conservationamong variant-specific surface proteins of Giardia lamblia. Molecular and BiochemicalParasitology 49: 215-227.

Mowatt,M.R., B.Y. Nguyen, J.T. Conrad, R.D. Adam, and T.E. Nash. 1994. Size heterogeneityamong antigenically related Giardia lamblia variant-specific surface proteins is due todifferences in tandem repeat copy number. Infection and Immunity 62: 1213-1218.

Nash,T.E., and A. Aggarwal. 1986. Cytotoxicity of monoclonal antibodies to a subset ofGiardia isolates. Journal of Immunology 136: 2628-2632.

Nash,T.E., S.M. Banks, D.W. Alling, J.W. Merritt, Jr., and J.T. Conrad,. 1990a. Frequency ofvariant antigens in Giardia lamblia. Experimental Parasitology 71: 415-421.

Nash,T.E., J.T. Conrad, and M.R. Mowatt. 1995. Giardia lamblia: Identification andcharacterization of a variant-specific surface protein gene family. Journal of EukaryoticMicrobiology 42: 604-609.

Nash,T.E., F.D. Gillin, and P.D. Smith. 1983. Excretory-secretory products of Giardia lamblia.Journal of Immunology 131: 2004-2010.

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Nash,T.E., D.A. Herrington, M.M. Levine, J.T. Conrad, and J.W. Merritt, Jr. 1990b. Antigenicvariation of Giardia lamblia in experimental human infections. Journal of Immunology 144:4362-4369.

Nash,T.E., D.A. Harrington, G.A. Losonsky, and M.M. Levine. 1987. Experimental humaninfections with Giardia lamblia. Journal of Infectious Diseases 156: 974-984.

Nash,T.E., and D.B. Keister. 1985. Differences in excretory-secretory products and surfaceantigens among 19 isolates of Giardia. Journal of Infectious Diseases 152: 1166-1171.

Nash,T.E., H.T. Lujan, M.R. Mowatt, and J.T. Conrad. 2001. Variant-specific surface proteinswitching in Giardia lamblia. Infection and Immunity 69: 1922-1923.

Nash,T.E., T. McCutchan, D. Keister, J.B. Dame, J.D. Conrad, and F.D. Gillin. 1985.Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humansand animals. Journal of Infectious Diseases 152: 64-73.

Nash,T.E., J.W. Merritt, Jr., and J.T. Conrad. 1991. Isolate and epitope variability insusceptibility of Giardia lamblia to intestinal proteases. Infection and Immunity 59: 1334-1340.

Nash,T.E., and M.R. Mowatt. 1993. Variant-specific surface proteins of Giardia lamblia arezinc- binding proteins. Proceedings of the National Academy of Science. USA 90: 5489-5493.

Papanastasiou,P., T. Bruderer, Y. Li, C. Bommeli, and P. Köhler. 1997a. Primary structure andbiochemical properties of a variant- specific surface protein of Giardia. Molecular andBiochemical Parasitology 86: 13-27.

Papanastasiou,P., A. Hiltpold, C. Bommeli, and P. Köhler. 1996. The release of the variantsurface protein of Giardia to its soluble isoform is mediated by the selective cleavage of theconserved carboxy-terminal domain. Biochemistry 35: 10143-10148.

Papanastasiou,P., M.J. McConville, J. Ralton, and P. Köhler. 1997b. The variant-specificsurface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein.Biochemical Journal 322: 49-56.

Pimenta,P.F., P.P. da Silva, and T.E. Nash. 1991. Variant surface antigens of Giardia lambliaare associated with the presence of a thick cell coat: Thin section and label fractureimmunocytochemistry survey. Infection and Immunity 59: 3989-3996.

Roberts-Thomson,I.C., and G.F. Mitchell. 1978. Giardiasis in mice. I. Prolonged infections incertain mouse strains and hypothymic (nude) mice. Gastroenterology 75: 42-46.

Singer,S.M., H.G. Elmendorf, J.T. Conrad, and T.E. Nash. 2001. Biological selection ofvariant-specific surface proteins in Giardia lamblia. Journal of Infectious Diseases 183: 119-124.

Singer,S.M., and T.E. Nash. 2000. T-cell-dependent control of acute Giardia lamblia infectionsin mice. Infection and Immunity 68: 170-175.

Stager,S., R. Felleisen, B. Gottstein, and N. Muller. 1997a. Giardia lamblia variant surfaceprotein H7 stimulates a heterogeneous repertoire of antibodies displaying differentialcytological effects on the parasite. Molecula and Biochemical Parasitology 85: 113-124.

Stager,S., B. Gottstein, and N. Muller. 1997b. Systemic and local antibody response in miceinduced by a recombinant peptide fragment from Giardia lamblia variant surface protein(VSP) H7 produced by a Salmonella typhimurium vaccine strain. International Journal forParasitology 27: 965-971.

Stager,S., B. Gottstein, H. Sager, T.W. Jungi, and N. Muller. 1998. Influence of antibodies inmother's milk on antigenic variation of Giardia lamblia in the murine mother-offspringmodel of infection. Infection and Immunity 66: 1287-1292.

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Stevens,D.P., D.M. Frank, and A.A. Mahmoud. 1978. Thymus dependency of host resistance toGiardia muris infection: studies in nude mice. Journal of Immunology 120: 680-682.

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Svärd,S.G., T.C. Meng, M.L. Hetsko, J.M. McCaffery, and F.D. Gillin. 1998. Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. MolecularMicrobiology 30: 979-989.

Touz,M.C., H.D. Lujan, S.F. Hayes, and T.E. Nash. 2003. Sorting of encystation-specificcysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires aconserved tyrosine-based motif. Journal of Biological Chemistry 278: 6420-6426.

Upcroft,P., N. Chen, and J.A. Upcroft. 1997. Telomeric organization of a variable andinducible toxin gene family in the ancient eukaryote Giardia duodenalis. Genome Research7: 37-46.

Yang,Y.M., and R.D. Adam. 1994. Allele-specific expression of a variant-specific surfaceprotein (VSP) of Giardia lamblia. Nucleic Acids Research 22: 2102-2108.

Yang,Y., and R.D. Adam. 1995a. A group of Giardia lamblia variant-specific surface protein(VSP) genes with nearly identical 5' regions. Molecular and Biochemical Parasitology 75:69-74.

Yang,Y.M., and R.D. Adam. 1995b. Analysis of a repeat-containing family of Giardia lambliavariant-specific surface protein genes: Diversity through gene duplication and divergence.Journal of Eukaryotic Microbiology 42: 439-444.

Yang,Y.M., Y. Ortega, C. Sterling, and R.D. Adam. 1994. Giardia lamblia trophozoitescontain multiple alleles of a variant-specific surface protein gene with 105-base pair tandemrepeats. Molecular and Biochemical Parasitology 68: 267-276.

Zhang,Y.Y., S.B. Aley, S.L. Stanley, Jr., and F.D. Gillin. 1993. Cysteine-dependent zincbinding by membrane proteins of Giardia lamblia. Infection and Immunity 61: 520-524.

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PATHOGENISIS AND IMMUNITY TOENTAMOEBA HISTOLYTICA

Jessica L. Tarleton and William A. Petri, Jr.University of Virginia Division of Infectious Diseases

ABSTRACTDiseases caused by the parasite Entamoeba histolytica

disproportionately affect residents of underdeveloped areas, afflicting placeslacking sufficient sanitation, hygiene, and water processing especially. Whilethe recent differentiation between Entamoeba histolytica and themorphologically identical but completely asymptomatic Entamoeba disparbased on genetic and biochemical analyses has enhanced the study of thedisease-causing parasite, a mystery still exists as to the factors which stillcause about 90% of Entamoeba histolytica infections to remain asymptomaticafter colonization. Researchers delving into these areas have identifiedseveral features of the parasite—including the galactose and N-acetylgalactosamine inhibitable adherence lectin, proteinases, andamoebapores, all virulence factors in Entamoeba—and of the host, includingthe association of anti-lectin IgA with resistance to disease, which maydetermine whether infection is invasive. As specific and easy diagnostics aredeveloped to aid in identifying more precisely the health burden caused by E.histolytica, vaccine candidates are under evaluation.Key words: Entamoeba histolytica, amebiasis, antigen detection test, amebiccolitis, amebic liver abscess, proteinase, amoebapore

INTRODUCTIONThe World Health Organization’s latest estimate implicates

Entamoeba histolytica in 50 million infections and 100,000 deaths each year(WHO 1997). Unfortunately, the majority of this burden falls on peoples ofunderdeveloped regions. Poverty, crowding, poor sanitation and hygiene—areality in many of the world’s developing areas—foster the growth and spreadof this parasite. In these endemic areas, such as Dhaka, Bangladesh, where amajor epidemiological survey is underway, as many as 1 in 10 children willdie before their birthday, 30% of those deaths attributed to diarrhealdiseases (Petri et al. 2000).

Tarleton and Petri

While research has recently made large advances towards a greaterunderstanding of this and other diseases that mainly plague the developingworld, it has yet to reveal a large-scale solution to for those afflicted by E.histolytica. Infection with the parasite may manifest itself in several symptompatterns—or usually, not at all: 90% of individuals colonized with the parasiteexhibit no symptoms of disease whatsoever (Ayeh-Kumi et al. 2001). Ofthose who do, amebic colitis and amebic liver abscess represent the mostcommon health problems caused by E. histolytica. Amebic colitis ischaracterized by a gradual, several-week onset of weight loss and diarrhea,with 70% of patients finding blood in stools (Petri and Singh 1999). Coloniculcers of these patients usually take on a flask shape characteristic of necrosiscaused by E. histolytica. Amebic liver abscess (ALA), which usually occursindependently of colitis, may present acutely or with a several week onset,with symptoms including weight loss, fever, and abdominal pain. Uncommonclinical symptoms of E. histolytica infection include acute necrotizing colitis,ameboma—masses of tissue in the colon, and rectovaginal fistulas, andextraintestinally, cutaneous amebiasis, splenic abscess, and brain abscess(Petri and Singh 1999; Devilliers and Durra 1998).

Only the newest epidemiological studies have accurately estimatedthe impact of E. histolytica on health, for only recently was E. histolyticadifferentiated from Entamoeba dispar, a morphologically identical butnonpathogenic species (Diamond and Clark 1993; Haque et al. 1998).Previously, E. dispar was simply identified as noninvasive infection by E.histolytica. While it remains virtually impossible to distinguish between E.histolytica and E. dispar microscopically, the two species have been found todeviate both genomically and biochemically (reviewed in Tanyuksel et al.2001). Four glycolytic enzymes, glucose-phosphate isomerase,phosphoglucomutase, hexokinase, and malic enzyme, vary between thespecies, which can be demonstrated by gel electrophoresis (Diamond andClark 1993). Furthermore, E. histolytica and E. dispar differ in DNArestriction patterns and can be differentiated by PCR analysis (Tachibana etal. 1991; Novati et al. 1996) and by E. histolytica-specific monoclonalantibodies (Haque et al. 1998).

Even with this recent differentiation it is clear that most E. histolyticainfections never induce symptoms. Now, with this recent reclassification,researchers can begin to unravel confusing issue of invasive and noninvasiveinfections by E. histolytica. Do variations between parasites, between hosts,or a combination of theses factors account for invasiveness and severity ofinfection?

Regions of endemicity for E. histolytica usually include those withinadequate sanitation and water supplies, poor hygiene, crowding, andwarmer, wetter climates (Walsh 1988); clearly, temperate developing areas, in

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Tarleton and Petri

combination with lack of health resources, are the hardest hit by this infection.In such areas, several host factors, such as sex, immunocompromised state,sexual activity, and even genetic susceptibility may make infection anddisease more likely (Jalan and Mailtry 1988).

First, the prevalence of amebic liver abscess (ALA) is at least 10times higher in males than females (Petri and Singh 1999; Hughes and Petri2000). Heavy alcohol consumption also may be a risk factor for ALA (Petriand Singh 1999); some researchers suggest that a higher tendency towardsheavy alcohol use in males could partially explain this observance (Seeto andRockey 1999; Hughes and Petri 2000). In addition, a large review of 86papers about E. histolytica written between 1925-1997 reports that,cumulatively, reviewed articles indicate a higher incidence in males thanfemales of all invasive E. histolyltica infections except fulminating amebiccolitis, but not a higher incidence of asymptomatic E. histolytica infection(Acuna-Soto et al. 2000).

Men who have sex with men are at a higher risk for E. histolyticainfection due to a higher frequency of fecal-oral contact (Seeto and Rockey1999). Recently more attention has focused on E. histolytica infection in HIVinfected persons, both in conjunction with higher observed risk in men whohave sex with men and also with regards to immunosuppression. Mostpatients with ALA in two San Francisco hospitals from 1979-1994 who hadnot lived in or traveled to an endemic area were immunosuppressed, includingHIV infection. While cases of amebiasis concurrent with HIV infectioncontinue to be reported (Ohnishi, Murata, and Okuzawa 1994; Fatkenheuer etal. 1997), and immunosuppression has been associated with higher incidenceof infection (Seeto and Rockey 1999), no study has conclusively explained alink between HIV and invasive E. histolytica infection.

Finally, recent research has revealed a genetic component to diseasesusceptibility. In children from Dhaka, Bangladesh, where a largeepidemiological survey in children is underway, a higher incidence ofinfection and reinfection correlated with presence of anti-trophozoite IgG inserum. Furthermore, the study showed that anti-trophozoite IgG can beinherited, with siblings of children with the antibody showing 4.8 timesgreater odds of possessing the same antibody. Finally, most children do notconvert from anti-trophozoite IgG negative to positive upon new infection.The authors suggest that serum anti-trophozoite IgG may serve as a marker ofgenetic deficiencies in innate or acquired immune responses (Haque et al.2002).

In the United States, where, in general, water sanitation and publichealth are optimal, immigration from an endemic area or recent travel to anendemic area prove to be high risk factors for E. histolytica infection (Seetoand Rockey 1999; Hughes and Petri 2000). Infection with E. histolytica

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usually presents clinically within a year of immigration to the U.S (Petri andSingh 1999).

In light of the differentiation of the species E. histolytica and E.dispar, diagnosis by microscopy is no longer sufficient. The TechLab E.histolytica II kit relies on antigen detection sensitive for a surface lectinunique to E. histolytica. Antigen detection tests may occur on either stool orserum samples, but serum testing has proven more sensitive and specific(Haque et al. 2000; 2001). In addition, PCR can be used to detect E.histolytica; though proven less sensitive than antigen detection, it carries thepotential advantage of distinguishing between strains (Mirelman et al. 1997;Tanyuksel et al. 2001). However, this technology is not feasible forwidespread use in developing countries where diagnosis is needed most. TheWorld Health Organization emphasizes the importance of using diagnosticsspecific for E. histolytica to improve treatment and management of thedisease (WHO 1997).

PATHOGENESISThe life cycle of the parasite consists of two stages: the invasive

trophozoite and the infectious cyst. In terms of human disease, thetrophozoite is responsible for tissue invasion and damage, while the cyst is themeans of human transmission (Lushbaugh et al. 1988). Cysts may reside incontaminated food in water, which human hosts ingest (WHO 1997). Whenthe ingested cyst, which can withstand harsh environmental conditions,reaches the small intestine, it excysts into a quadranucleate ameba, which thendivides into eight uninuclear trophozoites (Lushbaugh et al. 1988). Evidencefrom studies with the reptilian parasite Entamoeba invadens imply that thetrophozoite may monitor the galactose and N-acetylgalactosamine in itsintestinal environment to determine the most favorable time to encyst(Eichinger 2001).

A cell surface protein of the trophozoite plays a primary role in thissensory activity as well as critical adhesion activities of the trophozoite. TheGal/GalNAc lectin recognizes galactose (Gal) and N-acetylgalactosamine(GalNAc) (Ravdin and Guerrant 1981; Petri et al. 1987) found on humancolonic mucin glycoproteins. Interaction between the lectin of the trophozoiteand the host glycoproteins is required for adherence and cytolysis, so that ifeither is lacking or inhibited, the trophozoite is rendered benign (Ravdin andGuerrant 1981; Le et al. 1988). The Gal/GalNAc lectin is unique to E.histolytica, and it is, in fact, this antigen that the TechLab stool antigendetection test recognizes.

The Gal/GalNAc lectin has thus far been characterized in great detailbecause of its critical role in adherence and cytolysis and its vaccinecandidacy. It consists of three subunits (Petri et al. 1989); the heavy (Hgl)

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and light (Lg1) subunits are connected by disulfide bonds (Mai et al. 1999). Athird, intermediate subunit (Igl) is noncovalently associated with the Hgl/Lgldimer (Cheng et al. 1998) (Figure 1).

The carbohydrate recognition domain (CRD), the region of the lectinthat binds Gal and GalNAc has been localized to the Hgl (Dodson et al.1999). The importance of this region lies in its potential as a site for directinginhibitory vaccines or drugs (Petri et al. 2002). Much less is known about thefunctions of Lgl and Igl in virulence. One study used antisense RNA toinhibit production of Lgl in order to discern its role in virulence. Amebadeficient in Lgl demonstrated reduced cytotoxicty and cytopathogenicity.However, the loss of Lgl may have disrupted the Hgl/Lgl dimer enough toeffect virulence for reasons not isolated to the function of the Lgl (Ankri et al.1999). Because of our current inability to produce knockout mutant ameba,the antisense RNA approach may prove very useful in isolating the functionsof many ameba proteins (Ramakrishnan and Petri 2001).

The lectin is not only involved in adherence but has been directlyidentified as a player in cell cytolysis. While it has been shown that host-parasite contact via the Gal/GalNAc lectin is required for cytolysis (Ravdinand Guerrant 1981), the lectin itself contributes directly to the cytolyticactivity. Anti-lectin monoclonal antibody (mAb) against epitope 1 of Hgl

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inhibited cytotoxicity but not adherence, indicating disparate mechanisms ofthe two functions (Saffer and Petri 1991).

Among the lectin’s plurality of functions, its cytoplasmic tail is alsosuspected to have intracellular signaling functions. Amebae expressing afusion protein containing the cytoplasmic domain of the Hgl subunit have adecreased adherence and lysis ability in vitro as well as decreased severity ofliver abscess in an animal model (Vines et al. 1998).

The interplay between the extracellular sensory function of the lectinand the internal arrangement of the cytoskeleton may play a large role inpathogenicity. The E. histolytica trophozoite moves by forming a pseudopodin front, into which its streaming cytoplasm moves, and a posterior foot calleda uroid; the membrane moves towards the posterior uroid (Calderon et al.1980). This locomotion is most likely powered by movement and synthesis ofthe actin cytoskeleton (Bailey 1988). The mechanism called capping occurswhen the ameba, as its membrane streams backwards, collects surfaceantigens on the uroid and then releases the “cap” along with the collectedantigens, including the Gal/GalNAc lectin and a 96-kDa surface protein(Arhets et al. 1998). It has been suggested that this shedding of antigens andtheir attached host antibodies may aid the ameba in evading host immunedefenses (Arhets et al. 1995). However, more recent research shows that thecytoskeleton plays a more direct role in ameba pathogenesis: experimentsshow that ameba with defective cytoskeletons not only fail to form uroids orundergo capping but also fail to kill target cells in vitro, and Arhets et al.suggest that the cytoskeleton plays a critical role in contact-dependentcytotoxicity (Arhets et al. 1998).

The cysteine proteinases, secreted into the extracellular environmentof an amoeba, have also been identified as major virulence factors of E.histolytica. The proteinases have the ability to degrade elements of theextracellular matrix, including purified fibronectin, laminin, and type Icollagen, which would allow the parasite direct access to its target cell (Keeneet al. 1986). Furthermore, the cysteine proteinases interfere with both thecomplement pathway and humoral response of the human immune system.The enzymes can cleave the complement C3 in such a way as to activate thecomplement pathway, causing lysis of E. dispar but not E. histolytica (Reedand Gigli 1990). The Gal/GalNAc lectin inhibits complement-mediated lysisof E. histolytica by this specific mechanism by cross-reacting with CD59, amembrane inhibitor of C5b-9 in human blood cells (Braga et al. 1992). Thecontrasting responses of E. dispar and E. histolytica to the proteinases maypartially explain their contrasting invasiveness. The parasite does not onlyactivate but may conversely evade complement activity: the proteinases arecapable of degrading and inactivating C3 and C5 so as to circumvent this hostimmune response to the parasite (Reed et al. 1995). In addition, the

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proteinases are also primarily culpable for E. histolytica’s degradation ofsecretory IgA and IgG, which may limit host humoral immune responses(Kelsall and Ravdin 1993; Tran et al. 1998).

One particular proteinase, CP5, has been identified in E. histolyticabut is absent from E. dispar. Antisense RNA inhibition of CP5 did not causea significant decrease in the destruction of cell monolayers by intacttrophozoites, but interestingly decreased the trophozoites’ erythrophagocyticability for unknown reasons (Ankri et al. 1998)

E. histolytica also secretes a pore-forming protein, calledamoebapore, important in the pathogenesis of disease. Three isoforms, A, B,and C, exist. The peptide can insert ion channels into artificial membranesand depolarize and may also be cytolytic to eukaryotic cells (Lynch et al.1982; Rosenberg et al. 1989; Leippe et al. 1994). The amoebapores haveantibacterial properties, disrupting membrane integrity of Gram-positivebacteria (Leippe et al. 1994). It has been suggested that the primary functionof amoebapores is the killing of ingested bacteria (Leippe et al. 1994).

