BAcTKROLOGICAL REVIEWS, June 1977, p. 373-390Copyright ©) 1977 American Society for Microbiology

Vol. 41, No. 2Printed in U.S.A.

Fusobacterium necrophorum: Its Characteristics and Role asan Animal PathogenBARBARA F. LANGWORTH

American Cyanamid Company, Princeton, New Jersey 08540

INTRODUCTION.............................................................. 373CLASSIFICATION ......... 374DESCRIPTION OF F. NECROPHORUM. 376Cellular Morphology ................................... 376Lipopolysaccharide ................................... 376Toxins ................................... 377Biochemical Properties ........ ........................... 378Serological Properties .................................... 378Antibiotic Sensitivities ........ ............................ 381Isolation and Cultivation .................................... 381

ANIMAL DISEASES AND CHEMOTHERAPY .................................. 382Natural Infections .................................... 382

Calf diphtheria .................................... 382Liver abscesses .................................... 383Foot rot .......................................... 383Other diseases .................................... 384

Experimental Infections ......... ........................... 385IMMUNITY................................................................... 386CONCLUDING REMARKS.................................... 387LITERATURE CITED.................................... 387

INTRODUCTIONFusobacterium necrophorum is a gram-nega-

tive, nonsporeforming, nonmotile, strictly an-aerobic, pleomorphic bacterium in the familyBacteroidaceae. Recognized as an animal path-ogen since the late 1800s, F. necrophorum wasmainly described as occurring in liver abscessesin cattle, foot rot in many domestic animals,calf diphtheria (unrelated to the disease in hu-mans), and necrotic lesions in the oral cavity(see reference 82). F. necrophorum was alsoknown to survive in the soil of pastures, whichwas an enigma, considering that it was a non-spore-forming anaerobe (35).More recently, F. necrophorum has been iso-

lated from the normal flora in the oral cavity,gastrointestinal tract, and genitourinary tractof humans and animals (85). When involved indisease, it may cause localized necrotic lesionsand abscesses or be carried by the bloodstreamto internal tissues, causing necrosis and ab-scess formation (105).Although the incidence in human disease is

not yet well established, F. necrophorum hasbeen isolated and cultivated from abscesses,blood, body fluids, and oral infections (3).F. necrophorum was originally thought to be

a secondary invader requiring a previous infec-tion, wound, or other predisposing factor togain entry into the host. It has now been shownthat pure cultures ofF. necrophorum are inde-

pendently capable of causing disease. In natu-ral infections with F. necrophorum other orga-nisms are frequently isolated, but true syner-gism has been described in only a few instances(8, 74-76).The inability to subculture the organism

hampered early investigators, and descriptionsin the literature were mostly based on the fusi-form morphology observed under the micro-scope (see reference 57).The study of anaerobic bacteria has been

greatly facilitated by the advent of the culturaltechniques ofHungate (47), who first developeda method for handling and cultivating anaero-bic organisms by placing them under a streamof deoxygenated carbon dioxide. His "roll-tube"technique used a carbon dioxide-filled test tubecoated with agar, the bacteria either being inthe agar or streaked on the surface. The sealedtube was then placed in a standard incubator.This provided a closed anaerobic system underwhich the growth of colonies could be observedwithout their exposure to air. Holdeman andMoore (45) further refined these techniques.They recognized the importance of collectingspecimens in containers devoid of oxygen andprocessing the specimens as rapidly as possible.Holdeman and Moore (46) developed proceduresfor prereduced, anaerobically sterilized mediawhich, when used under a stream of deoxygen-ated carbon dioxide, supported the growth of

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374 LANGWORTH

most anaerobes. These techniques led to thereevaluation of the taxonomy of anaerobic bac-teria.

Descriptions of bacteria conforming to thecurrent definition of F. necrophorum have ap-peared under the following names, of whichSpherophorus necrophorus is perhaps the mostcommon (the name Spherophorus is now con-sidered invalid since it was initially used todefine two species of lichen [82]): Actinomycesnecrophorus, A. pseudonecrophorus, Bacillusfundibuliforms, B. funduliforms, B. necropho-rus, B. necrosus, Bacterium funduliforms, B.necrophorum, Bacteroides fundibuliformis, B.funduliformis, B. necrophorus, Corynebacte-rium necrophorum, Fusiformis hemolyticus, F.necrophorus, Necrobacterium necrophorus,Proactinomyces necrophorus, Pseudobacter-ium funduliformis, Sphaerophorus fundulifor-mis, S. necrophorus, S. pseudonecrophorus,Streptothrix necrophorus, and S. necupthora(14).The 7th edition of Bergey's Manual ofDeter-