IMMUNITYHuman immune defenses against Entamoeba histolytica begin with

the mucus lining the intestinal wall. Conflictingly, while mucus serves torestrict the ameba’s access to colonic epithelial cells (Chadee et al. 1987), italso provides a rich environment for colonization. Mucins, making up aportion of human colonic mucus, produce the O-linked proteins, galactose andN-acetylgalactosamine, to which the Gal/GalNAc lectin specifically binds(Ravdin et al. 1985; Chadee et al. 1988). The relative benefit to burden ratioof colonic mucin glycoproteins, as to whether they help in dispelling theparasite or encourage colonization and therefore invasive infection, is still notclear (Tse and Chadee 1991).

Inside the intestine, secretory antibodies are produced againstparasites. Specifically, IgA is produced in response to invasive infection byE. histolytica. Anti-lectin IgA has been found in human milk (Grundy et al.1983), serum (Abu-El-Magd et al. 1996), and saliva, and a saliva antibody testhas even been proposed, though not widely adopted, for diagnosis of infection(Del Muro et al. 1990). Recently, secretory IgA against the Gal/GalNAclectin has been associated with defense against infection (see below).

The inflammatory response provides a third mechanism of humandefense against the parasite, and recent research has taken us a step closer tounderstanding this means of protection as well. Until recently, the dynamicbetween leukocytes recruited by the immune system and amebic trophozoitesremained unclear: different research reported that polymorphonuclearneutrophils killed trophozoites but also that the reverse was true (Gillin et al.1988). In intestinally derived cell lines, Entamoeba histolytica induces the

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production of certain pro-inflammatory cytokines and chemokines such asand IL-6 and IL8 (Kim et al. 1995; Yu et al. 1997; reviewed in Stanley

2001). Eckmann et al. (1995) demonstrated the variety of cytokines, includingand IL-6, secreted from cultured human cells

upon contact with trophozoites; the release of these cytokines appear to bemediated by production of cytolytically released Yu and Chadee(1997) found that upon stimulation with amebic proteins, amebic secretoryproteins, or live trophozoites, colonic cells produced IL-8, even without cell-cell contact or colonic cell damage.

In order to determine whether such cytokine production occurs invivo, the severe combined immunodeficiency disease mouse with a humanintestinal xenograft (SCID-HU-INT mouse) provided an opportunity to use ananimal model to produce a human immune response in the region of humantissue, the intestine. Seydel et al.(1997) determined that when the humanintestinal tissue of these mice was infected with trophozoites, the xenograftsproduced the human inflammatory cytokines and IL-8 in directresponse to E. histolytica, and that cytokine production occurred in regionsother than those in direct contact with the parasites.

However, while this research showed definitively that cytokinescontributed to the inflammatory response seen in infected tissues, the exactcause of the inflammation and tissue damage for which E. histolytica earnedits name was still up in the air. Several more studies began to illuminate thisissue. By inhibiting transcription factor, which controls expression ofIL-1, IL-6, and IL-8, cytokine production and gut inflammation in E.histolytica infected tissue was greatly reduced. Interestingly, tissue damagewas also greatly reduced, possibly implicating the host inflammatory responsein its own tissue damage (Seydel et al. 1998). Employing the SCID-HU-INTmouse model again, it was shown that mice whose neutrophils had beendepleted before infection with E. histolytica suffered significantly less tissuedamage (Seydel et al. 1998). It appears as though trophozoites, when lysinghost neutrophils, spill their toxic contents into host tissues and create damagenot directly caused by the parasites (Jarumilinta and Kradolfer 1964; Guerrantet al. 1981).

Further elucidation of human immune response came with Campbellet al.’s (2000) findings that the 170 kDa subunit of the Gal/GalNAc lectininduced IL-12 production in human THP-1 macrophages; the researchers evenlocalized the immunogenic region of the subunit to between amino acids 596-998. As IL-12 promotes Th1 cytokine differentiation and in turn macrophageprotection, the induction of IL-12 by the lectin figures significantly in thevaccine search.

In the hopes of designing a vaccine for amebiasis, the role of acquiredimmunity to E. histolytica infection bears much importance, and huge strides

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have been made in this area recently. Clinical observations have noticed moresevere cases of amebiasis in patients in immunocompromised states, includingpatients taking corticosteroids (Ratcliffe 1988). Animal models in variousstates of immunosuppression have also displayed more severe infections(Ghadirian and Meerovitch 1981a; Ghadirian and Meerovittch 1981b;Ghadirian and Kongshavn 1985; reviewed in Huston and Petri 1998).

A recent study by Haque et al. (2001) on a large group of children inDhaka, Bangladesh, an endemic area, has greatly advanced our understandingabout protective immunity against amebiasis: they found that the presence ofanti-lectin IgA provides a marker of acquired immunity. Evidence of this liesin several specific findings: first, none of 64 children in the initial survey whowere found to have stool anti-lectin IgA were colonized with E. histolytica;second, after a one-year prospective study, children with stool anti-lectin IgAshowed 64% fewer new E. histolytica infections (while 39% of childrenshowed new infections during the one-year period). Finally, IgA appeared inthe system concurrently with resolution of infection. A subsequent study haspinpointed the area of the anti-lectin response to the carbohydrate recognitiondomain (CRD), refining the marker of acquired immunity to IgA producedagainst the CRD of the Gal/GalNAc lectin (see Fig. 2). In addition, thepossibility of anti-trophozoite IgG marking genetic insufficiency of immunedefenses will surely be filled out in much more detail in the future (Haque etal. 2002).

The human immune responses to Entamoeba histolytica infectionremain only generally defined, but the hope of a vaccine pushes researchfurther into knowledge of this subject. Vaccine development appears a distantbut feasible goal. (Table 1).

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New information on the existence of acquired immunity hassupported that hope, and growing knowledge of the highly- conserved andpathogenically critical Gal/GalNAc lectin has made it the object of severalvaccine tests (Petri and Ravdin 1991).

Several other amebic proteins present possible vaccine candidates aswell. The serine-rich Entamoeba histolytica protein (SREHP) is also well-conserved, immunogenic, and has not been found in other Entamoeba species,including E. dispar (Stanley et al. 1990). Immunization with recombinantSREHP protected 100% of gerbils from ALA when administered through asingle intradermal injection (Zhang 1994). A 29-kDa cysteine rich antigenhas also been shown to elicit a protective response in animal models (Soong1995). In addition, 150- and 170-kilodalton surface antigens of E. histolyticahave conferred protection against ALA in hamsters (Cheng and Tachibana2001). The amoebapore and a few cysteine proteinases have also beenconsidered as possible vaccine candidates (Huston and Petri 1998).

Clearly, many issues should be considered when seeking a solution tothe health problems caused by E. histolytica—it is imperative that we treat theoverarching health risks posed by poverty and poor sanitation, as well asresearch further the nature of human immunity, the variations in virulenceamong parasite strains, and vaccine candidates. Diseases of poverty posemultifaceted challenges encompassing both science and society. As researchinto each moves forward, the pool of information from which we can drawupon and combine will grow, leading us toward better health solutions toamebic diseases.

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Stanley, S.L. 2001. Pathophysiology of amebiasis. Trends in Parasitology 17:280-285.Stanley, S.L., A. Becker, C. Kunz-Jenkins, L. Foster, and E. Li. 1990. Cloning and expression

of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats.Proceedings of the National Academy of Sciences USA 87:4976-4980.

Tachibana H, Ihara S, Kobayashi S, Kaneda Y, Takeuchi T, and Watanabe Y. 1991.Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates ofEntamoeba histolytica identified by polymerase chain reaction. Journal of ClinicalMicrobiology 29:2234-2239.

Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Genomic DNA differencesbetween pathogenic and nonpathogenic Entamoeba histolytica. Proceedings of the NationalAcademy of Science USA 86:5118-5122.

Tanyuksel, M., H Tachibana, and W.A. Petri, Jr. 2001. Amebiasis, an emerging disease. InEmerging Diseases 5 ed., W.M. Scheld, W.A Craig, and J.M. Hughes (eds.), ASM Press,Washington, D.C., p.197-212.

Tran, V. Q., D. S. Herdman, B. E. Torian, and S. L. Reed. 1998. The neutral cysteineproteinase of Entamoeba histolytica degrades IgG and prevents its binding. Journal ofInfectious Diseases 177:508-511.

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Tse, S.K. and K. Chadee. 1991. The interaction between intestinal mucus glycoproteins andenteric infections. Parasitology Today 7: 163-172.

Vines, R.R., G. Ramakrishnan, J.B. Rogers, L.A. Lockhart, B.J. Mann, and W.A. Petri, Jr.1998. Regulation of adherence and virulence by the Entamoeba histolytica lectincytoplasmic domain, which contains a beta2 integrin motif. Molecular Biology of the Cell 9:2069-2079.

Walsh, J.A. 1988. Prevalence of Entamoeba histolytica infection. In Amebiasis, J.I. Ravdin(ed.). John Wiley & Sons, New York, p.93-105.

World Health Organization. 1997. Amoebiasis. W. H. O. Weekly Epidemiological Record72:97-100.

Yu, Y. and K. Chadee. 1997. Entamoeba histolytica stimulates interleukin 8 from humancolonic epithelial cells without parasite-enterocyte contact. Gastroenterology 112: 1536-1547.

Zhang, T., P.R. Cieslak, and S.L. Stanley. 1994. Protection of gerbils from amebic liverabscess by immunization with a recombinant Entamoeba histolytica antigen. Infection andImmunity 62:1166-1170.

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INNATE AND T CELL-MEDIATED IMMUNERESPONSES IN CRYPTOSPORIDIOSIS

Carol R. Wyatt1 and Vincent McDonald2

1 Department of Diagnostic Medicine/Pathogiology, Mosier Hall, Kansas State University,Manhattan, KS 66506-57052 Barts and the London School of Medicine and Dentistry, Department of Adult and PaediatricGastroenterology, DDRC, Turner St, London E1 2AD, UK

ABSTRACTA variety of innate immune responses may help to control early

parasite replication, initiate inflammation and generate signals for T cellactivation. Ultimately, elimination of infection involves cells that areof the Th1 (cell-mediated immunity) phenotype, but there may be a protectiverole for lymphocytes of the Th2 type (antibody-dependent responses).Intraepithelial lymphocytes have increased activity as a result of infection andmay be important in the anti-cryptosporidial immune response.Key words: Cryptosporidium, innate immunity, T cells, intraepitheliallymphocytes, cytokines, antibodies

INTRODUCTIONSince its recognition as an important zoonotic pathogen, much research on

Cryptosporidium parvum has focused on host responses to the parasite.Responses that lead to disease and that end the infection are important tounderstand because they offer clues that can lead to effective control ofcryptosporidiosis. This chapter describes our current understanding of thecontributions of the host to disease pathogenesis and to clearance of C. parvuminfection.

INNATE IMMUNITYInnate immunity comprises T cell-independent immune responses that

provide at least partial protection against infection. Non-lymphoid cells andnatural killer (NK) cells may be involved. Products from these cells such ascytokines, complement or antibiotic peptides may be important in microbicidalmechanisms.

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Infection of epithelial cells by C. parvum activates a keyinflammatory transcription factor (Chen et al. 2001). Expression of theproinflammatory chemokines IL-8, and RANTES, isdependent, and increased expression of these molecules has been observed ininfected cell lines (Gargalla et al. 1997; Laurent et al. 1997). Treatment ofcells with a specific inhibitor of reduced secretion of IL-8 (Chen et al.2001). also promotes production of cationic antimicrobial peptidessuch as defensins and, in bovine C. parvum infection, increased expression of a

was demonstrated (Tarver et al. 1998). Synthesized anti-microbialpeptides were previously shown to inhibit the viability of C. parvumsporozoites (Arrowood et al. 1991). The initial stimulus for activationis unclear but parasite products might interact with Toll-like receptor (TLR)molecules or intracellular proteins such as NOD-1 (Philpott et al. 2001) whichrecognize evolutionarily conserved bacterial products.

Cryptosporidial infection of epithelial cells also induces prostaglandinproduction (Laurent et al. 1998). Prostaglandins may have different protectiveeffects because they can modulate T cell activation and decrease inflammation.Prostaglandins might also act by promoting increased mucin production andcontributing to mechanisms of diarrhea.

An increased incidence of cryptosporidiosis has been reported in HIVpatients with mutations in the mannose-binding lectin (MBL) gene (Kelly et al.2001). MBL activates complement via lectin-associating serine proteases thatcleave C4 and C2. MBL and C4 adhered to the surface of sporozoites in vitroand complement components and MBL were detected in the gut lumen of AIDSpatients with diarrhea (Kelly et al. 2001). MBL may block parasite attachmentto host cells or induce the complement membrane attack complex.

A crucial innate response to intracellular pathogens is production byNK cells. NK cell activation is induced by proinflammatory cytokines fromaccessory cells including IL-12 and and is inhibited by IL-10 (Tripp etal. 1993). Cryptosporidial antigen can generate a similar response in splenicNK cells from SCID mice which lack T + B cells (McDonald et al. 2000).

can partially protect against C. parvum infection of SCID and T cell-deficient nude mice. Affected mice develop progressive, chronic, andsometimes fatal infections (Ungar et al. 1990; Mead et al. 1991; reviewed byTheodos, 1998). Treatment with neutralizing antibodies exacerbatesinfection and hastens the onset of morbidity (Ungar et al. 1991; Chen et al.1993; McDonald and Bancroft, 1994; Urban et al. 1996). micehave heavier infections and greater morbidity than do SCID mice with an intact

gene (Hayward et al. 2000). Induction of in neonatal SCID mice

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involves IL-12 since injection with the cytokine can suppress C. parvumdevelopment. IL-12 treatment increases expression in the ileum,and administration of neutralizing anti- IL-12 to SCID mice increases parasitereproduction. directly inhibits C. parvum reproduction in humanenterocyte cell lines by preventing sporozoite invasion and by depletingintracellular Fe availablility (Pollok et al. 2001).

It is not clear, however, if NK cells are the source of in theseinfection models, scid-nu/nu mice carrying the Beige mutation, making themdeficient in NK cell function, had more intense C. parvum infections than didSCID mice (Mead et al. 1991). Other attempts to demonstrate NK involvementwere unsuccessful, however. SCID mice given anti-ASGMl antibodies todeplete NK cells, or IL-2 to stimulate NK cell maturation, were not made moresusceptible to infection (Rohlman et al. 1993; McDonald and Bancroft, 1994).And, no role for in activation of production was seen, asinjection with neutralizing antibodies had no effect on parasitereproduction (Chen et al. 1993; McDonald and Bancroft, 1994).

IMMUNOPATHOGENESISDysregulation of the mucosal immune response is critical to the

pathogenesis of numerous intestinal diseases. For example, Crohn’s is a Th1(see later) inflammatory condition associated with T cell and macrophageinfiltration of the lamina propria and expression of the proinflammatorycytokines IL-12,IL-18, and (Garside, 2000) that can inducevillous atrophy and crypt hyperplasia. Indeed, treatment of Crohn’s with anti-TNF monoclonal antibodies can be a highly effective therapy. Interestingly, C.parvum infection increases susceptibility of mice to developspontaneous colitis similar to ulcerative colitis (Sacco et al. 1998).

Villous atrophy and crypt hyperplasia are common features duringcryptosporidial infection and lymphocytes, macrophages and neutrophilsinfiltrate the infection site (Tzipori, 1988). C. parvum-infected calves haveincreased numbers of villous and cell subpopulations(Abrahamsen et al. 1997). In mice, cattle and humans, infection leads toincreased intestinal expression of (Kapel et al. 1996; White et al. 2000;Wyatt et al. 2001). Induction of other proinflammatory cytokines such as IL-12 in cattle (Payer et al. 1998) and (Lacroix et al. 2001) in mice hasalso been described. Cytokines activate intestinal metalloproteases, whichdamage the tissue (MacDonald et al. 1999), and may also impair ion movementor induce epithelial cell apoptosis. Inflammation increases production ofsecretagogues, including prostaglandins, neural peptides and reactive nitrogen

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or oxygen intermediates (Gaginella et al. 1995). Secretagogue activity anddamage to the epithelium that prevents absorption are likely to be involved inthe development of cryptosporidial diarrhea.

The regulation of the inflammatory response is poorly understood. Incalves, the numbers of and cells subsides in parallel withrecovery from a primary C. parvum infection (Abrahamsen et al. 1997),suggesting the infection, at least in part, drives the inflammatory response. Inmurine C. parvum infection, plasticity in the Th response was reported as awaning of a Th1 response late in infection as a Th2 response emerged (Aguirreet al. 1998). This change in polarity may allow Th1 and Th2 cytokines todownregulate each other’s inflammatory functions.

Intestinal regulatory cells (Th3 or Tr1) that produce the anti-inflammatory cytokines or IL-10 prevent inflammatory bowel diseasein mice (Groux and Powrie, 1999). inhibits the severe intestinalinflammation normally observed in C57BL/6 mice infected orally withToxoplasma gondii (Buzoni-Gatel et al. 2001). Intraepithelial lymphocytes(IEL) produce and the cytokine may inhibit chemokine production byenterocytes. Significantly, expression is increased in intestinal biopsiesfrom humans infected with C. parvum (Robinson et al. 2000) and a similarfinding has been made in necropsies from infected mice (McDonald et al., inpreparation). In addition to its direct anti-inflammatory properties, canameliorate damage to epithelial barrier function caused by in vitrofollowing C. parvum infection (Roche et al. 2000).

T CELLS INVOLVED IN ADAPTIVE IMMUNITYMuch of the data on the role of T cells in immunity to cryptosporidial

infection has been obtained from murine infection models. T cells are essentialfor parasite clearance as chronic C. parvum or C. muris infections areobserved in T cell-deficient mice whereas infections in normal mice are self-limiting (reviewed by McDonald and Bancroft, 1998). Infections inimmunocompromised mice can be cleared by injection of T cell-containinglymphoid cells from normal mice (reviewed by Theodos, 1998). In addition, astudy with T cell receptor (TCR) knockout mice suggested that cellsare necessary for recovery whereas the cells are not essential but mayprovide some protection in neonates (Waters and Harp, 1996).

Within the cell population, the two major cell subsets are the T-helper cells and the cytotoxic cells. Cryptosporidial infection isexacerbated in mice deficient in cells through a mutation in MHCclass II expression, or by treatment with anti-CD4 (Aguirre et al. 1994; Ungar

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et al. 1991; McDonald et al. 1994). Also, SCID mice injected with lymphoidcells from normal mice do not recover from infection if cells aredepleted from the donor cells (Chen et al. 1993; McDonald and Bancroft 1994;McDonald et al. 1994; Perryman et al. 1994). In human disease, life-threatening infection is common in HIV patients with low cell counts(Blanshard et al. 1992). Restoration of cells following antiretroviraltherapy confers resistance to C. parvum infection (Farthing, 2000).cells are, therefore, essential for elimination of Cryptosporidium infection.

The cell subpopulation appears to be less important in immunity.Mice deficient in MHC-class I expression, and so lacking cells, were nomore susceptible to infection than control mice (Aguirre et al. 1994). Similarly,in mice depletion of cells by antibody, the ability to control infectionwas either unaffected or only mildly diminished (Ungar et al. 1990, 1991; Chenet al. 1993; McDonald and Bancroft, 1994; McDonald et al. 1994, 1996;Perryman et al. 1994). Splenic lymphocytes incubated with C. parvum antigenscontained proliferating but not cells (Harp et al. 1994).

cells are activated by MHC class I-restricted presentation of peptidesnormally originating from proteins present in the cell cytoplasm (Williams etal. 1996). The extracytoplasmic location of Cryptosporidium may mean thatthe class I presentation pathway is deficient.

TH1 AND TH2 RESPONSESIn response to infection, cells differentiate into Th1 or Th2 cells

that mediate cell-mediated (Th1) or humoral (Th2) responses are defined bypatterns of cytokine production; IL-2, IL-12, and are associatedwith Th1 and IL-4, IL-5, and IL-10 with Th2. Intracellular pathogens induceThl responses, while extracellular pathogens promote Th2 responses. Th1 andTh2 responses can cross-regulate each other, and the outcome of an infectioncan depend upon the timing and magnitude of each response.

C57BL/6 mice are susceptible to C. parvum, and either die orremain persistently infected, suggesting a role for in clearance. Ininfected neonatal C57BL/6 mice, mucosal IL-4 and IL-10 areexpressed, while in neonatal parental strain mice, which clear the infection,both Th1 and Th2 cytokines are expressed (Lacroix et al. 2001). Splenocytesfrom C57BL/6 mice stimulated in vitro with a sporozoite antigenpreparation express IL-5, but not IL-2, IL-4, or suggesting a biastoward production of IgA antibodies (Mead and You, 1998). In contrast,C57BL/6 mice have prolonged infections compared with the parentstrain (Aguirre et al. 1998). Administration of neutralizing results in

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increased numbers of shed oocysts but does not prolong infection in adultC57BL/6 mice while anti-IL-4 treatment prolongs the infection.

BALB/c mice infected with C. parvum clear the infection (Meadand You, 1998), and repeated IL-12 injection increases oocyst output andprevents recovery (Smith et al. 2001). Splenocytes from BALB/c micestimulated in vitro with sporozoite antigens express IL-2, IL-4, IL-5, and

and those stimulated with a recombinant 23 kDa sporozoite surfaceprotein express IL-2, IL-5, and but not IL-4 and IL-12, mRNA(Bonafonte et al. 2000), suggesting that C. parvum infection induces Th1- andTh2-like responses in BALB/c mice. Neonatal BALB/c micealso clear the infection; however, they shed more oocysts compared withparental strain mice (McDonald, unpublished observations).

GUT MUCOSAL LYMPHOCYTESMucosal lymphocytes are the first lymphoid cells to contact mucosal

pathogens. Intraepithelial lymphocytes (IEL) are located at the base of thevilli, beneath epithelial cell tight junctions, while lamina propria lymphocytes(LPL) lie within the villi beneath the lamina propria membrane (reviewed byKraehenbuhl and Neutra, 1992). Both IEL and LPL have been implicated inimmune responses to cryptosporidial infection.

induction of chemokine expression in intestinal epithelial cells fromC. parvum-infected mice results in recruitment of T cells and macrophages intothe mucosa (Lacroix-Lamande et al. 2002). Consistent with this observation,neonatal calf ileal explants inoculated in vitro with C. parvum oocystsaccumulate mucosal and lymphocytes around epithelial cells whencompared with control explants (Wyatt et al. 1999). IEL from 3 day infectedcalves express IL-10 mRNA in conjunction with the expression of severalsporozoite epitopes, and also express combinations of and(Wyatt et al. 2002). LPL have increased numbers of andlymphocytes compared with controls (Abrahamsen et al. 1997).

During cryptosporidial diarrhea, neonatal calf IEL have increasedpercentages of lymphocytes and lymphocytes andexpress (Wyatt et al. 1997). Calves that have recovered from diarrheahave elevated and and LPL that express but not IL-10, IL-4, or IL-2 (Wyatt et al. 2001).

Adoptive transfer of IEL from primed BALB/c donor mice to SCID micewas shown to clear C. muris infection in a response requiring cells and

(McDonald et al. 1996; Culshaw et al. 1997. Subsequent similar work

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using C. parvum confirmed that IEL from primed donor mice can clear apersistent infection in SCID mice (Adjei et al. 2000).

HUMORAL IMMUNITYAntibodies can be important in the immune response to a pathogen.

Antibodies that fix complement can promote lysis while those that bindmolecules critical to host cell invasion can neutralize the infectivity of thepathogen. However, the role of antibodies in immunity to C. parvum isunclear.

C. parvum infected mice excrete fecal antibodies to sporozoite antigens,and IgA isotype antibodies can reduce the severity of infection in neonatal mice(reviewed by Wyatt, 2000). However, HIV-1 infected patients with chroniccryptosporidiosis and low cell counts make serum IgM, IgG, and IgA,and salivary IgA, antibodies to C. parvum, but these antibodies do not clear theinfection (Cozon et al. 1994).

P23 is an immunodominant sporozoite surface glycoprotein that contains atleast two epitopes sensitive to antibody neutralization. In cattle, antibody-richcolostrum generated by immunizing cattle with recombinant p23 protectscalves against cryptosporidial diarrhea, but does not clear the infection(reviewed by Wyatt, 2000). C. parvum infected calves excrete antibodies top23 (Wyatt et al. 2000). IgA antibodies are prominent 5-6 days afterinoculation, and IgM and peak 7 days after inoculation while the calveshave diarrhea. antibodies peak as calves recover from diarrhea. Bovine

antibodies are regulated by IL-4, while antibodies are regulated by(reviewed by Wyatt, 2000), and calves recovering from cryptosporidial

diarrhea have LPL that contain and switched B cells, and expressbut not IL-4 (Wyatt et al. 2001). Thus, calves recovering from

cryptosporidiosis have LPL capable of generating an immune response thatincludes antibodies and

CONCLUSIONSThese observations raise several issues. First is the dual role of the Th1

response. Th1 cytokines mediate the inflammation that leads to disruption ofintestinal architecture and cell functions, resulting in diarrhea. Yet Th1cytokines are also necessary to clear C. parvum infection. Thus, anyintervention designed to stimulate a protective Th1 response must also controlthe inflammatory effects on the gut.

Second is the apparent protective role of Th2 cytokines such as IL-4. Asan intracellular pathogen, control of Cryptosporidium should not require a Th2

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response. However, antibodies that can prevent entry of the pathogen intoepithelial cells can decrease the level of infection in the gut, thereby lesseningdisease manifestations. Perhaps IL-4 induces production and elaboration intothe gut lumen of antibodies that prevent sporozoites and merozoites frominfecting additional epithelial cells.

Third is the role of cells that infiltrate the gut. If MHC Ipresentation is ineffective due to the extracytoplasmic location of the parasite,then what is the role of cells? An answer might lie in the type of T cellexpressing the CD8 molecule. In several models, gut mucosal cells expressCD8. These cells are skewed toward developing a Th1-like response (Yin et al.2000), and they can downregulate inflammatory responses (Egan and Carding,2000). Indeed, if gut mucosal cells can modulate inflammation within themucosa during C. parvum infection, then manipulation of these cells mightminimize inflammation while the infection is being controlled.

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defensin: molecular cloning and characterization of a gene with inducible intestinalepithelial cell expression associated with Cryptosporidium parvum infection. Infection andImmunity 66: 1045-1056.

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Tripp C.P., S.F. Wolf, and E.R.Unanue. 1993. Interleukin-12 and tumor necrosis factor alphaare costimulators of interferon gamma production by natural killer cells in severe combinedimmunodeficiency mice with listeriosis, and interleukin-10 is a physiologic antagonist.Proceedings of the National Academy of Sciences 90:3725-3729.

Tzipori S. 1988. Cryptosporidiosis in perspective. Advances in Parasitology 27:63-129.UngarB.L.P., J.A. Burris, C.A. Quinn, and F.D. Finkelman.1990. New mouse models for chronicCryptosporidium infection in immunodeficient hosts. Infection and Immunity 58: 961-969.

Ungar B.L.P., T.-C. Kao, J.A. Burris, and F.D. Finkelman. 1991 Cryptosporidium infection inan adult mouse model. Independent roles for IFN-g and CD4+ T lymphocytes in protectiveimmunity. Journal of Immunology 147:1014-1022.

Urban J.F. Jr., R. Fayer, S.-J. Chen, W.C. Gause, M.K. Gately, and F.D. Finkelman. 1996.IL-12 protects immunocompetent and immunodeficient neonatal mice against infection withCryptosporidium parvum. Journal of Immunology 156:263-268.

Waters W.R., and J.A. Harp. 1996. Cryptosporidium parvum infection in T-cell receptorand mice. Infection and Immunity 64:1854-1857.

White A.C., P. Robinson, P.C. Okhuysen, D.E. Lewis, I. Shahab, S. Lahoti, H.L. Dupont, andC.L. Chappell. 2000. expression in jejunal biopsies in experimental humancryptosporidiosis correlates with prior sensitization and control of oocyst excretion. TheJournal of Infectious Diseases 181:701-709.

Wyatt, C.R. 2000. Cryptosporidium parvum and mucosal immunity in neonatal cattle. AnimalHealth Research Reviews. 1:25-34.

Wyatt, C.R., W.J. Barrett, E.J. Brackett, D.A. Schaefer, and M.W. Riggs. 2002. Associationof IL-10 expression by mucosal lymphocytes with increased expression of Cryptosporidiumparvum epitopes in infected epithelium. Journal of Parasitology. 88:281-286.

Wyatt, C.R., E.J. Brackett and W.J. Barrett. 1999. Accumulation of mucosal T lymphocytesaround epithelial cells after in vitro infection with Cryptosporidium parvum. Journal ofParasitology. 85:765-768.

Wyatt, C.R., E.J. Brackett, P.H.Mason, J. Savidge, and L.E. Perryman. 2000. Excretionpatterns of mucosally delivered antibodies to p23 in Cryptosporidium parvum infectedcalves. Veterinary Immunology and Immunopathology. 76:309-317.

Wyatt, C.R., E.J. Brackett, L.E. Perryman, A. C. Rice-Ficht, W.C. Brown, and K.I. O’Rourke.1997. Activation of intestinal intraepithelial T lymphocytes in calves infected withCryptosporidium parvum. Infection and Immunity. 65:185-190.

Wyatt C.R., J. Bracket, and J. Savidge. 2001. Evidence for the emergence of a type -1-likeimmune response in intestinal mucosa of calves recovering from cryptosporidiosis. Journalof Parasitology 87:90-95.

Williams D.B., Vassilakos A., and W.K Suh. 1996. Peptide presentation by MHC class Imolecules. Trends in Cellular Biology 6:267-273.

Yin, Z., D.-H. Zhang, T. Welte, G. Bahtiyar, S. Jung, L. Liu, X.-Y. Fu, A. Ray, and J. Craft.2000. Dominance of IL-12 over IL-4 in cell differentiation leads to default production of

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RATIONALE APPROACHES TO TREATINGCRYPTOSPORIDIUM, CYCLOSPORA, GIARDIA

AND ENTAMOEBA

Jan R. Mead1 and Pablo Okhuysen2

1Atlanta Veterans Affairs Medical Center and Department of Pediatrics, Emory School ofMedicine, Atlanta, GA 300332Department of Medicine, Division of Infectious Diseases and School of Public Health,University of Texas Health Science Center in Houston, Houston, TX 77030

ABSTRACTTreatment options for diarreal illness caused by intestinal protozoa

differ depending on the particular protozoa. Cryptosporidium parvum, animportant parasite among the immunocompromised and young children ofdeveloping nations remains refractory to all conventional therapies.Cyclospora cayetanensis, an emerging pathogen can be treated withtrimethoprim-sulfamethoxazole, however the discovery of alternativetreatments remains hampered by the inability to cultivate the parasite orestablish an animal model. Despite being a frequent cause of diarrheal illnessthroughout the world, relatively few agents outside of metronidazole are usedin therapy against Giardia lamblia. While metronidazole is the agent ofchoice for the treatment of most amoebic infections, proper diagnosis isimportant to distinguish between pathogenic and non-pathogenic Entamoebaspecies.Key words: Cryptosporidium parvum, Cyclospora, Giardia, Entamoebahistolytica

CRYPTOSPORIDIUMSeveral groups could benefit from an effective therapy against

cryptosporidiosis: children, the elderly, and immunocompromised groupssuch as, organ transplant recipients, and cancer patients. Chemotherapy wouldalso be useful among the immunocompetent in more severe cases and inoutbreak situations to curb the spread of disease. Among the more importantgroups in need of therapy are individuals infected with humanimmunodeficiency virus (HIV). While cryptosporidiosis is serious inimmunocompetent people, it can be devastating to those with AIDS.

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The use of potent highly active anti-retroviral therapy (HAART) inpatients with advanced HIV infection can improve or lead to the clearance ofC. parvum from the stools. Patients treated with double anti-retroviral therapyor protease inhibitors have demonstrated excellent responses and sustainedtherapeutic effects after follow-up (Ives and Easterbrook, 2001; Miao, et al.,2000). Although HAART is not thought to have a direct effect on C. parvum,its indirect activity is apparently a result of improved immunological status,related to increased cell counts rather than to modulation of the viralload. Few options exist for patients with AIDS in whom highly activeantiretroviral therapy fails or is not an option. Controlled treatment trials haveprovided some useful information but have not resulted in identifying aneffective therapeutic agent. While many compounds have demonstratedactivity using in vitro assays, fewer therapeutic agents have demonstratedsignificant potency using animal models.

Paromomycin has been one of the most widely used agents to treatcryptosporidial infections in AIDS patients. It is a poorly absorbedaminoglycoside that is related to neomycin and kanamycin. It achieves highconcentrations in the gut, in part due to poor bio-availability. It has shownefficacy in animal models (Healey et al., 1995; Tzipori et al., 1995) and as aprophylactic treatment in neonatal calves (Fayer and Ellis, 1993), lambs (Viuet al., 2000) and goats (Johnson et al., 2000; Mancassola et al., 1995). Inhumans, the drug was considered partially effective in that decreases insymptoms (frequency of stools) were noted but the parasite was not eradicated(Flannigan and Soave, 1993). In a placebo-controlled, double blind study,treatment was found to be partially effective (White et al., 1994). However, amore recent study showed no significant difference between the treated andplacebo groups (Hewitt, et al., 2000). Consequently, many patients initiallyrespond to paromomycin treatment with a decrease in diarrhea but thenrelapse.

Combination therapy with paromomycin and azithromycin demonstratedsome efficacy in relieving symptoms and parasitic burden. In a open-label,combination study (Smith et al., 1998) patients with AIDS, chroniccryptosporidiosis, and low cell counts weregiven 1.0 gram of paromomycin b.i.d. plus 600 mg of azithromycin once perday for 4 weeks, followed by paromomycin alone for 8 weeks. Both partialclinical and parasitological responses were observed, as treatment ofcryptosporidiosis with azithromycin and paromomycin was associated withsignificant reduction in oocyst excretion and some clinical improvement.

Nitazoxanide, a broad-spectrum antiparasitic agent, was found to beeffective in a clinical trials performed in Mali, Mexico and Egypt (Doumbo etal., 1997; Rossignol et al., 1998; Rossignol et al., 2001); however, clinicaltrials in the U.S. have not been encouraging, and nitazoxanide has not been

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approved by the US Food and Drug Administration. In a double-blind,placebo-controlled study, patients were randomly treated with either 500 or1000 mg nitazoxanide, or placebo orally b.i.d. for 14 days (Rossignol et al.,1998). Patients on nitazoxanide then crossed over to placebo while theplacebo patients crossed over to nitazoxanide therapy at either the high or lowdose depending on their randomization. Both doses of nitazoxanide producedparasitological cure rates superior to the placebo responses (12/19 [63%, P =0.016] for patients receiving 1 g/d and 10/15 [67%, P = 0.013] for thosereceiving 2 g/d). In a recent study, a 3-day course of nitazoxanidesignificantly improved the resolution of diarrhea, parasitological eradicationand mortality in HIV-seronegative, but not HIV-seropositive children (Amadiet al., 2002).

Other therapies have been evaluated with various degrees of reportedefficacy. None have been proven totally efficacious. These includespiramycin, (Portnoy et al., 1984; Saez-Llorens et al., 1989; Vargas et al.,1993), clarithromycin, (Holmberg et al., 1998; Jordan et al., 1996), rifabutin,(Fichtenbaum et al., 2000), roxithromycin (Uip et al., 1998) and the use ofhyperimmune bovine anti-Cryptosporidium colostrum (Fries et al., 1994;Okhuysen et al, 1998). In the absence of an effective therapy forcryptosporidiosis, fluid support and maintaining electrolyte balance is ofparamount importance for severe cases among immunocompetent andimmunocompromised individuals.

A CHALLENGING PARASITE WITH INNATERESISTANCE

Why are so many therapies not effective against this parasite? It seemsthat C. parvum has a natural resistance to drug therapy. Several factors maycontribute to this lack of efficacy. These include: 1) the parasite’s uniquelocation in the host cell which may affect drug concentration (transportedfrom the host cell across to the parasite); 2) lack of specific targets ordifferences in targets either at the molecular or structural levels; 3) differencesin biochemical pathways; 4) existence of transport proteins or efflux pumpsthat transport drugs out of the parasite or into the host cell.

In the last few years the establishment of large scale sporozoite-expressedsequence tag (EST) and genomic sequence tag (GST) projects have aided inthe identification of C. parvum genes and in the understanding ofphylogenetic similarities and diversities within the genome. C. parvum, itappears, is more divergent and less related to the other coccidia. It has beenproposed that C. parvum is more closely related to the gregarine parasiteswithin the Apicomplexa. Comparisons of small-subunit ribosomal RNA genesequences have demonstrated that the gregarine/Cryptosporidium clade is

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separate from the other major apicomplexan parasites (Carreno et al., 1999).Another study compared six different proteins along with the SSU rRNA andconcluded that the parasite should be placed at an early emerging branch ofthe Apicomplexa (Zhu et al, 2000a).

Other structural differences have been noted. Unlike most of theApicomplexa, C. parvum appears to lack a plastid genome (Riordan et al,1999; Zhu et al., 2000b). In general, macrolide antibiotics have notdemonstrated consistent efficacy even after long-term administration. Whilethis lack of efficacy may or may not be related to the lack of a plastid, drugdevelopment targeting a plastid genome or metabolic pathway associated withit may not be useful. In addition, evidence suggests that C. parvum has aputative mitochondrion and mitochondrion-associated enzymes markedlydifferent in structure from those of its nearest relatives (Riordan et al, 1999)but lacks much of the electron transport chain.

Some parasite targets (e.g. enzymes, structural proteins) are different thanthose of other related parasites. For example, the dihydrofolate reductase(DHFR) gene of C. parvum differs from the DHFR of Plasmodium.Sequencing of the dihydrofolate reductase gene indicated that the enzymemay be intrinsically resistant to 2,4-diaminopyrimidine inhibitors (Vasquez etal., 1996). The C. parvum DHFR active site contained novel residues atseveral positions analogous to those at which point mutations have beenshown to produce antifolate resistance in other DHFRs (Vasquez et al.,1996). This may in part explain why C. parvum is resistant to clinically usedantibacterial and anti-protozoal anti-folates. It has also been determined thatC. parvum differs fundamentally in polyamine metabolism from othereukaryotes (Bacchi and Yarlett, 1995). Polyamine biosynthesis in C. parvumoccurs via a pathway used by plants and certain bacteria, in which arginine isconverted to agmatine by the action of arginine decarboxylase (Keithly et al.,1997). Neither arginine decarboxylase nor agmatine is found in other parasiticprotozoa, which make this a unique target.

A factor that may contribute to the ineffectiveness of many drugs is theunusual location of the parasite in the host cell. The parasite has a uniqueniche inside the cell in that it is intracellular but extracytoplasmic. It has beenpostulated that the basal membranes modulate the transport of certain drugs,so drugs entering the cytoplasm of the host cell may not be transported acrossto the parasite. There is evidence to suggest that this is the case for geneticinand paromomycin, a clinically relevant drug (Griffiths et al., 1998). In cellculture studies, apical but not basolateral exposure of these drugs led tosignificant parasite inhibition.

The existence of multi-drug resistant (MDR) transporters might facilitateresistance and may also be involved in the rapid efflux of drugs as well asnutrient uptake. P-glycoproteins and MDRs are members of the ATP-binding

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cassette (ABC) superfamily that are responsible for drug resistance byextruding drugs against a concentration gradient. ABC transporters withconsiderable homology to the mammalian MDR-associated protein (MRP)and at least one grouped with the multidrug resistance protein (MDR) havebeen identified in C. parvum (Perkins et al., 1999; Zapata et al., 2002; Strongand Nelson, 2000).

NEW AREAS OF DRUG RESEARCHA number of drug targets have been proposed over the years but have not

been pursued either because the presumed drug target could not be found,toxicity of the drug was too great, or because the drug lacked efficacy inanimal models or in clinical trials. Some of the following therapies remainpromising because the target is selective or unique and efficacy has beendemonstrated either in vitro or in vivo against the target.

The shikimate pathway is an important pathway in plants, bacteria, andfungi for the synthesis of aromatic compounds, amino acids, ubiquinonecompounds, and folate. Several apicomplexans, including C. parvum,demonstrated evidence for the presence of enzymes of the shikimate pathway(Roberts et al., 1998).

A fatty acid synthase gene (CpFAS1) identified in C. parvum encodes amultifunctional polypeptide which differs from the organellar type II fattyacid enzymes identified in T. gondii and P. falciparum (Zhu et al., 2000c).

As mentioned above, arginine, an essential amino acid and precursor ofthe polyamine pathway, is converted by C. parvum to agmatine by the actionof arginine decarboxylase (Keithly et al., 1997). Inhibitors of argininedecarboxylase (ADC) significantly reduce intracellular growth of C. parvum,whereas inhibitors of ornithine had no effect upon ADC activity or upongrowth of the parasite (Water et al., 1997; Waters et al., 2000).

Anti-tubulin agents such as the benzimidazoles and the dinitroanilineherbicides have demonstrated efficacy against a number of parasitic agents(Roos et al., 1997; Stokkermans et al., 1996; Traub-Cseko, et al. 2001).Although benzimidazoles are widely used against helminth infections andsome protozoa, C. parvum did not have the predicted amino acids forbenzimidazole drug sensitivity in the tubulin-coding gene (Katiyar et al.,1994; Edlind, et al., 1994). In a subsequent study, benzimidazole treatmentwas not effective against C. parvum when evaluated in mice (Fayer et al,1995). Conversely, dinitroaniline herbicides were effective against C. parvumin vitro (Arrowood et al, 1995). The efficacy of this class of compounds hasbeen determined in neonatal mice (Armson et al., 1999).

In addition, several newly synthesized drugs have recently demonstratedanticryptosporidial activity. These include several lipophilic DHFR inhibitors(Brophy et al., 2000) and acridinic thioethers and aurone analogs (Kayser et

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al., 2001). These latter compounds have structural similarities to the plant-derived phenolic chalcones that are active against other parasites such asPlasmodium, Leishmania and Trypanosomes.

Despite the lack of response with immune colostrum in clinical trials,immunotherapy may still be useful in conjunction with conventional drugtherapy or as a mechanism to decrease the severity of infection in neonatalanimals or moderately immunocompromised individuals. The ability ofmonoclonal antibodies to neutralize C. parvum infectivity and controlinfection in vivo has been demonstrated (Langer et al., 2001; Riggs et al.,1997). Colostrum or monoclonal antibodies directed at neutralizing epitopesor antigens involved in attachment or invasion of host cells may be moreeffective than broadly generated immunotherapies.

Not only is it important to develop better, more efficacious drugs, but alsoto improve drug transport and delivery to the parasite in vivo.Nanosuspensions are drug nanoparticles dispersed in a liquid phase, leading toincreased solubility and dissolution velocity. Alternatively, the use ofmucoadhesive polymers may help to increase drug retention time in the gut.Mucoadhesive drug delivery systems are also attractive since they attach tothe intestinal wall and are in close proximity to the parasite. It was shownthat a mucoadhesive nanosuspension bupravaquone formulation cleared theinfection more effectively from the intestinal tract than the unmodifiednanosuspension (Kayser et al., 2001a).