minative Bacteriology (11) separated the familyBacteriodaceae (gram-negative, anaerobic,nonsporeforming rods) into four genera on thebasis of morphological criteria. Bacteroideswere simple rod-shaped cells with roundedends, Fusobacterium had pointed ends, Dialis-ter had a diameter of less than 0.15 Mm, andSpherophorus were pleomorphic rods. The Bac-teroidaceae have now been divided into threemain genera based on the amount and type ofvolatile fatty acids produced. Fusobacteriumare defined as producing major amounts of bu-tyric acid, Leptotrichia as producing majoramounts of lactic acid, and Bacteroides as pro-ducing a mixture of acids. F. necrophorum isfurther differentiated from other species ofFu-sobacterium by both the formation of indoleand the production of propionic acid from lac-tate (14). Previously, F. necrophorum strains ofanimal origin and strains ofhuman origin werethought to be different species (5), but resultsbased on gas chromatographic analyses havenot substantiated this division (46).The 16 species ofFusobacterium listed in the

eighth edition of Bergey's Manual (14) includeseven species that are encountered in clinicalspecimens, F. gonidaformis, F. mortiferum, F.naviforme, F. necrophorum, F. nucleatum, F.russii, and F. varium, and nine other speciesthat are infrequently isolated from infectiousmaterial (3, 14, 85). The species may be differ-entiated on the basis of sugar fermentationsand biochemical reactions.With contemporary techniques for isolation,

cultivation, and identification of anaerobes, it

seems appropriate to examine the literature onthe organisms that are now considered F. nec-rophorum in order to collate the many diversi-fied data and correlate various observations.

CLASSIFICATIONIn the past, isolation and cultivation of an-

aerobic bacteria were hampered because oftheir extreme susceptibility to oxygen. Cul-tures were difficult to preserve, and thereforelittle exchange of organisms between differentlaboratories occurred. As a result, most descrip-tions of anaerobes were based only on origin ofthe culture and morphology. Newly isolatedand named fusiforms were frequently the sameorganisms as described previously, but becausethey were reported by different investigatorswho did not have access to similar strains forcomparison studies, they were mislabeled orgiven a new name.The first published description of F. necro-

phorum was by Loeffler who, in 1884, discussedthe importance of microorganisms in diphthe-ria of humans, calves, and doves (58). He in-jected mice subcutaneously with caseous mate-rial from the larynx of a calf with diphtheria.From the abscesses that formed he was able toisolate the organism on a calf serum mediumbut could not further subculture the bacteria.In 1891 Bang isolated a fusiform bacillus fromabscessed livers of cattle, and Flugge, in a de-scription of microorganisms in 1886, named theorganism Bacillus necrophorus (see reference62). In 1905 Mohler and Washburn (see refer-ence 26) thought that foot rot in cattle was alsocaused by this "necrotic bacillus." Other de-scriptions of fusiform bacilli reported in theearly literature include an account of orga-nisms isolated from necrotic lesions of thethroat of humans by Vincent in 1896 and fromdiseased appendixes by Veillon and Zuber in1898. The organisms described were typical ofwhat is now called F. fusiforme (see reference14).

In 1923 Knorr suggested the genus Fusobac-terium for all gram-negative, anaerobic fusi-form bacteria.

In 1955 Wilson and Miles (105) did not thinkthere was sufficient information to subdividethe gram-negative, nonsporeforming, anaero-bic bacteria and lumped them together underthe species F. fusiformis (Table 1).Morphology was still the main criterion for

separation in 1957 in the 7th edition ofBergey'sManual (11). The family Bacteroidaceae wasdivided into three genera: the Bacteroides de-fined as rods, Fusobacterium defined as pleo-morphic filamentous bacteria with pointed

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F. NECROPHORUM AS AN ANIMAL PATHOGEN 375

TABLs 1. Nomenclature of anaerobic, gram-negative, nonsporeforming, rod-shaped bacteria'

Reference

Prevot, 1938 (72) Family Ristellaceae SpherophoraceaeGenus Ristella Zuberella Fusiformis Fusocillus

I Sphaerophorus SphaerocillusWilson and Miles, 1955 (106) Fusiform groupBreed, Murray, and Smith, 1957 (11) Family Bacteroidaceae

Genus Bacteroides Sphaerophorus FusobacteriumPrevot, Turpin, and Kaiser, 1967 (74) Family Ristellaceae Spherophoraceae

Genus Ristella Capsularis Zuberella Sphaerophorus SphaerocillusBuchanan and Gibbons, 1974 (14) Family Bacteroidaceae

Genus Bacteroides Fusobacterium Leptotrichia0 Modified from Aalbaek.

ends, and Spherophorus defined as pleo-morphic filamentous bacteria with roundedends.