How can we develop efficacious drugs? The continuing increase ingenome sequence data should aid in the identification of new enzymes andbiochemical pathways and increase our understanding of host/parasiteinteractions. However, the inability to cryopreserve the parasite andpropagate the parasite continuously in vitro hinders many avenues ofchemotherapeutic research. Cryopreservation of the parasite would establishstandard isolates that could be used for multiple studies with consistency andreduced variability. The ability to propagate the parasite continuously in vitrocould lead to establishment of a genetic model that would be useful formutant/transfection studies. These studies would then facilitate identifyingmolecules as viable drug targets. In addition, the lack of effective drugsagainst these targets severely hampers the ability to validate these targets.Identification of unique targets in this challenging parasite is an importantfirst step in developing effective therapeutic agents.

CYCLOSPORACyclospora cayetanensis is a protozoan parasite of humans causing

diarrheal illness after colonizing the mucosal epithelium of the small intestine.C. cayetanensis is a parasite that has been described world-wide andidentified in water and food. Cyclosporiasis causes “flu-like” symptoms

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including profound diarrhea lasting 1 to 3 weeks. Clinical signs includeweight loss, nausea, anorexia, vomiting, and abdominal cramping (Herwalt,2000). Among immunocompromised individuals (e.g. HIV-infected),cyclosporiasis symptoms may be exacerbated. However, drug treatment isgenerally effective in controlling infection and relapse (or reinfection) can beaverted by maintenance or prophylactic drug dosages (Pape et al., 1994;Verdier, et al., 2000).

Several studies have demonstrated that trimethoprim-sulfamethoxazole(TMP-SMX) is the drug of choice. In a placebo-controlled study in Nepal,TMP-SMX (160/800 mg) taken twice daily for 7 days was effective attreating Cyclospora infections (Hoge et al., 1995). Treatment was also foundto be effective in a study of HIV-infected patients, when TMP-SMX wasgiven 4 times a day for 10 days, followed by secondary prophylaxis (Pape etal., 1994). Among individuals that cannot be treated with TMP-SMZ (e.g.sulfa drug allergies), treatment options are limited and infections may beprolonged. Most alternative treatments such as albendazole, TMP alone,azithromycin, nalidixic acid, norfloxacin, tinidazole, metronidazole,quinacrine, tetracycline, and diloxanide furoate, do not seem to be effectiveagainst the parasite (Pape et al., 1994).

One alternative, ciprofloxacin, has demonstrated moderate activity inHIV-infected patients when a 500 mg dose was administered twice daily. Ina randomized, controlled study comparing trimethoprim-sulfamethoxazole orciprofloxacin, patients who received trimethoprim-sulfamethoxazole, diarrheaceased in 3.0 days, whereas those patients treated with ciprofloxacin hadcessation of diarrhea at 4.0 days (Verdier et al., 2000). While active,ciprofloxacin was not as effective as TMP-SMZ in this population and has notshown comparable activity in immunocompetent patients (Herwalt, 2000).

Little is known about potential virulence, pathogenicity or drugsusceptibility differences among isolates involved in outbreaks and inendemic environments. One of the major challenges for research into thebiology of Cyclospora is the lack of in vitro or in vivo models. Until aninfectivity model is available that supports the entire life cycle of the parasite,many of these characteristics will remain obscure. Attempts to establish ananimal model of cyclosporiasis using human-derived oocysts has not yet beenachieved (Eberhard et al., 2000).

GIARDIAThe antiparasitic agent of choice is metronidazole administered 250 mg

t.i.d. for 5 days with an efficacy approximating 80 to 85% (Garner 2002).Patients should be advised of the disulfiram-like reaction that may occur withthe concomitant use of alcohol. Isolates with decreased susceptibility tometronidazole have been described in vitro (Upcroft 2001) and clinical

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experience suggests that this may be relevant in practice. Albendazole at adose of 400 mg daily for 5 days is an alternative to therapy and appears to beeffective. Other effective agents not available for use in the US are tinidazoleadministered as a single dose (also effective in 90% of cases) and quinacrine100 mg orally three times a day after meals for 5 days. Treatment of pregnantwomen poses special problems due to the potential mutagenicity ofmetronidazole although this has not been well documented in humans. Inthese cases, treatment should be delayed until at least after the first trimester,provided the patient’s hydration and nutritional status can be maintained. Iftherapy is indicated in this setting then paromomycin, a non-absorbableaminoglycoside can be considered for use at a dose of 500 mg four timesdaily if the patient’s renal function is normal. Uncommonly, one facesrefractory cases in which a combination of metronidazole and quinacrine canbe used for a longer period of time (14 day course). Nitazoxanide, a broadspectrum antiparasitic agent and its metabolite tizoxanide are more potentthan metronidazole in vitro even in metronidazole resistant isolates and can bean alternative treatment when used at a dose of 1.5 gm po bid for 30 days inareas of the world where this drug is approved for use (Abboud, 2001; Adagu,2002; Rossignol 2001; Ortiz 2001). Newer targets will undoubtedly beidentified as the Giardia genome is analyzed (Adam, 2000).

Given the ubiquity of giardiasis in certain environments such as day carecenters, treatment of asymptomatic Giardia infections is not routinelyrecommended since healthy children do not appear to suffer deleteriouseffects with chronic asymptomatic cyst passage and therapy may expose themto unnecessary medication related side effects.

ENTAMOEBA HISTOLYTICATherapy of Entamoeba infection should take in consideration not only the

location and severity of the infection but the species causing infection. It isnow well recognized that infections with E. dispar are asymptomatic and thattherapy is not warranted. For asymptomatic cyst passers of E. histolytica,paromomycin at a dose of 500 mg tid for 7 days has shown to be moreeffective than diloxanide furoate when administered for 10 days (Blessman,2002).

Symptomatic infections should always be treated. E. histolytica can causesymptomatic intestinal syndromes including the following: 1) a dysentericsyndrome with production of small volumes of bloody, mucoid stools withoutfecal leukocytes, 2) colitis characterized by ulcerations of the colonicmucosa with typical flask-shaped abscesses, or 3) the formation of a fibroticmass in the intestinal wall (ameboma). Metronidazole is the agent of choicefor the treatment of amoebic colitis. Tinidazole is a reasonable alternativewith the advantage that single dose therapy can be equally efficacious.

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Intestinal infections should also be treated with an intraluminal agent toprevent future invasion of remaining cysts.

Chronic amoebic colitis is clinically indistinguishable from inflammatorybowel disease and those receiving corticosteroids are at risk for toxicmegacolon and perforation and may sometimes necessitate parenteral therapywhen patients are unable to tolerate the oral route. Infective trophozoites canmigrate hematogenously to the right lobe of the liver, causing abscessformation, abdominal pain, jaundice and fever. Adjacent anatomicalstructures, such as the pulmonary parenchyma, peritoneum and pericardiumcan become involved. Amoebae can also disseminate to the brain.Immunosuppressed or malnourished individuals, those at the extremes of age,patients with malignancy, and women during pregnancy and post-partumstages are especially at risk for invasive amebiasis. Metronidazole followedby a luminal agent is the therapy of choice in extraintestinal disease. Sinceamebomas can mimic adenocarcinoma, a biopsy may be needed todifferentiate disease. Indications for surgical drainage of an amoebic abscessinclude large dimensions, impending rupture, left lobe location, or lack oftherapeutic response to metronidazole.

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Hoge, C. W., D. R. Shlim, R. Rajah, J. Triplett, M. Shear, J. G. Rabold, and P. Echeverria.1993. Epidemiology of diarrhoeal illness associated with coccidian-like organism amongtravellers and foreign residents in Nepal. Lancet 341: 1175-1179.

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Jordan, E. C. 1996. Clarithromycin prophylaxis against Cryptosporidium enteritis in patientswith AIDS. Journal of the National Medical Association 188: 425-427.

Katiyar, S. K., V. R. Gordon, G. L. McLaughlin, and T. D. Edlind. 1994. Antiprotozoalactivities of benzimidazoles and correlations with beta-tubulin sequence. AntimicrobialAgents and Chemotherapy 38:2086-2090.

Kayser, O. 2001. A new approach for targeting to Cryptosporidium parvum usingmucoadhesive nanosuspensions: research and applications. International Journal ofPharmaceutics 214: 83-85.

Kayser, O., R. Waters, K. Woods, S. Upton, J. Keithly, and A. Kiderlen. 2001. Evaluation of invitro activity of aurones and related compounds against Cryptosporidium parvum. PlantaMedica 67: 1-4.

Keithly, J., G. Zhu, S. Upton, K. Woods, M. Martinez, and N. Yarlett. 1997. Polyaminebiosynthesis in C. parvum and its implications for chemotherapy. Molecular and BiochemicalParasitology 88: 35-42.

Langer, R., D. Schaefer, and M. Riggs. 2001. Characterization of an intestinal epithelial cellreceptor recognized by the Cryptosporidium parvum sporozoite ligand CSL. Infection andImmunity 69: 1661-1670.

Lopez, A. S., D. R. Dodson, M. J. Arrowood, P. A. Orlandi Jr, A. J. da Silva, J. W. Bier, S. D.Hanauer, R. L. Kuster, S. Oltman, M. S. Baldwin, K. Y. Won, E. M. Nace, M. L. Eberhard,and B. L. Herwaldt. 2001. Outbreak of cyclosporiasis associated with basil in Missouri in1999. Clinical Infectious Diseases 32: 1010-1017.

Mancassola, R., J. Reperant, M. Naciri, and C. Chartier. 1995. Chemoprophylaxis ofCryptosporidium parvum infection with paromomycin in kids and immunologicalstudy.Antimicrobial Agents and Chemotherapy 39: 75-78.

Miao, Y., F. Awad-El-Kariem, C. Franzen, D. Ellis, A. Muller, H. Counihan, P. Hayes, and B.Gazzard. 2000. Eradication of cryptosporidia and microsporidia following successfulantiretroviral therapy. AIDS 25: 124-129.

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INACTIVATION AND REMOVAL OF ENTERICPROTOZOA IN WATER

F.W. Schaefer, III1, M.M. Marshall2 and J.L. Clancy3

1U.S. Environmental Protection Agency, Cincinnati, OH 452682University of Arizona, Tucson, AZ 857213Clancy Environmental Consultants, Inc., St. Albans, VT 05478

ABSTRACTProtozoan parasites including Giardia, Cryptosporidium, and

Entamoeba can be transmitted through water and cause disease in humans andanimals. Control of waterborne infection can be accomplished through avariety of physical and chemical means, resulting in the production of safedrinking water and protection of public health. Coagulation and filtration arethe most commonly employed methods for physical removal of parasites, whilechlorine-based compounds, ozone, and ultraviolet light are used forinactivation. Combinations of treatment technologies can result in parasiteremoval/inactivation greater than 6-log, resulting in reliable public healthprotection.Key words: Cryptosporidium, Giardia, Entamoeba, coagulation, slow sandfiltration, sedimentation, diatomaceous earth filtration, multi-medium filtration,dissolved air flotation, chlorine, chloramine, chlorine dioxide, ozone, ultravioletlight

INTRODUCTIONGiardia, Entamoeba, and Cryptosporidium are enteric pathogenic protozoaknown to infect the intestinal tract of numerous mammals including man.Unlike many parasites, these organisms have direct life cycles involvingvegetative, developmental stages that ultimately encyst in the host intestinaltract before passage in the fecal material. The Giardia and Entamoeba cyst aswell as the Cryptosporidium oocyst are environmentally resistant transmissionstages known to be transmitted by ingestion of water. These transmissionstages can be detected in most surface and finished water at some time or othernecessitating control to assure public health (LeChevallier et al., 1991a, b).Cysts and oocysts are known to be more resistant to disinfection thanpathogenic bacteria. Theory suggests that the wall of the cyst and oocyst

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protects the parasite from the disinfectant.In developed nations, waterborne outbreaks of giardiasis and

cryptosporidiosis have occurred numerous times over the latter part of thecentury. Waterborne amebiasis, on the other hand, rarely has been reported,with the largest outbreak occurring in Chicago in 1933 (Craun, 1986).However, this outbreak was the result of a sewage cross-connection to a hotelwater system rather than inadequate water treatment. Besides studies onphysical removal, studies on the efficacy of free chlorine, hypochlorite,chloramine, chlorine dioxide, ozone, and ultraviolet light (UV) have been doneon Giardia cysts and Cryptosporidium oocysts.

PHYSICAL REMOVAL OF PARASITESTwo important factors in physical removal of cysts and oocysts in the

water treatment process are size and composition of the wall. The spherical E.histolytica cysts range in size from 5 to The oval G. lamblia cystsrange in size from 8 to in length and 7 to in breadth. SphericalC. parvum oocysts range in size from 4 to The general rule is that thesmaller the cyst or oocyst the more difficult it is to remove using conventionalwater treatment technology. This concept has been documented over the latterpart of the century (Logsdon and Hoff, 1986). Consequently, using thisrationale, C. parvum oocysts are the most difficult of the aforementionedparasites to physically filter from water. Detailed information on the exactcomposition of cysts and oocysts is lacking. Little is known about theEntamoeba cyst wall. Giardia lamblia cysts contain significant amounts ofcarbohydrate and protein in a 3:2 ratio (w/w). The carbohydrate componenthas been identified as (Gerwig et al.,2002). Although studies on the oocyst wall of C. parvum have been done,about all that can be said is the oocyst wall is composed of glycoprotein(Bonnin et al., 1991). While protein differences and their uneven distributionthroughout the oocyst wall have been explored as a way to speciateCryptosporidium, definitive studies defining the exact composition of theoocyst wall remain to be completed (Harris and Petry, 1999; Jenkins et al.,1999). Cysts and oocysts are naturally electronegative (Ongerth and Hutton,1997). However, if they are not purified from fecal material properly avoidingextremes of pH, inactivation with formaldehyde, and exposure to potassiumdichromate, their surface charge will not mimic the natural condition (Schaefer,2001). Experiments designed to study the interaction of oocyst surface charge(zeta potential), glass bead packed columns, natural organic matter, biofilm,and alum as coagulant demonstrated that both biofilm on the glass bead packedcolumns and natural organic matter significantly decreased the column removal

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efficiency from 51% to 14% (Dai and Hozalski, 2002). The electronegativezeta potential of natural organic matter treated oocysts was significantlyincreased in addition to increasing their hydrophobicity. When alum was usedas a coagulant, it counteracted the high electronegative oocyst surface chargeresulting in a removal efficiency of 73%. The physical removal resultsreported below must be viewed with caution, for many of the investigators didnot use properly prepared or treated transmission stages.

The most popular techniques to physically remove particulates andmicroorganisms from water include rapid granular media (sand, dual media, ormixed media) filters, membranes, slow sand filters, or diatomaceous earth (DE)filters. Coagulation, flocculation, sedimentation or some combination thereofusually precedes the rapid granular media filters (Logsdon and Hoff, 1986).The physical removal of cysts and oocysts and the way in which they interactwith filter media and coagulants is related to a number of factors including thesurface characteristics of the cyst or oocyst, the amount of other particulates inthe water, the physical characteristics of the water, the size of the filter media,design of the filter, operation of the filter, and the size of the cyst or oocyst.

Slow sand filters, as the name implies, are beds of sand through whichwater slowly passes usually at a velocity around 0.1 m/hr in a 1 m deep filter.As the filter matures, the schmutzdecke, a microbiologically active scum layer,develops on the top and increases the efficiency of the filter. Better than 2-log(99.0%) removal of Giardia cysts is possible with slow sand filters (Bellamy etal., 1985). Cryptosporidium oocyst are efficiently removed using slow sandfiltration with better than 99.997 % reduction (Timms et al., 1995).

Jar tests of gravel pit water with turbidities ranging between 1 and 2nephelometric turbidity units (ntu) showed about 90% or higher removal ofGiardia cysts in the pH range of 6 to 9. In an experiment with the alumcoagulant at 22 mg/L, the Giardia cysts passing the filter were at a density of80 cysts/L. However, when the alum coagulant feed was interrupted, theGiardia cyst density passing through the filter rose to 1,760 cysts/L(Aronzarena, 1979). Turbidity breakthrough at the end of a rapid filter run hasbeen shown to be indicative of passage of large numbers of Giardia cysts(Logsdon et al., 1981). Clearly the optimal operation of coagulant feed and therapid filter are critical to removal of Giardia cysts. The Mobile WaterPurification Unit, Model 1940, was tested by the U.S. Army for removal of E.histolytica cysts. When alum and soda ash were used for coagulation 98.5 %and 99.8% of the applied E. histolytica cysts were removed duringsedimentation (U.S. Army, 1944). Several studies have studied optimizationof coagulation and rapid filtration processes for Cryptosporidium oocystremoval. In the first study, the best oocyst removals were ~3-log and were

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achieved with ferric chloride coagulant in combination with pre-oxidation,cationic polymer and filter aid addition and tri-media filtration (Yates et al.,1997). The second study investigated the effect of pH on the coagulants ferricchloride, alum, and polyaluminum chloride. The three coagulants wereeffective in achieving 4.3-log removal of oocysts (States et al., 2001).

Ongerth and Pecorano (1995) conducted trials with multiple runs overseveral months in the northwestern US using river sources to examine oocystremoval. The treatment system characteristics were direct filtration with in-lineflocculation; multi-media filters; low-turbidity, low-alkalinity raw water withoptimal alum coagulation; and constant rate operation at 5 gallons per minute persquare foot They achieved 2.7- to 3.1-log removal of Cryptosporidiumoocysts in this pilot-scale direct filtration plant, which was only slightly lower thanthe concurrent removal of Giardia cysts.

Dissolved air flotation (DAF) was shown to achieve a >2-log removalof oocysts in bench-scale trials under a variety of operating conditions(Plummer et al., 1995). The objective of the DAF study was to determine theeffect of specific design and operating variables on oocyst removal. Thesource water was from a reservoir in upstate New York. The study showedthat coagulant dosage had a significant effect on oocyst removal by DAF. At adose of 5 mg/L ferric chloride, oocyst removal was 3.7-log, but was reduced to2.0-log at 3 mg/L, and to 0.38-log at 2 mg/L. Acceptable turbidity, anddissolved organic carbon (DOC) were noted at a dosage of 3.5 mg/L,indicating that higher coagulant doses may be needed to provide optimal oocystremoval. In another DAF study, Cryptosporidium removals ranged from 2.9-log to 4.0-log, with granular activated carbon (GAC) and dual media filtersproviding the highest removals (Hall et al., 1995).

Nieminski and Ongerth conducted a two-year study at both a pilot- andfull-scale plant treating river water in Utah. The pilot plant processes includeda flash mixer, four-stage flocculation basins, and two sedimentation basinswith dual media filtration. The pilot plant had two treatment trains, one ofwhich was converted to direct filtration. The full scale plant was a 900 gpmplant which operated both as a conventional plant for some runs, and as adirect plant for others by bypassing the settling basin and routing flocculatedwater to the filters. For each operational mode, 10 seeding runs wereconducted for the pilot plant and four seeding runs were conducted for the full-scale plant. Results showed that Cryptosporidium oocyst removal was 2.9-log in both the conventional and direct modes in the pilot plant; at full scale,oocyst removal was 2.3-log by conventional treatment to 2.8-log by directfiltration. Cyst-sized particles, as determined by optical particle counters andturbidity were shown to be indicators of oocyst removal.

DE filters have been shown to remove certain viruses, bacteria, andprotozoa. The removal efficiency of this filter type is dependent upon the poregrade of the diatomaceous earth, precoat rate, body feed rate, whether thediatomaceous earth has been coated with hydrous oxides of iron or cationicpolymer and the filtration rate. Studies have shown more than 99% of G.muris cysts are removed by DE filters provided the filter septum is precoatedproperly (Logsdon et al., 1981). Similar results were obtained for G. lambliacyst removal (DeWalle et al., 1984). Entamoeba histolytica cysts arecompletely removed by a properly operated DE filter (U.S. Army, 1944).When properly operated, DE filters can produce up to 6-log Cryptosporidiumoocyst removal (Ongerth and Button, 1997; Ongerth and Hutton, 2001).

Membrane processes - ultrafiltration and microfiltration - have beenshown to provide high levels of cyst and oocyst removal (>6-log). In thesestudies, cysts and oocysts are rarely seen in the filter permeate, and removalsare generally determined based upon the initial seed concentration (Jacangelo etal., 1995). Physical straining of cysts and oocysts from the feed water appearsto be the removal mechanism. These processes are widely used in thepharmaceutical, electronics, and food industries for removal of sub-micronparticles so highly effective cyst and oocyst removal is expected.

DISINFECTIONThe effectiveness of chemical disinfection is dependent upon the

disinfectant, pH, temperature, disinfectant demand in the water, and theorganism being inactivated. Free chlorine, the most frequently useddisinfectant in water treatment, has several species in water depending on thepH. The more active species, HOCl, is found around pH 6 to 7, while the lessactive species, is present at higher pH of 8 to 10. In contrast to HOCl,

is most effective as an undissociated gas in the pH range 6 to 9.Chloramine, the weakest of the water treatment disinfectants, requires a pHaround 8 or higher to ensure monochloramine is the predominant species, whenchlorine and ammonia are mixed together in equimolar proportions. Ozone isindependent of pH in the ranges encountered in water treatment. Because ofthe high reactivity and volatility of ozone, controlling water disinfectionpresents challenges. As water temperature increases, ozone disinfectionefficacy increases. The effect of temperature and pH on the effectiveness ofdisinfectants on Giardia and other microorganisms is shown in Table 1.Compared to the other microorganisms listed in the Table 1, Giardia andEntamoeba cysts are more resistant to water treatment disinfection thanbacteria or viruses. G. muris appears to be more resistant to ozone andchlorine than G. lamblia.

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UV light is the most recent addition to the arsenal of disinfectants forprotozoan parasites. Earlier studies of UV effectiveness on Giardia andCryptosporidium indicated that very high UV doses were required forinactivation, but these studies were misleading. In vitro excystation was usedas the indicator of inactivation, and data showed that little inactivationoccurred at reasonable UV doses (Rice and Hoff, 1981; Karanis et al., 1992;Ransome et al., 1993; Finch et al., 1997; Clancy et al., 1998). However, whenanimal infectivity was used to assess UV efficacy, a dramatic reduction ininfectivity was noted (Bukhari et al., 1999). Animal infectivity measures theability of the cyst or oocyst to complete the infection cycle, whereas in vitroexcystation measures metabolic activity. It is now understood that althoughcysts and oocysts treated with UV may still exhibit metabolic activity, they areunable to cause infection in a susceptible host, and therefore, have beensuccessfully inactivated. Once this was known, a rapid succession of studieswas undertaken to determine the susceptibility of Cryptosporidium inparticular. Studies on UV inactivation of Giardia and Cryptosporidium arepresented in Table 2.

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Chlorine is effective against Giardia, and water treatment processesare designed to provide sufficient contact time such that cysts remaining in thewater after filtration will be inactivated by chlorine or other disinfectantexposure. However, Cryptosporidium is resistant to chlorine-baseddisinfectants and oocysts escaping filtration could remain viable aftertreatment.

Ozone can also be used to control Cryptosporidium in water, but thelevels of inactivation are nowhere near those seen with UV. Temperature playsan important role in ozone inactivation, and water systems in colder climatescannot achieve the required levels of Cryptosporidium inactivation with ozone.The CT for ozone inactivation of Cryptosporidium is approximately 25 to 35timers higher than that required for ozone inactivation of Giardia.

Ozone has a high potential for bromate ion formation, a potentialhuman carcinogen, when employed at the significantly higher CT levels toinactive C. parvum oocysts (Federal Register, 1996). Goals of 0.5- to 1-loginactivation of Cryptosporidium may be achievable depending on many factorsincluding water quality, plant design and operational flexibility, limiting ozoneas a choice for Cryptosporidium control to specific sites. UV remains the mosteffective choice for Cryptosporidium control, both in terms of efficacy andcost effectiveness.

CONCLUSIONSCurrent water treatment processes can be highly effective for control

of protozoan parasites in drinking water. Combinations of physical removalthrough coagulation and filtration, coupled with disinfection can result in logreductions ranging from 2- to over 6-log. Protozoan parasites are resistant tostandard chemical disinfection using chlorine-based compounds, so physicalremoval plays an important role in the treatment process for parasite control.UV light is the latest and most effective method for control of both Giardia andCryptosporidium, and is being implemented worldwide for control of thesepathogens. Production of safe drinking water relies on the multiple barrierapproach to drinking water treatment. This begins with source waterprotection to prevent pollution, followed by appropriate treatment, andmaintenance of water quality through proper storage and distribution to theconsumer. This holistic approach permits water suppliers to provide safedrinking water and a high level of public health protection.

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Gerwig, G.J., J.A. Kuik, B.R. Leeflang, J.P. Kamerling, J.F.G. Vliegenthart, C.D. Karr, andE.L. Jarroll. 2002. The Giardia intestinalis filamentous cyst wall contains a novel beta (1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology12:499-505.

Hall, T., J. Pressdee, R. Gregory, and K. Murray. 1995. Cryptosporidium removal duringwater treatment using dissolved air flotation. Water Science and Technology 31:125-135.

Hargy, T.M., J.L. Clancy, Z. Bukhari, M.M. Marshall. Shedding UV light on theCryptosporidium threat. Journal of Environmental Health, July/August 2000, 19-22.

Harris, J.R., and F. Petry. 1999. Cryptosporidium parvum: structural components of theoocyst wall. Journal Parasitology 85:839-849.

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Hayes, S.L., E.W. Rice, M.W. Ware, and F.W. Schaefer. 2003. Low pressure ultravioletstudies for inactivation of Giardia muris cysts. Journal of Applied Microbiology 94:54-59.

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Jacangelo, J.G., S.S. Adham, and J.-M. Laine. 1995. Mechanism of Cryptosporidium,Giardia and MS2 virus removal by MF and UF. Journal American Water WorksAssociation 87(9):107-121.

Jenkins, M.C., J. Trout, C. Murphy, J.A. Harp, J. Higgins, W. Wergin, and R. Fayer. 1999.Cloning and expression of a DNA sequence encoding a 41-kilodalton Cryptosporidiumparvum oocyst wall protein. Clinical Diagnostic Laboratory Immunology 6:912-920.

Karanis, P., W.A. Maier, H.M. Seitz, D. Schoenen. 1992. UV sensitivity of protozoanparasites. Journal Water Supply Research and Technology-Aqua. 41(2):95-100.

Ketzenelson, E., B. Kletter, and H.I. Shuval. 1974. Inactivation kinetics of viruses andbacteria in water by use of ozone. Journal American Water Works Association 66(12):725-729.

Klevens, C.M. 2001. Parameters for UV disinfection of surface water. Poster Session, FirstInternational Congress On Ultraviolet Technologies. Washington, D.C.

Leahy, J. G. Inactivation of Giardia muris cysts by chlorine and chlorine dioxide. 115 p.1985. Ohio State University; Dept. of Civil Engineering. Thesis/Dissertation

LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991a. Giardia and Cryptosporidium spp.in filtered drinking water supplies. Applied and Environmental Microbiology 57:2617-2621.

LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991b. Occurrence of Giardia andCryptosporidium spp. in surface water supplies. Applied and Environmental Microbiology57:2610-2616.

Logsdon, G.S. and J.C. Hoff. 1986. Barriers to the transmission of waterborne disease, pp.255-276. In G.F. Craun (ed.), CRC Waterborne Diseases in the United States CRC Press,Boca Raton, Florida.

Logsdon, G.S., J.M. Symons, R.L. Hoye Jr, and M.M. Arozarena. 1981. Alternative filtrationmethods for removal of Giardia cysts and cyst models. Journal American Water WorksAssociation 73(2):111-118.

Mackey, E.D., H.B. Wright, T.M. Hargy, J. Malley, Jr., and R.S. Cushing. 2002. ComparingCryptosporidium and MS2 bioassays – implications for comparing UV reactor validation.Journal American Water Works Association 94(2): 62-69.

Nieminski, E. and J. Ongerth. 1995. Removing Giardia and Cryptosporidium by conventionaltreatment and direct filtration. Journal American Water Works Association 87(9): 96-106.

Ongerth, J. and J.P. Pecoraro. 1995. Removing Cryptosporidium using multimedia filters.Journal American Water Works Association 87(12): 83-89.

Ongerth, J.E. and P.E. Hutton. 1997. Diatomaceous earth filtration for removal ofCryptosporidium oocysts. Journal American Water Works Association 89 (12):39-46.

Ongerth, J.E. and P.E. Hutton. 2001. Testing of diatomaceous earth filtration for removal ofCryptosporidium oocysts. Journal American Water Works Association 93 (12): 53-56.

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Ransome, M.E., T.N. Whitmore, E.G. Carrington, 1993. Effect of disinfectants on theviability of Cryptosporidium parvum oocysts. Water Supply (Amsterdam) 11: 75-89.

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MONITORING OF GIARDIA ANDCRYPTOSPORIDIUM IN WATERIN THE UK AND US

J.L. Clancy1 and P.R. Hunter2

1Clancy Environmental Consultants, Inc., PO Box 314, St. Albans, VT 054782School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ,United Kingdom

ABSTRACTRegulatory agencies in the UK and US approach monitoring water for

Giardia and Cryptosporidium quite differently. Water suppliers in the US arenot required to monitor for Giardia and Cryptosporidium in water, but large-and most medium-sized water utilities monitor regularly. Monitoring is donebecause there are testing methods available, the public expects testing to bedone, and utilities may be vulnerable to litigation if outbreaks occur in theabsence of testing. Consumers, regulators, and elected officials often viewparasite monitoring as a proactive way to protect public health. Routineparasite monitoring provides no public health protection, as it is not sensitiveenough to predict or detect contamination events leading to negative publichealth outcomes. In England and Wales there is a legal requirement for allwater utilities to conduct a risk assessment of each supply. If a particularsupply is deemed to be at risk of contamination by Cryptosporidium oocysts,then continuous monitoring of the supply for oocysts needs to be carried outwhere water is filtered at a rate of not less than 40 L/hr. It is a criminaloffense for a supply to exceed 1 oocyst/10L over 24 hours. The regulationsapply only to treated water systems where an adequate treatment system is inplace. There is no particular requirement for testing for Giardia, though somewater utilities have tested their supplies.Key words: Giardia; Cryptosporidium; water supplies; monitoring; publichealth; regulations

INTRODUCTIONIn this chapter, we review the different approaches used in the United

Kingdom (UK) and United States (US) for monitoring water supplies forCryptosporidium and Giardia. Although there are similarities between theapproaches of the two countries, the UK has developed a more legalisticapproach involving continuous monitoring of supplies considered to be at

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significant risk. We first consider the situation in the US and then turn ourattention to the UK setting.

EARLY HISTORY OF WATER TESTING FOR GIARDIAAND CRYPTOSPORIDIUM IN THE US

Microbiologists have been concerned with monitoring for pathogensin water for over a century. Suckling (1910) noted that the search forpathogenic organisms is “...beset with difficulties and is seldom successful.”He noted low pathogen numbers, large sample volumes, cumbersomeprocedures, and the time required to complete analyses as relevant issues.Nearly 100 years later, in spite of the advances made in microbiologicaltesting, these same concerns remain. This has not, however, dampened thedevelopment of methods for the recovery and detection of Giardia cysts andCryptosporidium oocysts in water, nor impeded widespread testing in theabsence of regulatory requirements.

From 1971 to 1985, 92 outbreaks of waterborne giardiasis occurred inthe US (Craun, 1986). The first detection of Giardia cysts in water was madeduring an outbreak in Rome, NY in 1975 by filtering ~1 million L of Romedrinking water through a swimming pool filter, extracting the sediment, andfeeding it to two beagle pups. The pups developed giardiasis, and a singlecyst was noted microscopically in the sediment. This method of samplecollection was impossible to use routinely, and in 1989, the United StatesEnvironmental Protection Agency (USEPA) developed a portable system forsample collection that consisted of a nominal porosity string wound filter forsample collection (Jakubowski and Hoff, 1979). The processing involvedwashing the filter with distilled water; centrifugation or overnight settling ofthe extracted material; centrifugation of the settled material; furtherconcentration of the sediment by centrifuging in Lugol’s iodine; separation ofthe cysts from the sediment using zinc sulfate flotation; and microscopicexamination. This method was not used routinely, but was used to examinewater supplies suspected of causing a giardiasis outbreak.

Over the next decade the method was greatly improved byintroducing a Percoll-sucrose separation step to replace the zinc sulfate, andincorporating an immunofluorescent monoclonal antibody (mAb) stain forcyst detection. Since there was also a mAb for Cryptosporidium, this wasincorporated into the method and it became a single analysis that couldrecover and detect both parasites (LeChevallier et al., 1990). LeChevallierand colleagues used this new method, nicknamed the IFA method as it usedimmunofluorescence for parasite detection, in national surveys of source andfinished drinking water samples from 66 surface water plants. Giardia and

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Cryptosporidium were found in the raw waters at frequencies of 87% and97%, respectively (LeChevallier et al., 1991a). Giardia was detected in 17%and Cryptosporidium in 27% of the finished water samples (LeChevallier etal., 1991b). From 1988 to 1993, a survey of 347 samples from 72 surfacewater treatment plants showed Giardia present in 54% of the samples with a60% prevalence of Cryptosporidium (LeChevallier et al., 1995). This periodcoincided with a number of outbreaks of waterborne cryptosporidiosis in boththe US and UK, and included the 1993 Milwaukee outbreak affecting over400,000 people.

Although few water utilities had done testing of their supplies forGiardia and Cryptosporidium, the Milwaukee outbreak brought attention tothe possibility of widespread oocyst contamination of source waters. Due tothe high level of water industry interest in determining the concentrations ofGiardia and Cryptosporidium in source and finished water, the IFA methodcame into widespread use. This was the beginning of voluntary nationaltesting for Giardia and Cryptosporidium. The IFA method was used inseveral studies and became the standard method for protozoa analysis,although it had never been collaboratively tested to determine its precisionand bias. Clancy et al (1994) were the first to conduct a collaborative study ofthe IFA method, demonstrating in an evaluation of 16 laboratories that overallperformance was poor. Recoveries were low for both parasites, and falsepositives and negatives were reported commonly. Additional studiesfollowed that confirmed these findings; samples seeded with >9,000 cysts andoocysts were reported as non-detects by expert laboratories (Clancy et al.,1999). The IFA method suffered from poor reproducibility, poor sensitivity,high detection limit (>100 organisms/L), inability to differentiate cysts oroocysts using IFA-based technology, high false positive rate and high falsenegative rate.

Although testing for protozoan parasites is not mandated, Giardia isregulated under the 1986 amendments to the Safe Drinking Water Act, theSurface Water Treatment Rule (SWTR). The USEPA set a maximumcontaminant level of zero for Giardia, but regulates this contaminant throughtreatment technology and not by direct measurement. The SWTR requiresthat surface water treatment plants achieve a 99.9% or 3-log removal andinactivation of Giardia cysts. Conventional filtration receives 2.5-log creditand the additional 0.5-log is achieved through disinfection. Plants meetingthese operational standards are said to be in compliance with the regulation.

THE USEPA INFORMATION COLLECTION RULEIn 1997, the USEPA instituted a monitoring regulation, the

Information Collection Rule (ICR). The ICR required utilities serving greaterthan 100,000 people and using surface water to monitor source waters for

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Giardia cysts and Cryptosporidium oocysts for 18 months. Those with sourcewaters found to be positive for either protozoan at a concentrationL were required to initiate finished water monitoring. In spite of the lack ofrobustness of the IFA method, it was altered slightly and was the specificmethod required by USEPA for this monitoring. The IFA method is nowreferred to as the ICR method (USEPA, 1996).

Analysis of the 18 months of ICR data showed that of 5,829 samplesanalyzed for the protozoa, 93 percent of the samples were non-detects forCryptosporidium and 81 percent were negative for Giardia. When cysts oroocysts were detected, it was generally one or two organisms observed in asmall subsample. These observed numbers were then extrapolated to 100 Lfor source water or 1000 L for treated water. The final result was often a veryhigh reported number based upon this extrapolation. The ICR data fallgenerally into two broad categories – either non-detects or very high reportedlevels based on low analyzed sample volume. For example, from a 100 Lsample, a portion equivalent to ~2.5 L is actually examined microscopicallydue to method limitations. If 1 oocyst is observed, then the reported value is40 oocysts per 100 L, assuming incorrectly that oocysts are evenly distributedin a sample. The non-detect data are equally absurd. Using the sameexample, if no oocysts were detected in the 2.5 L equivalent volume, thecount is reported as <40 oocysts per 100 L. This means the count could be 39or 0 or any number in between. The method is so poor that actual count dataas well as non-detect data are unreliable (Allen et al. 2000). The ICR data arein contrast to the high levels noted in the previous studies by LeChevallier etal (1991a,b; 1995). One possible explanation is that the early versions of theIFA method did not require confirmation of cysts and oocyst, but relied onIFA identification alone, and so overestimates (false positives were likely tobe reported.

USEPA METHODS 1622 and 1623Over the years, several laboratories tried to improve the ICR method,

but these attempts were futile. Shortly after the ICR monitoring began, theUSEPA began working on an improved method for Cryptosporidium analysis.This effort did not focus on improving the ICR method; rather it abandonedthe ICR method altogether and used a new approach. The approach taken indeveloping this new method was to evaluate each step in the method -sampling, processing, and assay - for its ability to permit recovery of spikedoocysts in reagent water. Each individual step was optimized in reagentwater, and after optimization, the steps were combined into a full method thatwas single laboratory validated in two laboratories (Clancy et al., 1999).Important advances in the new method include: 1) use of sampling filters thatpermit 100% capture of oocysts and permit high and consistent oocyst

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recovery (~80%), 2) replacement of buoyant density gradient centrifugationwith immunomagnetic separation (IMS) for significantly improved recoveryof target organisms from sediment, 3) use of well slides in place ofmembranes for sample preparation for microscopy, 4) inclusion of 4’-6’-diamidino-2-phenylindole (DAPI) staining in conjunction with mAb stainingfor enhanced confirmation, and 5) the requirement of observing oocysts at1000 X using epifluorescence, UV, and Nomarski differential interferencecontrast optics for better identification. When a Giardia IMS kit becameavailable, the assay was expanded to include both parasites. These methodshave been improved slightly since their initial development and are nowfinalized as USEPA Method 1622: Cryptosporidium in Water byFiltration/IMS/FA and Method 1623: Giardia and Cryptosporidium in Waterby Filtration/IMS/FA (USEPA, 2001a, b). These methods are available atwww.epa.gov/microbes.

Methods 1622 and 1623 are still characterized by high variability, butare much more sensitive than previous methods. In laboratories with well-trained analysts, recoveries can be 50% or greater routinely with a seed doseof 100 organisms. With the improvements in sampling, separation, andstaining, false positive and false negative rates are lower. These methods aresignificant improvements over the ICR and other methods, and recognizingthis, many countries have adopted them for use as the standard for protozoananalysis of water. Originally developed for 10 L source water samples, themethods have been adapted for higher volume source (50 L) and finishedwater (1000 L) monitoring (McCuin and Clancy, 2003). LeChevallier et al(2003) repeated the earlier studies of source water occurrence and added a cellculture- polymerase chain reaction (CC-PCR) assay to determine infectivityof recovered oocysts. The addition of the infectivity assay allows riskassessment for the first time since water supply monitoring began. Withoutinfectivity, risk assessment was based on IFA staining and microscopy alone,and included measurements of true positives, false positives, and non-viableoocysts. The USEPA plans to use Method 1622 in another temporarymonitoring program slated for 2004 under the Long Term 2 Enhanced SurfaceWater Treatment Rule (LT2). Like Giardia in the SWTR, Cryptosporidiumwill be regulated through treatment technology and not through testing. In theLT2 monitoring scheme, utilities serving greater than 10,000 people will berequired to measure the Cryptosporidium concentration in their source watersin order to next determine the level of treatment needed to protect publichealth. Utilities with higher Cryptosporidium levels will need to provideadditional treatment through removal or inactivation of the parasite. Themonitoring is for the presence of oocysts only, detected using IFA andconfirmed with DIC and DAPI staining. For regulatory purposes, the totalnumber of oocysts will be used to determine occurrence and hence risk; no

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differentiation as to viability or infectious potential of oocysts will bemeasured.

CURRENT US MONITORING PRACTICESAlthough method development has progressed significantly, the

usefulness of routine monitoring data - a sample collected monthly or lessfrequently - is limited. However, this has not stopped most US water utilitiesfrom monitoring their raw and finished drinking water for Giardia andCryptosporidium in the absence of regulatory requirements. While significantimprovements, Methods 1622 and 1623 still have limitations: 1) the samplevolumes are relatively small (10-50 L for source water, up to 1000 L forfinished water), 2) the method does not predict viability of cysts or oocysts, sorisk assessment is not possible, 3) variability is high due to sample matrixeffects, 4) a negative result does not mean cysts or oocysts are not present inthe water, 5) a positive result does not mean the water presents a public healthconcern, and 6) the data are not real-time, often unavailable to the utility fordays or weeks. For these reasons the data cannot be used to make publichealth decisions, but are used to satisfy public expectations and managementdirectives.

THE BACKGROUND TO WATER TESTING FORGIARDIA AND CRYPTOSPORIDIUM IN THE UK

The approach to testing treated waters in the UK differs significantlyfrom that in the US. Before discussing the UK approach, it is important tounderstand the different legal and political environment found in the UK. TheUK legislation on the quality of water intended for consumption is itselfgoverned by European legislation. Until recently this was the Directive80/778/EEC (Anon 1980). A more recent directive is now being enacted intoUK legislation (Anon 1998). Neither of these European directives has setstandards for either Cryptosporidium or Giardia. However, both require thatwater intended for human consumption “is free from any micro-organismsand parasites and from any substance which, in numbers or concentrations,constitute a danger to human health”. The problem was and remains that it isdifficult to agree on a health standard for Cryptosporidium.

What effectively changed water quality legislation in the UK was anoutbreak of cryptosporidiosis that occurred in the South West Region ofEngland in August 1995 (Harrison et al., 2002; Waite and Jiggins, 2002).Some 575 cases were identified as being part of this outbreak. This number isprobably an underestimate as the area affected was a popular touristdestination and increased reporting was noted from many regions in the UKcoincident with this outbreak (Nichols, 2002). Descriptive epidemiology

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linked the outbreak to drinking water from a particular water treatment works.This same treatment works had been associated with another outbreak somethree years earlier and the UK Drinking Water Inspectorate (DWI) consideredthat there were grounds for prosecution for providing water unfit for humanconsumption. The evidence on which the case was brought was the report ofthe Outbreak Control Team (OCT). This prosecution failed over theadmissibility of the epidemiological evidence. In brief the presiding judgeruled that the report of the OCT was not admissible as evidence asepidemiological studies do not allow a complete forensic chain of evidence(Waite and Jiggins, 2002). This left the DWI in the position that it may neverbe able to prosecute water utilities for providing unfit water where the onlyevidence came for epidemiological studies. This was the main driver for thenew legislation, discussed below, for monitoring drinking water supplies forCryptosporidium oocysts. The background for this legislation is discussed inmore detail by Waite and Jiggins (2002).