Several species ofSphaerophorus were iden-tified and separated according to their origin.S. necrophorus was considered an animal path-ogen and S. funduliformis a human pathogen(5).Dack et al. in 1938 published a comparison of

F. necrophorum strains isolated from the ulcer-ated colon of humans and monkeys and fromliver abscesses in cattle, pigs, and lambs (18).Since no significant biochemical differencesamong the strains were observed and inocula-tion into rabbits indicated varying degrees ofpathogenicity, they suggested that all the iso-lates were one species. Prevot and Kirchheimer(74) were in disagreement with Dack et al. as aresult of serological studies of the two speciesand decided that even though a common anti-gen was shared, the isolates were indubitablytwo distinct species.

In 1944 Boe and Thjotta (10) examined therelationship between Leptotrichia and Fuso-bacterium, which they thought had been con-fused by previous authors due to the similarmorphological characteristics. They deter-mined that the strong fermentation of carbohy-drates and lack of indole production by theLeptotrichia compared to weak carbohydratefermentation and strong indole production bythe Fusobacterium were valid differentiatingcharacteristics. Serological studies, however,demonstrated that the Fusobacterium and Lep-totrichia were related.

In 1945 Lahelle and Thjotta (57) published astudy comparing the Fusobacterium, whichwas defined as being of human origin, withActinomyces necrophorus (which they sug-gested be renamed Necrobacterium), whichwas an animal pathogen. Based on the results

of serological studies, Lahelle and Thjotta de-termined that the human and animal isolateswere closely related.The separation of strains based on a host

preference was again examined in 1954 by Be-erens (5), who supported the distinction basedon his results of a hemagglutination study.Four strains of S. necrophorus (animal iso-lates) were tested and were found to aggluti-nate chicken, sheep, and human erythrocytes.None of 14 strains of S. funduliformis (humanisolates) agglutinated chicken or sheep erythro-cytes, and only two agglutinated human eryth-rocytes.

In a later paper Beerens et al. (6) included astudy ofS. pseudonecrophorus and ascertainedthat each of three putative species was actuallya different phase of one species. The character-istics for each were as follows: S. necrophorusproduced a hemagglutinin and a hemolysin andwas pathogenic for mice; S. funduliformis didnot produce a hemagglutinin, produced a he-molysin, and was nonpathogenic for mice; andS. pseudonecrophorus did not produce a he-magglutinin or hemolysin and was nonpatho-genic for mice. They considered the three var-iants as phases A, B, and C, respectively, basedon observations of changes in these propertiesduring subcultures of strains over severalyears, which to them suggested mutation fromthe A to B to C phase.The use of gas chromatography for the sepa-

ration of volatile fatty acids was described in1952 by James and Martin (49) and recom-mended for the identification of microorga-nisms (on Mars) by Oyama in 1963 (67), afterwhich followed many papers analyzing thefatty acid and metabolic products of variousbacteria.Werner et al. (101) examined 27 strains of

fusiform bacteria and were able to separate

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Fusobacterium from Leptotrichia based on theproduction of butyric acid. They were of theopinion that fermentations of various sugarswere not adequate to differentiate the species ofFusobacterium. They also determined thatmorphologically similar clinical isolates ofBac-teroidaceae that produced large amounts of bu-tyric acid had more biochemical characteristicsin common with strains considered Fusobacte-rium than with strains labeled Bacteroides orLeptotrichia. In a subsequent paper, Werner(98) noted the butyric acid ratio among F. nec-rophorum, F. varium, and F. mortiferumranged from 1.05 to 1.9, 1.8 to 5.0, and 3.5 to 6.0,respectively.Comparing morphological and biochemical

criteria and results of fatty acid analysis, Wer-ner and Reichertz (102) determined that theseparation of Fusobacterium and Bacteroidescould be based solely on the formation of isoval-eric and isobutyric acids by the Bacteroidesstrains. Fritsche (27) studied strains of Bacte-roides that produced butyric acid and observedthat the amount formed was much less thanthat produced by Fusobacterium. Further in-vestigations (29, 100) led to the conclusion thatproduction of major amounts of isovaleric andisobutyric acids should take precedence in theclassification ofBacteroides, and that Fusobac-terium could be distinguished by the productionof major amounts of butyric acid.The taxonomic position of species within the

genus Fusobacterium is still dubious. Ameri-can authors follow the biochemical distinctionsfrom Bergey's Manual, whereas many Euro-pean authors favor Prevot's more morphologi-cally based nomenclature. Slight discrepanciesin sugar fermentations as published by differ-ent authors may result, not from strain varia-tion, but from insufficient control of the testmedia as to pH, etc. For example, Simon (80)discovered the optimal initial pH ofhis mediumfor maximum acid production of F. necropho-rum was 7.7, whereas the initial pH of mostmedia commonly used was much lower.