Unlike the situation for Cryptosporidium there is currently nolegislative pressure for monitoring treated water supplies for Giardia in theUK. In large part this reflects the very different experience with waterbornegiardiasis in the UK compared to the US. The UK has only ever seen oneoutbreak of giardiasis linked to mains drinking water compared to over 20outbreaks of cryptosporidiosis (Hunter, 1997). The one outbreak of giardiasiswas in 1985 and, although a case control study found an association with awater supply, the exact problem was not identified (Jephcott et al., 1986).

UK MONITORING OF CRYPTOSPORIDIUM IN TREATEDWATER UNDER THE REGULATIONS

The regulations for testing water supplies for Cryptosporidium wereoriginally set out in the Water Supply (Water Quality) (Amendment)Regulations (1999). These regulations were then incorporated into the WaterSupply (Water Quality) Regulations (2000). These regulations require waterutilities to undertake a number of steps. The first of these is to conduct a riskassessment.

Under the regulations a water undertaker is required to conduct a riskassessment to determine whether each supply is at significant risk fromCryptosporidium. The report of that risk assessment then has to be submittedto the Secretary of State who will then make a judgment on whether the riskassessment has been adequately carried out. The regulations do not proscribehow the risk assessment is carried out though guidance is available (DrinkingWater Inspectorate, 1999). However, there are certain circumstances thatwould always mean that the water treatment works is at significant risk:

Direct abstraction or with an average storage of seven days orless from a river or stream.

i)

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ii)

iii)

Evidence of rapid river or surface water connection to theaquifer demonstrated by the confirmed presence of faecalcoliform bacteria in the raw water.Past history of an outbreak of cryptosporidiosis associatedwith the water supply where the reason is unexplained and nospecific steps have been taken to prevent a recurrence.

If a water supply is deemed to be at increased risk ofCryptosporidium oocysts, then the water undertaker is required to put in placea water treatment process that will guarantee an oocyst count of <1/10 L andhave monitoring in place to demonstrate that this standard is being met. Itshould be noted that the standard was chosen not because of health criteria,but that it was considered that this standard should be achievable in anyadequately managed water treatment works.

Under these regulations the sampling process is continuous and atleast 40 L/hr needs to be filtered. A gap in monitoring of no more than onehour is allowed while filters are changed. Usually the collection device shouldbe changed at least once a day. The regulations allow three days to analyzeeach filter, though if there is a significant rise in turbidity, or if there are otherreasons to suspect that oocyst counts may have risen, the filter should bechanged and examined as soon as possible.

The analysis has to be carried out in accredited laboratories usingapproved standard operating procedures (SOPs). Laboratories have toparticipate in an external quality assurance scheme. These SOPs cover aspectsof sample collection, transport, storage, analysis and reporting. The SOPs areavailable on the DWI website(http://www.dwi.gov.uk/regs/crypto/mainindex.htm). As the results may beused to support a criminal prosecution, the guidance documents specify aforensic degree of chain of evidence, using tamper proof containers and highlevels of documentation. All results should be reported to the DWI on amonthly basis, though abnormally high results must be reported withoutdelay. These results are also in the public domain and are available toconsumers should they wish to see them.

Abnormally high results should also be reported to the relevantHealth Authority who will then make an appropriate assessment of whetherthis poses a threat to Public Health and take the necessary steps to mitigatethat threat. The UK Public Health Laboratory Service issued advice on theinterpretation of these results when the regulations came into force (Hunter,2000).

Under these regulations, risk assessments were conducted on 1,481treatment works and some 332 (22.4%) (158 treated surface waters and 174treated groundwaters) were deemed to be at significant risk. Many of these at-risk treatment works have subsequently been taken out of service. In the year

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from April 2000, 36,916 samples were taken from 188 treatment works ofwhich 2,755 (7.5%) were positive with one or more oocysts/1000 L and sevenexceeded the standard of <1/10 L. In the following year, 51,168 samples weretaken from 166 sites and only 1,676 (3.3%) were positive with at least oneoocyst. None have exceeded the standard (Information available fromhttp://www.fwr.org/crypnote.htm). As yet there have been no outbreakslinked to drinking water in England and Wales since the regulations wereimplemented.

UK MONITORING OF CRYPTOSPORIDIUM ANDGIARDIA IN TREATED WATER NOT UNDER THEREGULATIONS

The discussion so far has been concerned with analyses under the“Cryptosporidium regulations”. These regulations apply only to England andWales, though both Scotland and Northern Ireland have similar regulations ordirections for their water undertakers.

Even in England and Wales, not all water supplies are covered by thislegislation. For example, some large surface water supplies still remainunfiltered and, as there is no process for removing oocysts, the regulations donot require continuous monitoring. All of these supplies should haveappropriate water treatment plants in place in the near future. In many cases,however, the water undertaker still monitors the supply on a continuous basisas if it were covered by the regulations. This type of monitoring is known asoperational as opposed to regulatory monitoring.

In addition, many water utilities monitor Cryptosporidium andGiardia in raw waters from time to time using 10 L “grab samples”. Grabsamples are sometimes used to assist in the investigation of outbreaks ofwaterborne disease (Howe et al., 2002), in the examination of raw water andoccasional other purposes. Alternate analytical methods for bothCryptosporidium and Giardia are recommended by the UK StandingCommittee of Analysts (1999).

CONCLUSIONSAs has been seen, there are both similarities and differences between

the US and UK approaches. The main difference is the use to which mostresults are put. In the UK, monitoring for Cryptosporidium is largely coveredby legislation and follows risk assessment. In the US, monitoring of oocystlevels is done to inform risk assessment. Much of the technology and methodsare the same and differ largely because of the requirement by the UKauthorities for continuous monitoring. The focus in the US is on source watertesting while the UK prescribes finished water monitoring. A major problem

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remains with oocyst monitoring that reflects the situation with pathogenmonitoring of water in general. Although significantly improved over the lastten years, the methods are still not robust and lack sensitivity and specificity.Information on viability, and hence potential to affect public health, is notknown. Also methods in common use are unable to distinguish between C.parvum and many other species that have little public health significance.

It is important to have sensitive and specific methods forCryptosporidium and Giardia detection in water, but their application is mostappropriate for research studies, watershed evaluations to target point sourcesof fecal contamination, outbreak investigations, etc. Even under thesecircumstances, the limits of the method must be fully understood and the datareported to reflect specificity, sensitivity, and reproducibility of the method.

Allen, M.J., J.L. Clancy, and E.W. Rice. 2000. The plain, hard truth about pathogen monitoring.Journal American Water Works Association 92 (9):94-76.

Anon. 1980. Council Directive 80/778/EEC of 15 July 1980 relating to the quality of waterintended for human consumption. Official Journal of the European Community L229:11-29.

Anon. 1998. Council Directive 98/83/EC of 13 November 1998 relating to the quality of waterintended for human consumption. Official Journal of the European Community L330:32-54.

Clancy, J.L., W.D. Gollnitz, and Z. Tabib. 1994. Commercial labs: how accurate are they?Journal American Water Works Association 86 (9):89-97.

Clancy, J.L., Z. Bukhari, R.M. McCuin, Z. Matheson, and C.R. Fricker. 1999. USEPA Method1622. Journal American Water Works Association 91 (9): 60-68.

Craun, G.F. 1986. Waterborne disease in the United States. CRC Press, Boca Raton, FL.Drinking Water Inspectorate. 1999. Guidance on assessing risk from Cryptosporidium oocysts

intreated water supplies to satisfy the Water Supply (Water Quality) (Amendment)Regulations. 1999. SI 1524. Department of the Environment, Transport and the Regions,London.

Harrison, S.L., R. Nelder, L.Hayek, I.F. Mackenzie, D.P. Casemore and D. Dance. 2002.Managing a large outbreak of cryptosporidiosis: how to investigate and when to decide to lifta ‘boil water’ notice. Communicable Disease and Public Health 5: 230-239.

Howe, A.D., S. Forster, S. Morton, R. Marshall, K. Osborn, Wright, P. and Hunter, P.R. 2002.Cryptosporidium oocysts in a water supply associated with an outbreak of cryptosporidiosis.Emerging Infectious Diseases 8: 619-624.

Hunter, P.R. 1997. Waterborne Disease: Epidemiology and Ecology. John Wiley, Chichester,UK.

Hunter, P.R. 2000. Advice on the response to reports from public and environmental health tothe detection of cryptosporidial oocysts in treated drinking water Communicable Disease andPublic Health 3:24-27.

Jakubowski, W. and T.H. Erickson. 1979. Methods for detection of Giardia cysts in watersupplies. In Waterborne transmission of giardiasis, W. Jakubowski and J.C. Hoff (eds.). USEnvironmental Protection Agency, 600/9-79-001, Cincinnati, OH.

Jephcott, A.E., N.T. Begg and I.A. Baker. 1986. Outbreak of giardiasis associated with mainswater in the united kingdom. Lancet i: 730-732.

LeChevallier, M.W., T.M. Trok, M.O. Burns, and R.G. Lee. 1990. Comparison of the zincsulfate and immunofluorescence techniques for detecting Giardia and Cryptosporidium.Journal American Water Works Association 82 (9):75.

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LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991a. Occurrence of Giardia andCryptosporidium spp. in surface water supplies. Applied and Environmental Microbiology57:2610-2616.

LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991b. Giardia and Cryptosporidium spp. infiltered drinking water supplies. Applied and Environmental Microbiology 57:2617-2621.

LeChevallier, M. W. and W.D. Norton. 1995. Giardia and Cryptosporidium in raw and finishedwater. Journal American Water Works Association 87 (9): 54-68.

LeChevallier, M.W., G.D. DiGiovanni, J.L. Clancy, Z. Bukhari, S. Bukhari, J.S. Rosen, J.Sobrinho, and M.M. Frey. 2003. Comparison of Method 1623 and cell culture-PCR fordetection of Cryptosporidium in source waters. Applied and Environmental Microbiology 69:971-979.

McCuin, R.M. and J.L. Clancy. 2003. Modifications to USEPA methods 1622 and 1623 fordetection of Cryptosporidium oocysts and Giardia cysts in water. Applied and EnvironmentalMicrobiology 69:267-274.

Nichols, G. 2002. Using existing surveillance-based data. In: Drinking Water and InfectiousDisease: Establishing the Links. CRC Press, Boca Raton, FL., 131 -141.

Standing Committee of Analysts. 1999. Isolation and Identification of Cryptosporidium oocystsand Giardia cysts in waters: SCA Blue Book No 172. Environment Agency London.(http://www.environment-agency.gov.uk).

Suckling, E.V. 1910. The Examination of Waters and Water Supplies. ed. The BlakistonCo, Philadelphia.

USEPA. 1996. Information Collection Rule. United States Environmental Protection Agency.Office of Research and Development, Washington, DC. ICR Microbial Laboratory Manual.EPA/600/R-95/178.

USEPA. 2001a. USEPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U. S.Environmental Protection Agency. Office of Water, Washington, DC. EPA 821-R-01-026.

USEPA. 2001b. USEPA Method 1623: Cryptosporidium and Giardia in Water byFiltration/IMS/FA. U.S. Environmental Protection Agency. Office of Water, Washington,DC. EPA 821-R-01-025.

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ENTAMOEBA HISTOLYTICA GENOME

ABSTRACTE. histolytica is a human parasite that is a significant cause of worldwide

morbidity and mortality. This parasite is also of interest because it is an earlybranching amitochondriate eukaryote. The genome project should be completedby the end of 2003, however, the current release and preliminary annotation hasalready confirmed some previous observations and revealed new interestingaspects of the genome. The genome size is estimated to be 20 Mb with anoverall AT content of 78%. The exact number of chromosomes is not resolved.Estimates range from 6 to 14. The genome includes numerousextrachromosomal circular elements including an episome with the rDNA genes.The chromosomal genome also contains a variety of other repeated elementsincluding degenerate transposon-like elements. The promoter elements of E.histolytica have some unique features including a novel "GAAC" element.Introns are estimated to occur in 15% of protein encoding genes. A limitednumber of genomic sequences of other Entamoeba species have also beenreleased. A comparison of these genomes, along with the nonpathogenic"cousin" of E. histolytica should help to discern the basis of virulence and thephylogenetic relationship of these and other species.Key words: Entamoeba histolytica, E. dispar, E. terrapinae, E. invadens, E.moshovskii, genome, rDNA, episomes

J.J. McCoy1 and B.J. Mann1,2

1Departments of Internal Medicine and 2Microbiology, University of Virginia School ofMedicine

INTRODUCTIONThe human intestinal protozoan parasite E. histolytica is estimated to

infect 50 million people and result in approximately 100,000 deaths per yearfrom ulcerative colitis and amebic liver abscesses (WHO, 1995). Infection withE. histolytica, or amebiasis, is found worldwide, but is most common indeveloping countries. In most cases, E. histolytica infections are symptomless,but approximately 10% of infected individuals develop colitis (Reed, 2000).Most cases are seen in the very young and the very old, pregnant women,corticosteroid-treated individuals, and the malnourished (Seeto et al., 1999).Human to human transmission of E. histolytica occurs by a fecal-oral routebeginning with the ingestion of the tetra-nucleated cyst in contaminated food or

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water. The trophozoite form colonizes the bowel lumen and can invade throughintestinal epithelium to cause colitis, liver abscesses, or form cysts that areexcreted to begin a new round of infection (Clark et al., 2000).

GENOME PROJECTThere are two E. histolytica Genome Projects. One, funded by the

National Institutes of Health, is at the Institute for Genomic Research (TIGR)(www.tigr.org/tdb/e2k1/eha1/) and the other is at the Wellcome Trust SangerInstitute Pathogen Sequencing unit (www.sanger.ac.uk/Projects/E_histolytica/).In October 2002 sequence information from the two projects was merged tocreate a 7-fold coverage of the genome. Automated annotation for the E.histolytica assemblies of >10 kb, representing approximately 12 Mb of uniquesequence coverage and a majority of the E. histolytica coding regions, becameavailable in January 2003. A database of the upstream and downstreamsequences between predicted coding regions from the automated annotation ofthe 7X assembly is also available on the TIGR website for searching anddownload.

A comparative Entamoeba genome project is also underway at theWellcome Trust Sanger Institute in collaboration with Graham Clark, LondonSchool of Tropical Medicine and Hygiene. Sequences of E. terrapinae, aparasite of turtles, E. moshovskii, a free-living organism, and E. invadens, areptilian parasite, are available on the Sanger site. The eventual plan is tosequence 21,000 shotgun clones of each parasite. Entamoeba invadens has beenused a model of encyst- and excystment because its lifecycle can be completed inculture, and E. histolytica does not readily form cysts in culture (Eichinger 2001).A dataset of 16,000 Entamoeba invadens sequence reads is also available at theTIGR site.

The E. histolytica genome is ~78 % AT rich overall and highlyrepetitive. These features of the genome have made assembly difficult (BrendanLoftus, TIGR, Neil Hall, personal communication). It is likely that the genomewill not be completely closed, although investigators in the field have discussedclosing at least one chromosome (Mann, 2002).

CHROMOSOMESThe chromatin organization, karyotype and ploidy of E. histolytica have

been difficult to determine and somewhat controversial. Analysis of chromatinspreads by electron microscopy revealed nucleosome-like structures (Torres-Guerrero et al., 1991). The chromatin contains basic DNA-binding proteins thatare different from other known eukaryotic histones (Torres-Guerrero et al.,1991), however genes encoding homologs to histones H1 (Scharfetter et al.,

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1997), H2B (Sanchez et al., 1994), H3 (Fodinger et al., 1993), and H4 (Binder etal., 1995) have been identified.

The failure of the E. histolytica genome to condense at metaphase hashindered karyotypic characterization, however light microscopy studies estimate6 chromosomes in E. histolytica nuclei (Arguello et al., 1992; Gomez-Conde etal., 1998) while electron microscopy suggests 9-12 chromosome-like structures(Torres-Guerrero et al., 1991). The pulsed-field gel electrophoresis technique

GENE ORGANIZATIONBased on published sequence, intergenic regions appear to be relatively

short, ranging in size from 400 bp-2.3 kb (Bruchhaus et al., 1993; Petter et al.,

size from 0.3 to 2.2 Mb. These authors also identified 14 linkage groups using68 independent cDNA probes. Although size polymorphisms were identified,linkage groups were conserved between the different isolates. Several probesbound to as many as four different bands, suggesting a ploidy of 4n (Figure 1).By adding the sizes of the largest chromosomes from each of the 14 linkagegroups, the haploid genome size was estimated to be approximately 20 Mb.

has not unambiguouslyresolved E. histolyticachromosomes. This maypossibly be due toabundant endogenousamebic nucleases, and themolecular mixture oflinear and circularmolecules, whichgenerates broad bands(Orozco et al., 1993;Petter et al., 1993;Riveron et al., 2000).Willhoeft and Tannichused rotating-fieldelectrophoresis (ROFE) toestablish electrophoretickaryotypes for threedifferent E. histolyticaisolates, HM1-IMSS,200:NIH, and HK-9(Willhoeft et al., 1999b).Using this technique, 31-35 chromosomes wereidentified that ranged in

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1992; Willhoeft et al., 1999a). One pair of genes, pak and mcm3, haveoverlapping transcripts, with the 3’ untranslated region (UTR) of pac sharing 40nucleotides with the 5’ UTR of mcm3 (Gangopadhyay et al., 1997). Initiallyintrons were found in only a few of the early reported gene sequences (Lohia etal., 1993; Plaimauer et al., 1994; Sanchez-Lopez et al., 1998; Urban et al., 1996;Willhoeft et al., 1999a) suggesting a paucity of introns in E. histolytica.However, a more thorough and recent study identified an additional nine intron-containing genes in E. histolytica and the closely-related species Entamoebadispar (Wilihoeft et al., 2001). The identified introns contain between 46 and115 nucleotides, and have an average AT content of 83%, higher than the 73%found in the corresponding coding sequences and 78% in the E. histolyticagenome overall. Conserved motifs were identified at the 5’ (GTTTGT) and 3’(TAG) ends of the introns, corresponding to the GT and AG intron splice signalsof most eukaryotic genes. Evidence for functional splicing machinery in E.histolytica was the identification of a U6 small nuclear RNA gene (Miranda etal., 1996). The U6 gene was expressed and present as a single copy in the E.histolytica genome. An analysis of an estimated 2-fold coverage of the E.histolytica genome found introns in 15% of genes with homologies to knownproteins, an unexpectedly high percentage based on previously reported intron-containing genes (Loftus, unpublished, Mann, 2002). The final analysis of theentire genome should eventually give the precise percentage.

The sequences of 5’ and 3’ UTRs of protein-encoding genes in E.histolytica have been analyzed for their influence on gene expression (Bruchhauset al., 1994; Gilchrist et al., 1997; Ortiz et al., 2000; Purdy et al., 1996; Hidalgoet al, 1997; Schaenman et al., 1998). Conserved 5’ UTR sequences that havebeen identified include an unusual TATA-like site 30 nucleotides upstream of thetranscription start site, an initiator element at the transcription start site, and anovel “GAAC” element, located at variable distances between the TATA-likesite and the transcription start site (Singh et al. 1997). The 3’ UTRs contain aconserved pentanucleotide TAA/TTT, which acts as the transcription terminationsignal and a polypyrimidine region at the end of transcribed sequences(Bruchhaus et al., 1993; Singh et al., 1997). It has been proposed that thesetranscribed sequences determine mRNA secondary structure, which in turn,contributes to the binding of proteins that regulate gene expression (Ortiz-Garciaet al., 1997).

The gene encoding a homolog of the 54 KDa subunit of the signalrecognition particle (SRP54) gene provided evidence that E. histolytica capsmRNAs in a manner similar to other eukaryotes (Ramos et al., 1997). Cappingof the SRP54 transcript was supported by the identification of an extra G residueat the cDNA transcription start site that was not present in the genomic sequence.

Another unusual characteristic of E. histolytica is that transcription ofprotein-coding genes is (Lioutas et al., 1995b), suggesting

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either that E. histolytica RNA polymerase II is different from other eukaryoticRNA polymerases II, or that in E. histolytica, protein-encoding genes may betranscribed by RNA polymerase I, such as is the case for the PARP and VSGgenes in trypanosomes.

EPISOMESEarly attempts to resolve individual E. histolytica chromosomes by

PFGE revealed a smear of fuzzy bands and several distinct high molecularweight bands. It was proposed that at least some of these high molecular weightbands represented circular DNA. Electron microscopy verified the presence ofcircular episomes of 4-50 kb in E. histolytica strain HM-1:IMSS (Dhar et al.,1995; Lioutas et al., 1995a).

The most abundant and best-characterized episome is the 24.5 kb rDNA(EhR1) molecule that is present in about 200 copies per haploid genomeequivalent. The EhR1 molecules account for over 80% of E. histolytica circularDNAs and 5-10% of total cellular DNA. Each EhR1 molecule in strain HM-1:IMSS contains two 5.2 kb inverted repeats encoding ribosomal RNA(Bhattacharya et al., 1989). A 3.7 kb downstream spacer and a 9.2 kb upstreamspacer separate the two units (Sehgal et al., 1994). The rRNA genes are believedto be present exclusively on the EhR1 plasmid, because no chromosomal copiesof ribosomal genes have been identified. No proteins appear to be encoded onEhR1, although a single polyadenylated 0.7 kb RNA was identified by Northernblots. In contrast to naturally occurring plasmids in most prokaryotes andeukaryotes, two-dimensional gel electrophoresis and electron microscopic studiesidentified multiple replication sites on the EhR1 episome (Dhar et al., 1996).

The smaller episomes do not appear be derivatives of the rDNA plasmidbecause EcoR1 fragments of the rDNA plasmid do not hybridize with theseepisomes on Southern blots (Dhar et al., 1995). The DNA sequences of thesesmaller episomes have not been determined so their coding potential andfunctions are unknown.

The genome of E. histolytica is highly repetitive and contains severaltypes of repetitive DNA. The most abundant repeated DNA is found on theribosomal plasmid EhR1. Two families, the 170 bp DraI repeats and the 144 bpScaI repeats, comprise the 3.7 kb downstream intergenic spacer, while the 9.2 kbupstream spacer contains ScaI, 145 bp PvuI, and HinfI repeats (Bhattacharya etal., 1998).

Long interspersed elements (LINEs) and short interspersed elements(SINEs) are autonomous and non-autonomous transposable elements,respectively. By analyzing sequences generated by the E. histolytica genome

REPEATED SEQUENCES

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project, Van Dellen et al described three families of LINEs, named EhLINEs,which include the previously reported EhRLE (Sharma et al., 2001) and HMc(Mittal et al., 1994) repeated sequences, and two families of SINE-like sequencesnamed EhSINEs (Van Dellen et al., 2002). One family, EhLINE1, shared acommon 3’ end with IE, a previously reported highly transcribed 0.55 kbrepetitive element (Cruz-Reyes et al., 1995). Individual EhLINE/EhRLEelements do not contain any open reading frames but a comparison of a numberof elements suggests that they contain degenerate nucleic acid-binding motifs,restriction enzyme-like endonuclease domains, and reverse transcriptase domains(Sharma et al., 2001; Van Dellen). They also share 3’ end sequences withEhSINEs. It is estimated that LINEs and SINEs comprise 6% of E. histolyticaDNA, second only to rDNA episomes in their abundance in the genome.

Zaki and Clark isolated two other novel multicopy loci, designated locus1-2 and locus 5-6, that contain internal repeats from E. histolytica, in an effort toisolate microsatellites (Zaki et al., 2001). While microsatellites were not found,these loci may have the potential to be used as polymorphic molecular markers toinvestigate E. histolytica epidemiology and intraspecies variations. Examinationof sequences from the E. histolytica Genome project has identified many othergroups of previously unidentified repetitive DNA (Van Dellen et al., 2002). Thefunctions of these repetitive sequences are not yet known.

EVOLUTIONARY POSITIONIn the colonic lumen E. histolytica is an obligate fermentor, lacking

proteins of the mitochondrial electron transport chain and tricarboxylic acidcycle. Organelles of higher eukaryotes, such as mitochondria, roughendoplasmic reticulum, Golgi apparatus, centrioles, and microtubules, have notbeen identified by electron microscopy (Clark et al., 2000). Because of thisapparent lack of higher eukaryotic machinery, combined with the presence ofbacterial-like fermentation enzymes, early evolutionary theories considered E.histolytica to be a transitional eukaryote that diverged from other eukaryotesprior to the acquisition of mitochondria and other organelles. (For a morecomplete review, see Samuelson, 2002). However, evidence supportingsecondary loss of mitochondria has been presented and may explain the apparentrelative simplicity of E. histolytica compared to higher eukaryotes. The E.histolytica genome contains at least three genes, pyridine nucleotidetranshydrogenase (PNT), the mitochondrial chaperonin cpn60, and amitochondrial-type hsp70, normally found on the mitochondrial genome(Bakatselou et al., 2000; Clark et al., 1995). The identification of these genes inE. histolytica supports the secondary loss hypothesis. The loss may have resultedfrom adaptation of the parasite to the gut (Boore et al., 1999; Clark et al., 2000).In addition, the identification of a mitochondrion-derived organelle, (the cryptonor mitosome) (Mai et al., 1999; Tovar et al., 1999) and Golgi (Ghosh et al.,

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1999), along with genes of higher eukaryotes such as ras-family signaltransduction proteins (Lohia et al., 1994; Lohia et al., 1996), ABC-family drugtransporters (Descoteaux et al., 1995), and introns (Wilihoeft et al., 2001),suggest a common eukaryotic ancestor.

BACTERIAL-LIKE GENES IN E. histolyticaThree hypotheses have been proposed that might explain the presence of

bacterial-like anaerobic fermentation enzymes in E. histolytica and otherprotozoans. The amitochondriate fossil hypothesis suggests that genes encodingfermentation enzymes were present prior to the acquisition of mitochondrion, andthen lost in most eukaryotes (Reeves, 1984). The hydrogen hypothesis suggeststhat fermentation genes were acquired simultaneously with the mitochondrialendosymbiont (Martin et al., 1998; Rotte et al., 2000). The lateral transferhypothesis proposes that the fermentation genes were directly acquired fromanaerobic prokaryotes (de Koning et al., 2000; Doolittle, 1999; Field et al., 2000;Rosenthal et al., 1997). Phylogenetic evidence supports the lateral transferhypothesis in that 1) the sequences of several E. histolytica fermentation genesare more similar to those of bacteria than to those of phylogenetically similarprotozoan parasites, 2) E. histolytica fermentation enzyme gene homologs areabsent from most higher eukaryotes, and 3) E. histolytica possesses several otherbacterial genes besides those involved in anaerobic fermentation (Rosenthal etal., 1997). Molecular techniques have made it possible to test evolutionaryhypotheses based on phenotypic traits. Completion of the E. histolytica genomeproject is certain to lead to a better understanding of the parasite’s evolution andphylogenetic relationship with other protests.

Entamoeba histolytica vs. Entamoeba disparE. dispar is closely related to E. histolytica, both morphologically and

genetically, yet it has never been associated with disease (Diamond, 1993). Thegenomes of these two organisms are highly similar and apparently syntenic(Willhoeft, 1999a). Homologs for several of the better-studied E. histolyticavirulence factors are also found in E. dispar, but some differences exist. E.dispar contains at least two genes encoding the galactose/N-acetyl D-galactosamine (Gal/GalNAc)-inhibitable lectin heavy subunit and four genesencoding the lectin light subunit (Dodson, 1997). The Gal/GalNAc lectin is themajor surface adhesin of the parasite. Lectin heavy and light subunit homologsshowed 86% and 79% amino acid identity between the two species. Homologsfor all three isoforms (A,B,C) of the amebic pore-forming protein, amoebaporeare also present in E. dispar (Leippe et al., 1993). Analyses of amebic cysteineproteinases found that two of the most highly expressed and active cysteineproteinase genes (ehcp1 and ehcp5), which account for over 70% of cysteineproteinase activity in E. histolytica, are not expressed in E. dispar (Bruchhaus et

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al., 1996; Jacobs et al., 1998). A pseudogene corresponding to ehcp5 is presentand positionally conserved in E. dispar (Willhoeft et al., 1999a). A directcomparison of these two genomes, along with microarray analysis should help touncover differences in homologous genes and gene expression.

CONCLUSIONThe E. histolytica genome project along with genomic sequence from

other Entamoeba species should provide the tools to identify key virulencefactors and the mechanisms pathogenicity. This should lead to betterunderstanding of host-parasite relationships, and potentially new drugs andvaccines. The genome sequence should also help to establish phylogeneticrelationships between early branching eukaryotes and a greater understanding ofthe evolution of eukaryotic organisms.

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Boore, J. L., and S. I. Fuerstenberg. 1999. Entamoeba histolytica: a derived, mitochondriateeukaryote? Trends in Microbiology 7:426-428.

Bruchhaus, I., T. Jacobs, M. Leippe, and E. Tannich. 1996. Entamoeba histolytica andEntamoeba dispar: differences in numbers and expression of cysteine proteinase genes.Molecular Microbiology 22:255-263.

Bruchhaus, I., M. Leippe, C. Lioutas, and E. Tannich. 1993. Unusual gene organization in theprotozoan parasite Entamoeba histolytica. DNA Cell Biology 12:925-933.

Bruchhaus, I., and E. Tannich. 1994. Induction of the iron-containing superoxide dismutase inEntamoeba histolytica by a superoxide anion-generating system or by iron chelation.Molecular and Biochemical Parasitology 67:281-288.

Clark, C. G., M. Espinosa Cantellano, and A. Bhattacharya (2000). Entamoeba histolytica: AnOverview of the Biology of the Organism. In Amebiasis, J. I. Ravdin, ed. (London, ImperialCollege Press), pp. 1-45.

Clark, C. G., and A. J. Roger. 1995. Direct evidence for secondary loss of mitochondria inEntamoeba histolytica. Proceedings of the National Academy of Sciences U S A 92:6518-6521.

Cruz-Reyes, J., T. ur-Rehman, W. M. Spice, and J. P. Ackers. 1995. A novel transcribed repeatelement from Entamoeba histolytica. Gene 166:183-184.

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de Koning, A. P., F. S. Brinkman, S. J. Jones, and P. J. Keeling. 2000. Lateral gene transfer andmetabolic adaptation in the human parasite Trichomonas vaginalis. Molecular Biology andEvolution 17:1769-1773.

Descoteaux, S., P. Ayala, J. Samuelson, and E. Orozco. 1995. Increase in mRNA of multipleEh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoebahistolytica parasites. Gene 164:179-184.

Dhar, S. K., N. R. Choudhury, A. Bhattacharaya, and S. Bhattacharya. 1995. A multitude ofcircular DNAs exist in the nucleus of Entamoeba histolytica. Molecular and BiochemicalParasitology 70:203-206.

Dhar, S. K., N. R. Choudhury, V. Mittal, A. Bhattacharya, and S. Bhattacharya. 1996.Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of theprotozoan parasite Entamoeba histolytica. Molecular Cell Biology 16:2314-2324.

Diamond, L. S., and C. G. Clark. 1993. A redescription of Entamoeba histolytica Schaudinn,1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. Journalof Eukaryotic Microbiology 40:340-344.

Dodson, J. M., Clark, C. G., Lockhart, L. A., Leo, B. M., Schroeder, J, W., Mann, B. J. 1997.Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoebahistolytica and Entamoeba dispar. Parasitology International 46:225-235.

Doolittle, W. F. 1999. Phylogenetic classification and the universal tree. Science 284:2124-2129.

Eichinger, D. 2001. A role for a galactose lectin and its ligands during encystment ofEntamoeba. Journal of Eukaryotic Microbiology 48:17-21.

Field, J., B. Rosenthal, and J. Samuelson. 2000. Early lateral transfer of genes encoding malicenzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes toEntamoeba histolytica. Molecular Microbiology 38:446-455.

Fodinger, M., S. Ortner, B. Plaimauer, G. Wiedermann, O. Scheiner, and M. Duchene. 1993.Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primarystructure. Molecular and Biochemical Parasitology 59:315-322.

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CRYPTOSPRORIDIUM PARVUM GENOMICS:IMPACT ON RESEARCH AND CONTROL

G. Zhu1 and M. S. Abrahamsen2

1Department of Veterinary Pathobiology, Texas A & M University, College Station, TX2Department of Veterinary Pathobiology, University of Minnesota, St. Paul, MN

ABSTRACTCryptosporidium parvum is a well-recognized cause of diarrhea in

humans and animals throughout the world, and is associated with a substantialdegree of morbidity and mortality in patients with acquired immunodeficiencysyndrome (AIDS). Despite intensive efforts over the past 20 years, there iscurrently no effective therapy for treating or preventing infection by C.parvum. Until recently, the development of effective anticryptosporidialtherapies has been hindered by the paucity of biological targets for structure-based drug design. With the pending completion of sequencing of the entireC. parvum genome, there should be no lack of potential biological targets fordevelopment of specific inhibitors. What are needed are focused efforts toidentify essential parasite biochemical pathways for which specific inhibitorscan be developed that are safe to the host. Analysis of the currently availablesequences has identified specific aspects of C. parvum biochemistry that areunique relative to mammals. In addition, the ongoing genome efforts areproviding a clearer picture of the biology of the apicomplexan parasites. It isbecoming evident that C. parvum uses distinct approaches to solve basicmetabolic needs as compared to Plasmodium falciparum and Toxoplasmagondii. These differences suggest that therapies developed for these modelapicomplexans may not necessarily be effective against cryptosporidiosis, andemphasizes the importance of understanding the unique biology of C. parvum.Key Words: Genomics, DNA replication, fatty acid biosynthesis, glycolysis,purine salvage, metabolism

INTRODUCTIONThe life cycle of C. parvum is similar to that of other apicomplexans,

and is composed of multiple asexual and sexual developmental stages (Fayeret al., 1997). Currently, it is not known precisely how the genome contentchanges in the different developmental stages, nor are the basic mechanisms

DNA REPLICATION PROTEINSThe life cycles of all apicomplexans consist of at least 3 distinct

developmental processes: sporogony, merogony, and gamogony. Bothsporogony and merogony are cell multiplication processes that produce morethan 2 daughter cells in each cell cycle and differ from the host cell somaticduplication, which suggests a unique mechanism may be involved in the DNAreplication in apicomplexan parasites. However, little was known about themolecules that are involved in the process and regulation of the apicomplexanDNA replication. Recently, the rapid development in molecular biology andgenomics has provided us opportunities in the discovery and characterizationof DNA replication proteins in C. parvum and other apicomplexans.

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of DNA replication or translocation during development understood. It isevident, however, that for C. parvum to complete its life cycle, it mustfaithfully replicate its genome a significant number of times and thus musthave a high metabolic demand for nucleotides throughout its development.Further, as an obligate intracellular organism C. parvum must have uniquemeans of generating energy and acquiring or synthesizing the basic cellularcomponents required to accomplish this rapid cell multiplication andextensive remodeling of the parasite’s cell structure during completion of itslife cycle.

A major limitation to understanding the basic biology of C. parvum isthe inability to obtain purified samples of the various asexual, sexual, andintracellular developmental stages of the parasite. As a consequence, fewmolecular or biochemical studies have been directed toward understanding themechanisms associated with the pathogenesis of C. parvum or thecomplicated developmental biology during its intracellular development. Toaddress this limitation, several large-scale sequencing projects have beeninitiated (Liu et al., 1999; Strong and Nelson, 2000), including a project tosequence the complete C. parvum genome (CpGP) that is nearing completion(Abrahamsen, 1999; Widmer et al., 2002). These efforts have dramaticallyincreased our understanding of this important pathogen and havedemonstrated that C. parvum differs from the related apicomplexan pathogensToxoplasma gondii and Plasmodium falciparum in several basic biologicalprocesses. The molecular divergence of C. parvum from other apicomplexansis also congruent with the phylogenetic position of the Cryptosporidium genusas an early emerging branch within the Apicomplexa (Zhu et al., 2000a), orfurthermore, as a sister to the Class Gregarina (Carreno et al., 1999). In thischapter, we will discuss key insights into DNA replication, purinemetabolism, fatty acid biosynthesis and energy metabolism that are unique toC. parvum relative to its mammalian host that may ultimately provide newbiological targets for development of specific inhibitors.

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One important class of DNA replication molecules is the replicationprotein A (RPA) that belongs to the eukaryotic single stranded DNA (ssDNA)binding proteins involved in the DNA replication, repair and recombination(Wold, 1997; Iftode et al., 1999). In animals, plants and fungi, RPA is aheterotrimeric protein of approximately 70, 32 and 14 kDa subunits. The largesubunit (RPA1) consists of an N-terminal domain for interacting with variousfactors involved in the regulation of DNA replication, repair orrecombination, a middle section with two ssDNA-binding sites, and a C-terminal region for binding the other two subunits. We first discovered theCpRPA1 gene from the parasite genome by sequencing the downstreamregion near the CpFAS1 locus. Surprisingly CpRPA1 encodes a short-type54-kDa RPA1 subunit that differs from its host and lacks the N-terminalprotein-interacting domain that is present in animal, plant and fungal RPA1s,indicating that C. parvum may utilize a distinct pathway for regulatory factorsto interact with RPA during DNA metabolism (Zhu et al., 1999).

Interestingly, from the ongoing CpGP we have identified a secondRPA1 gene (CpRPA1B) that differs significantly not only from its host, butalso from CpRPA1 (Millership and Zhu, 2002). Although CpRPA1B appearsto encode a “full-size” RPA1 (i.e., its ORF predicts a 75.5 kDa protein),western blotting analysis detects only a ~45 kDa band, indicating that eitherCpRPA1B is translated at an alternative start codon, or the protein is cleavedfollowing translation. Therefore, both RPA1 proteins in C. parvum belong tothe short-type subunits. Short-type RPA1 subunits have also been identifiedfrom the genomes of P. falciparum (PfRPA1) and T. gondii (TgRPA1). LikeCpRPAlB, the native PfRPAl protein is short (54 kDa), although its ORFpredicts a 134-kDa polypeptide (Voss et al., 2002). In contrast, TgRPA1 isapparently encoded by a short ORF (data not shown). In addition, theCpRPA1 and CpRPA1B proteins are differentially distributed in C. parvumsporozoites and during its life cycle, suggesting different roles for these twoproteins in the parasite DNA metabolism.

In addition, a homologue to the middle subunit (CpRPA2) wasrecently identified from the CpGP. Despite the low homology among allRPA2 subunits, we were able to confirm that CpRPA2 protein was indeed anssDNA-binding protein and could be regulated by phosphorylation. Thephosphorylation of CpRPA2 completely abolished the ssDNA-bindingproperty. Similar to other DNA replication proteins, the expression ofCpRPA2 gene is regulated by the cell cycle and its protein is distributed moreintensively in the C. parvum oocysts, meronts and gamonts than other lifecycle stages (Millership and Zhu, unpublished data).

RPA proteins are essential to all eukaryotes and several anticancercompounds have been shown to target these proteins (Basilion et al., 1999;Peters et al., 2001). As apicomplexan RPA proteins differ from their hosts in

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both structure and function, data-mining of the CpGP and functional analysisto identify other elements in the RPA-associated DNA metabolic pathwaycould provide us new opportunities to study the process and regulation of theunique parasite life/cell cycles, and to explore this distinct pathway as a noveldrug target against apicomplexans

PURINE METABLOISM/SALVAGEThe enzymes of the nucleotide biosynthesis pathway are essential for

supporting cell proliferation. In mammals, nucleotides may be synthesizedthrough one of two pathways. In the de novo synthesis pathway, the purinering system is assembled in a step-wise manner. In the salvage pathway,preformed nucleotides, nucleosides and nucleobases are recycled. The extentof utilization of each pathway in mammals is dependent on the cell type andgrowth state of the cell. In contrast to their mammalian hosts, all parasiticprotozoa examined to date are incapable of synthesizing the purine ring denovo, but depend on preformed nucleotides that they purportedly obtain fromthe host using specific salvage pathways (Berens et al., 1995). Theapicomplexans P. falciparum and T. gondii share similar purine salvagepathways with other human protozoan pathogens including Entamoebahistolytica and Giardia lamblia. In the case of P. falciparum and T. gondii ithas been demonstrated that both hypoxanthine–guanine–xanthine phospho-ribosyltransferase (HXGPRT) and adenosine kinase are key enzymes forpurine salvage and that disruption of this pathway is inhibitory to parasitegrowth (Shi et al., 1999; Pfefferkorn et al., 2001; Kicska et al., 2002).

To date, few studies have investigated purine metabolism inCryptosporidium. A recent study presented biochemical evidence that C.parvum lacks the ability for de novo purine nucleotide synthesis (Doyle et al.,1998). The authors hypothesized that similar to Toxoplasma, C. parvum mayhave a single HXGPRT enzyme involved in purine salvage. Recently, weattempted to clone the putative C. parvum HXGPRT gene by geneticcomplementation in T. gondii (Striepen et al., 2002). This system takesadvantage of a T. gondii line in which the HXGPRT gene hasbeen inactivated (Donald et al., 1996). Without HXGPRT activity, parasitesurvival is dependent on inosine monophosphate dehydrogenase (IMPDH) toconvert IMP to XMP to supply GMP for DNA synthesis. In the presence ofmycophenolic acid (MPA), a potent inhibitor of IMPDH, only T. gondiitachyzoites that have been transformed with a vector containing a functionalHXGPRT gene are viable. To isolate the putative C. parvum HXGPRT gene,randomly sheared C. parvum genomic DNA was used to construct acomplementation library in an empty T. gondii expression vector. tachyzoites were transfected with this heterologous library andcultured in the presence of MPA. Following selection, drug resistant parasites

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emerged, however, the conceptual translation of the ORF contained in therecovered C. parvum genomic sequence revealed clear similarity to IMPDH,not HXGPRT. Surprisingly, the similarity of the isolated CpIMPDH gene wasmuch greater to prokaryotic than to eukaryotic IMPDH. All phylogeneticmethods support the specific grouping of C. parvum IMPDH with the

homologs (Striepen et al., 2002). CpIMPDH was nevergrouped with other eukaryotic IMPDHs including the IMPDH from theapicomplexan P. falciparum. Our findings indicate that CpIMPDH is not ofeukaryotic nuclear descent or even mitochondrial origin (since it is notspecifically related to the but instead was likely obtained bylateral gene transfer (LGT) from a Since bacterial IMPDHenzymes are much less sensitive to MPA, the relationship ofCpIMPDH also clarifies its insensitivity to MPA used in selective medium.

Moreover, subsequent experimental approaches and extensivesearching of the available C. parvum genomic and EST databases using T.gondii and P. falciparum HXGPRT failed to identify any C. parvumhomologs, strongly suggesting that C. parvum lacks this enzyme. In contrast,in silico analysis identified putative C. parvum homologs for all of theenzymes necessary for the salvage and conversion of adenosine to GMP: a 5’nucleotidase, an adenosine transporter, adenosine kinase, AMP deaminase,IMPDH and GMP synthase. These findings indicate that C. parvum purinesalvage is distinct from other apicomplexans and the only source of purinesfor C. parvum is dependent on the adenosine salvage pathway. Therefore, itwould be predicted that all of the enzymes within the adenosine salvagepathway, including IMPDH, are essential for C. parvum. Thus, inhibition ofthese enzymes would lead to a depletion in the supply of nucleotides for DNAsynthesis, resulting in the inhibition of parasite growth. CpIMPDH would beparticularly attractive target due to its apparent bacterial origin and cleardifference from eukaryotic IMPDH homologs.

ENERGY METABOLISMEarly biochemical analyses indicated that C. parvum relies mainly on

glycolysis as an energy source (Denton et al., 1996; Entrala and Mascaro,1997). A great number of enzymatic activities within the pathway have beendetected in oocysts and free sporozoites, almost all of which were present inthe cytosol.

There are many enzymes necessary for glycolysis. Malate and lactatedehydrogenases (MDH and LDH), both of which are cytosolic, have beenreported as partial sequences in the C. parvum EST and GSS projects (Liu etal., 1999; Strong and Nelson, 2000). The complete CpLDH1 gene wasdetermined by an independent cloning approach (Zhu and Keithly, 2002),while CpMDH1 was identified from a contig in the ongoing CpGP. Unlike

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LDH from T. gondii and Plasmodium spp. that contain a short amino acidinsertion at the active site loop, CpLDH1 does not possess such an insertion.Phylogenetic analysis indicates that these two enzymes in C. parvum andother apicomplexans are sisters to MDH, differing from themajority of other eukaryotic MDH and LDH enzymes. This observationsuggests that apicomplexan MDH and LDH enzymes might have beenacquired from (Zhu and Keithly, 2002).

In addition to MDH and LDH, a number of other enzymes within theglycolytic pathway have been identified by the complete EST and GSSprojects. In fact, by data-mining the C. parvum contigs deposited in theGenBank by the ongoing CpGP, we were able to identify almost all genes(except for one) encoding C. parvum glycolytic enzymes previously detectedwith biochemical assays (Entrala and Mascaro, 1997). These enzymesinclude: phosphoglucomutase (PGM), phosphoglucose isomerase (PGI),pyrophosphate-dependent phospho-fructokinase (PPi-PFK, two homologues),triosephosphate isomerase (TIM), glyceraldehyde 3-phosphate dehydrogenase(GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM),enolase, pyruvate kinase (PK), LDH, phosphoenolpyruvate carboxylase(PEPCL), MDH, malic enzyme (ME), and adenylate kinase (AK). Amongthem, the PPi-PFK is typical to anaerobic microorganisms and has beenproven to be a potential drug target for the phosphonic acid analogs in T.gondii (Peng et al., 1995). The only enzyme absent in the GenBank C. parvumcontigs at the moment is the aldolase. In contrast to P. falciparum, but inagreement with early observations that C. parvum lacks an activetricarboxylic acid (Krebs’) cycle (Denton et al., 1996; Entrala and Mascaro,1997), no enzymes within the Krebs’ cycle could be identified from allavailable C. parvum genomic data.

Another important enzyme associated with anaerobic metabolism isthe oxygen-sensitive pyruvate-ferredoxin oxidoreductase (PFO) that convertspyruvate to acetyl CoA and with the reduction of ferredoxin.Cryptosporidium PFO homologues were first found in the GSS entries fromGenBank. Subsequent cloning and sequencing efforts revealed that the C.parvum PFO was part of a pyruvate-NADP oxidoreductase PNO (CpPNO)fused with a cytochrome P450 reductase (CPR) (Rotte et al., 2001). This typeof gene fusion was coincidently found in another unrelated protist, Euglenagracilis. It is interesting that the E. gracilis PNO contains a mitochondrialtransit sequence. However, CpPNO lacks such a mitochondria transitsequence, suggesting it could be cytosolic. Phylogenetic analysis hassuggested a common ancestry of PFO in amitochondriate protists withEuglena mitochondrial PNO and apicomplexan CpPNO, which provides anew insight at the evolution of these unique proteins (Rotte et al., 2001).Furthermore, it has been suggested that CpPNO might be responsible for the

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parasite’s sensitivity to 5’-nitrothiazole (NTZ) that has been in human clinicaltrials for treating chronic cryptosporidiosis (Coombs and Muller, 2002). Thecomplete sequencing of CpPNO gene makes it possible to truly test thishypothesis.

FATTY ACID SYNTHETIC PATHWAYFatty acids are one of the major components of biomembranes. At

least 7 enzymes are involved in the synthesis of fatty acids (Smith, 1994):ketoacyl synthase (KS), acyl transferase (AT), ketoacyl reductase (KR),dehydrase (DH), enoyl reductase (ER), acyl carrier protein (ACP) andthioesterase (TE). In animals and fungi, these enzymes are fused into one ortwo multifunctional polypeptides, referred to as Type I fatty acid synthase(FAS). In bacteria and plants, these proteins are discrete, monofunctionalenzymes, termed Type II FAS. In eukaryotes, Type I FAS proteins arecytosolic, while Type II FAS enzymes are localized in the plant plastid.

Our understanding of fatty acid metabolism in the Apicomplexabegan with the discovery of several Type II FAS enzymes in T. gondii and P.falciparum and a giant 25-kb Type I FAS gene in C. parvum (CpFAS1)(Waller et al., 1998; Zhu et al., 2000c). Type II FAS genes were originallyidentified from T. gondii and P. falciparum EST by the presence of plastidtransit sequences, and subsequent analyses confirmed that these enzymeswere indeed targeted to the apicoplast. In contrast, CpFAS1 polypeptide waslocalized in the cytosol. It contains 21 domains distributed as a loading unit(ligase-ACP), 3 chain elongation modules (KS-AT-DH-ER-KR-ACP), and aC-terminal reductase. This architecture differs from animal FAS, which isfused with 7 enzymes (KS-AT-DH-ER-KR-ACP-TE) (Smith, 1994).

Does C. parvum differ from other apicomplexans by lacking anyType II FAS genes? Although this question cannot be ultimately answereduntil the entire C. parvum genome is sequenced and annotated, currentmolecular, biochemical and genomic data apparently support this hypothesis.Firstly, in vitro drug tests indicated that C. parvum was insensitive tothiolactomycin (a Type II FAS-specific inhibitor that could inhibit the growthof T. gondii and P. falciparum) (Zhu et al., 2000c). Secondly, C. parvumapparently lacks the plastid genome, suggesting that a plastid is likely absentto harbor Type II FAS (Zhu et al., 2000b). Thirdly, from all currentlyavailable C. parvum genome sequences in the GenBank, we are unable toidentify any Type II FAS homologues, nor the plastid genome.

In addition to CpFAS1, a 40-kb ORF encoding the first known protistpolyketide synthase (CpPKS1) gene was identified and characterized from C.parvum (Zhu et al., 2002). It was originally presented as 3 separate GSSentries homologous to CpFAS1 in the GenBank. About 38-kb nucleotides ofCpPKS1 were sequenced from overlapping gDNA clones, and the N-terminal

SUMMARYThe impact on basic research and target discovery of a complete C.

parvum genome project (CpGP) will be dramatic. It is predictable that allclassical or specific energy metabolic enzymes and isoenzymes will beidentified from the complete parasite genome. However, some enzymesexclusive to C. parvum and/or the Apicomplexa may require additionalfunctional studies to determine their biochemical features. The availability ofthe complete genome sequence will clearly accelerate the discovery andreconstruction of various pathways, both in silico and in vitro. From thecomplete genome, one can identify genes of interest to study their functionsand pharmaceutical values, determine if there are alternative pathways for thedrug targets, and study the dynamics of metabolic enzymes during theintracellular life cycle stages (which is almost impossible by traditionalbiochemical analysis of crude parasite extract). Moreover, the complete C.

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~2-kb gap was closed by taking advantage of the CpGP. Like CpFAS1,CpPKS1 contains a loading unit, 7 elongation modules and a reductase.However, most of the elongation modules in CpPKS1 contain only 2-6enzymes (incomplete sets), suggesting that their intermediate and/or finalproducts may retain keto, hydroxyl groups and/or double bands. This ischaracteristic to the Type I PKS identified from bacteria and fungi (Rawlings,1997; Staunton and Weissman, 2001).

The discovery of CpFAS1 and CpPKS1 genes provides us with a newopportunity to study the fatty acid/polyketide biosynthesis in C. parvum. Inaddition, these distinct mega-synthases may also serve as novel targets in drugdiscovery against cryptosporidiosis. Indeed, FAS enzymes have beenrecognized as a new class of drug target against a variety of tumors (Pizer etal., 1996; Pizer et al., 1998) and pathogens that include bacteria (Mdluli et al.,1998; Heath et al., 1999; McMurry et al., 1999) and fungi (Broedel et al.,1996; Zhao et al., 1996). This notion is further supported by the inhibitorystudies of FAS inhibitors in C. parvum, T. gondii and P. falciparum (Waller etal., 1998; Zhu et al., 2000c).In addition to CpFAS1 and CpPKS1, several other genes encoding fatty acidmetabolic enzymes have been identified from the ongoing CpGP. Theseinclude: 1) the Ppant transferase that activates ACP by transferring a 4’-phosphopantethein prosthetic group from CoA to the Ser residue in ACP; 2)three independent fatty acyl-CoA ligases (ACL) for the activation of fattyacids to form fatty acyl-CoA; 3) acyl-CoA binding protein (ACBP)responsible for the transport of fatty acyl-CoA; 4) acetyl-CoA carboxylase(ACC) for the synthesis of malonyl-CoA; and 5) fatty acyl elongase that istypically associated with microsomes in other eukaryotes. It is clear that amore complete picture will soon arise with the completion of the CpGP.

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parvum genome will ultimately clarify whether a speculative gene or drugtarget is absent or present, and will allow for a better understanding of theevolution and relationship of apicomplexan pathogens.

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INDEX

Acquired immunodeficiency syndrome(AIDS)

amebiasis in, 77cryptosporidiosis in, 30, 92, 104–105cyclosporiasis in, 47, 109

Acridinic thioethers, in cryptosporidiosis,107–108

AIDS. See Acquired immunodeficiencysyndrome (AIDS)

Albendazole, in giardiasis, 110Alcohol use, amebiasis and, 77Amebiasis, 75–84. See also Entamoeba

dispar; Entamoeba histolyticain AIDS, 77alcohol use and, 77antibody production in, 81, 83cytokines in, 81–82death from, 75, 141diagnosis of, 78extraintestinal, 111IgA in, 81, 83immune evasion in, 80immune response to, 81–84inflammatory response in, 81–82interleukin-12 in, 82lectin-based test in, 19–20lectin-glycoprotein interaction in, 78–79microscopy in, 18, 19mucus in, 81pathogenesis of, 78–81polymerase chain reaction test in, 20proteinases in, 80–81risk factors for, 76–78, 141–142in SCID mouse, 82serology in, 18–19serum anti-trophozoite IgG and, 77symptoms of, 76treatment of, 19, 110–111vaccine against, 83–84virulence factors in, 79–81

Amoebapore, 81Antibody

in amebiasis, 81, 83in cryptosporidiosis, 97

A in cyclosporiasis, 47in giardiasis, 67–68

Arginine decarboxylase inhibitors, incryptosporidiosis, 107

ATP-binding cassette transporters, inCryptosporidium parvum, 106–107

Aurone analogs, in cryptosporidiosis,107–108

Azithromycin, in cryptosporidiosis, 104

BBasil, cyclosporiasis with, 50Benzimidazoles, in cryptosporidiosis, 107Blackberries, cyclosporiasis with, 50Bupravaquone, in cryptosporidiosis, 108

CCat, giardiasis in, 6–7, 9–10Chromosomes, of Entamoeba histolytica,

142–143Ciprofloxacin, in cyclosporiasis, 109Clarithromycin, in cryptosporidiosis, 105CpFAS1 gene, 156–160CpPKS1 gene, 156–160Cryptosporidiosis. See also Cryptosporidium

parvumadaptive immunity in, 94–95age-related incidence of, 29–30in AIDS, 30, 92, 104–105antibody in, 97cytokines in, 95–96drug delivery systems in, 108drug resistance in, 105–107foodborne, 28immunopathogenesis of, 93–94inflammatory response in, 93–94innate immunity to, 91–93

in, 92–93, 95–96mucosal lymphocytes in, 96–97natural killer cell response in, 92–93

in, 92p23 in, 97

166 Index

pathogenesis of, 35prostaglandin production in, 92risk factors for, 35in SCID mice, 92–93, 96–97seasonal incidence of, 30in T cell–deficient mice, 94–95T cells in, 94–96transforming growth in, 94transmission of, 28–29treatment of, 103–108villous atrophy in, 93waterborne, 29, 34, 129–138. See also

Water monitoringCryptosporidium spp., 33–34

DNA fingerprinting of, 35–36water monitoring for, 129–138. See also

Water monitoringCryptosporidium hominis, 32, 33, 34, 35

DNA fingerprinting of, 35–36Cryptosporidium parvum, 27–37

animal type, 32ATP-binding cassette transporters in,

106–107CpFAS1 gene of, 156–160CpPKS1 gene of, 156–160dehydrogenases of, 157–158dihydrofolate reductase of, 106DNA fingerprinting of, 35–36DNA replication proteins of, 154–156drug resistance of, 105–107energy metabolism of, 157–159enzymes of, 107, 156–160fatty acid metabolism in, 159–160fatty acid synthase gene of, 107genome of, 153–161glycolytic enzymes of, 157–159host-adapted genotypes of, 32human type, 32life cycle of, 153–154molecular studies of, 30–37polymerase chain reaction test of, 31–32purine metabolism in, 156–157pyruvate-ferredoxin oxidoreductase of,

158–159transmission of, 28–29

CXXC motif, of Giardia, 61, 62Cyclospora cayetanensis, 43–53

detection of, 51–53identification of, 43–44life cycle of, 45–46taxonomy of, 44–45vs. Eimeria, 52–53

Cyclosporiasis, 43–53

in AIDS, 47, 109ciprofloxacin in, 109diagnosis of, 51–53foodborne, 45, 48, 49–51immunity to, 46, 47microscopy in, 51polymerase chain reaction test in, 52seasonality of, 48symptoms of, 46timethoprim-sulfamethoxazole in, 109treatment of, 47, 108–109waterborne, 48, 49

Cytokinesin amebiasis, 81–82in cryptosporidiosis, 95–96

DDehydrogenases, of Cryptosporidium

parvum, 157–158Dihydrofolate reductase, of

Cryptosporidium parvum, 106Dinitroaniline herbicides, in

cryptosporidiosis, 107DNA fingerprinting, of Cryptosporidium.

35–36DNA replication proteins, of

Cryptosporidium parvum, 154–156Dogs, giardiasis in, 6–7, 9–10Drug resistance, in cryptosporidiosis,

105–107

EEimeria, vs. Cyclospora, 52–53Energy metabolism, of Cryptosporidium

parvum, 157–159Entamoeba dispar, 15–22. See also

Amebiasisbacterial ingestion by, 21–22electron microscopy of, 21–22enzymes of, 17–18, 20–21, 76genome sequencing of, 21proteinases of, 80–81vs. E. histolytica, 20–22, 147–148

Entamoeba histolytica, 15–22. See alsoAmebiasis

amoebapore of, 81antibody to, 81bacterial ingestion by, 21–22bacterial-like genes in, 147–148chromosomes of, 142–143electron microscopy of, 21–22

Index 167

enzymes of, 17–18, 20–21, 75episomes of, 145evolutionary position of, 146–147Gal/GalNAc lectin of, 78–80, 82gene expression in, 144–145genome of, 21, 141–148locomotion of, 80long interspersed elements of, 145–146proteinases of, 80–81repeated sequences of, 145–146serine-rich protein of, 84short interspersed elements of, 145–146vs. E. dispar, 20–22, 147–148

Enzymesof Cryptosporidium parvum, 107, 156–160of Entamoeba dispar, 17–18, 20–21, 76of Entamoeba histolytica, 17–18, 20–21,

75Episomes, of Entamoeba histolytica, 145

FFatty acid metabolism, in Cryptosporidium

parvum, 159–160Fatty acid synthase gene, of

Cryptosporidium parvum, 107

GGal/GalNAc lectin, of Entamoeba

histolytica, 78–80, 82Genome

of Cryptosporidium parvum, 153–161of Entamoeba histolytica, 21, 141–148

GGCY motif, of Giardia, 61Giardia, 1–10

antigenic variation in, 60, 66–70assemblage A, 4–5, 60assemblage B, 4–5, 60CXXC motif of, 61, 62encystation of, 66excystation of, 66GGCY motif of, 61host specificity of, 2, 3–5life cycle of, 2post-translation modification in, 62–63RING motif of, 61–62secretory system of, 2species of, 2, 3–5transmission of, 5–7variant-specific surface proteins of,

59–70. See also Variant-specificsurface proteins; vsp genes

water monitoring for, 129–138. See alsoWater monitoring

Zn finger motif of, 62Giardiasis. See also Giardiasis

animal role in, 2–3antibody response to, 67–68biological selection in, 68–69in domestic dogs and cats, 6–7, 9–10experimental, 67–69in immunodeficient gerbils, 69in livestock, 6, 8non-immunological mechanisms in, 68–69pathogenesis of, 2in SCID mice, 69T cell response in, 68transmission of, 5–7treatment of, 109–110VSP expression in, 67waterborne, 7, 129–138in wildlife, 7–8

GP60 gene, 36

HHighly active anti-retroviral therapy, in

cryptosporidiosis, 104

IImmune system

in amebiasis, 81–84in cryptosporidiosis, 91–95in cyclosporiasis, 46, 47

Immunoglobulin Ain amebiasis, 81, 83in cryptosporidiosis, 97

Immunoglobulin Gin amebiasis, 77in cryptosporidiosis, 97in cyclosporiasis, 47

Immunoglobulin M, in cyclosporiasis, 47Immunotherapy, in cryptosporidiosis, 108Inflammatory response

in amebiasis, 81–82in cryptosporidiosis, 93–94

in cryptosporidiosis, 92–93,95–96

Interleukin-12, in amebiasis, 82

LLectin-based test, in amebiasis, 19–20Livestock, giardiasis in, 6, 8

168 Index

Long interspersed elements, of Entamoebahistolytica, 145–146

M

NNatural killer cells, in cryptosporidiosis,

92–93in cryptosporidiosis, 92

Nitazoxanidein cryptosporidiosis, 104–105in giardiasis, 110

PP23, in cryptosporidiosis, 97Paromomycin

in cryptosporidiosis, 104in giardiasis, 110

Polymerase chain reaction testin amebiasis, 20of Cryptosporidium parvum, 31–32in cyclosporiasis, 52

Prostaglandins, in cryptosporidiosis, 92Proteinases

in amebiasis, 80–81of Entamoeba dispar, 80–81of Entamoeba histolytica, 80–81

Purine metabolism, in Cryptosporidiumparvum, 156–157

Pyruvate-ferredoxin oxidoreductase, ofCryptosporidium parvum, 158–159

RRaspberries, cyclosporiasis with, 45, 49–50Replication protein A, of Cryptosporidium

parvum, 155–156Rifabutin, in cryptosporidiosis, 105RING motif, of Giardia, 61–62Roxithromycin, in cryptosporidiosis, 105

SSCID mouse

amebiasis in, 82

cryptosporidiosis in, 92–93, 96–97giardiasis in, 69

Serine-rich Entamoeba histolytica protein, 84Short interspersed elements, of Entamoeba

histolytica, 145–146Spiramycin, in cryptosporidiosis, 105

TTinidazole, in amebiasis, 110–111Transforming growth in

cryptosporidiosis, 94Trimethoprim-sulfamethoxazole

in cyclosporiasis, 47, 109

VVaccine, against amebiasis, 83–84Variant-specific surface proteins, 59–70

antibodies to, 67–68biological function of, 66–69CXXC motif of, 61during encystation, 66genes for, 63–65. See also vsp genesin gerbils, 68–69GGCY motif of, 61identification of, 60in mice, 67–68, 69post-translation modification of, 62–63protease effect on, 68regulation of, 65–66RING motif of, 61–62selection of, 66–69trafficking of, 62–63Zn finger motif of, 62

Villous atrophy, in cryptosporidiosis, 93vsp genes, 63–65

allele loss from, 66allele-specific expression of, 65–66alleles of, 63–64encystation and, 66expression of, 64–66similarities among, 64tandem repeats of, 64

WWater monitoring, 129–138

grab samples in, 137Information Collection Rule in, 131–132Surface Water Treatment Rule in, 131in United Kingdom, 134–137in United States, 130–134

Mesclun lettuce, cyclosporiasis with, 50Metronidazole

in amebiasis, 110–111in giardiasis, 109–110

Mucus, in amebiasis, 81

Index 169

USEPA Method 1622 in, 133–134USEPA Method 1623 in, 133–134Water Supply Regulations in, 135–137

Water treatment, 117–124bromate ion formation with, 124chemical disinfection for, 121–124chloramine disinfection for, 121, 122chlorine disinfection for, 124coagulants for, 119–120DE filters for, 121dissolved air flotation for, 120

membranes for, 121ozone disinfection for, 121, 122, 124physical processes for, 118–121rapid granular filter for, 119–120slow sand filters for, 119ultraviolet light disinfection for, 122, 123

Wildlife, giardiasis in, 7–8

ZZn finger motif, of Giardia, 62