Deoxyribonucleic acid homology studies havebeen adjunctive in determining the relation-ships of many organisms and may certainlyhave application here. Even though Prevot etal. (73) list guanine-plus-cytosine ratios, therelatedness of similar deoxyribonucleic acidshas not been established.Another useful tool for identification is phage

typing, such as is done with staphylococci. Al-though there have been no reports of phageactivity with F. necrophorum, a phage specificfor F. varium and two phages specific for B.fragilis have been isolated (65).

DESCRIPTION OF F. NECROPHORUMCellular Morphology

F. necrophorum is a gram-negative, nonspore-forming, pleomorphic bacillus from 0.5 to 1.75gtm in diameter, ranging from small, almostcoccoid bodies to filaments greater than 100 pmlong, with parallel sides and blunt or taperingends. The morphology will be affected by thetype of media used and the age of the culture.Filamentous forms are usually seen more fre-quently in young cultures and in a broth me-dium, and bacilli are more common in oldercultures and when growth is on agar. Swellingalong the filaments or at one end has also beenobserved. With standard stains, e.g., carbolfuchsin, irregular staining or beading may beseen (105). Older cultures appear to stain moreirregularly, and Beveridge (9) attributed theloss of staining ability to aging and degenera-tion of the cells.

L-forms ofF. necrophorum were first isolatedby Klieneberger-Nobel (55) and Dienes (19).The former was of the opinion that the formsobtained were symbionic or virus-like bodiessince she could propagate them without rever-sion to bacilli. Smith et al. (86) carefully stud-ied the filaments under electron microscopyand observed that, in addition to division bybinary fission, the bacteria swelled, forminground bodies from which delicate filamentsarose and segmented to yield L-forms. On sub-culturing, they observed reversion back to thepleomorphic filaments. It was noted that L-forms could be isolated more easily if penicillinwas added to the medium.Ernst reported seeing branched forms of F.

necrophorum in 1902 (20). This reference iscited by many investigators who state thatbranching was never observed by them andthat this phenomenon must have been an arte-fact. In 1970 Teresa et al. (91) published a pho-tograph showing a branched filament, al-though they acknowledged that branching wasseen too infrequently to be a significant charac-teristic.

LipopolysaccharideBiochemical studies of cell wall lipopolysac-

charide (LPS) components by Hofstad et al. (43,44) resulted in the identification of heptose and2-keto-deoxyoctonate (KDO) from water andphenol extracts. Further investigating the LPScomposition, Hofstad (42) was able to determinethe additional presence of galactose, glucose,and glucosamine. In both investigations KDOwas not found in Bacteroides strains. On theother hand Sonnenwirth et al. (87) were able to

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F. NECROPHORUM AS AN ANIMAL PATHOGEN 377

detect KDO in the four strains of Bacteroidestested. Possible procedural differences havebeen suggested to explain this discrepancy.(KDO and heptose are also important compo-nents ofthe LPS core ofSalmonella and Esche-richia coli [59].)Further characterizing the LPS fraction,

Garcia et al. (32) determined that it contained23.5% hexose, 6.4% heptose, 8.8% hexosamines,0.8% KDO, 2.4% protein, and no nucleic acids.

ToxinsStrains ofF. necrophorum have been shown

to possess a classical cell wall LPS endotoxin.Investigators have also demonstrated the pres-ence of exotoxins: hemolysin, leucocidin, and acytoplasmic toxin. However, whether each exo-toxin is a separate component has yet to bedetermined.

Orcutt (66) demonstrated the presence of anexotoxin in bovine isolates ofF. necrophorum.Intraperitoneal or intravenous injection of cul-ture filtrates into mice and rabbits producedillness and frequently death.

Beveridge (9) studied the toxins of F. necro-phorum strains isolated from cattle and walla-bies. Intradermal inoculation of culture fil-trates (prepared by several different methods)into rabbits produced an inflammatory reac-tion, and an intravenous inoculation in sheepproduced diarrhea and anorexia. No responsewas observed in guinea pigs by either route ofadministration. Endotoxin activity of thestrains was demonstrated in rabbits and guineapigs using a carbolized or formalinized prepara-tion. Intradermal inoculation produced necrosisof the superficial and deep layers of the skin.Dack et al. (18) also produced abscesses in

rabbits using the supernatant from cultures ofbovine, human, and monkey strains. Theynoted that a more severe response resultedfrom the bovine strain preparations.

Hofstad and Kristofferson (43) isolated theLPS fraction of F. necrophorum from labora-tory strains. When injected intradermally, in-oculation of preparations into rabbits did notresult in a pronounced Schwartzman reaction.Warner et al. (95), however, using a bovine

isolate ofF. necrophorum, were able to elicit aSchwar zman reaction in rabbits. They pre-pared a cell wall fraction and a soluble fraction.Both were toxic for mice. The soluble fractionwas heat stabile and insensitive to treatmentwith formalin, but was not toxic after treat-ment with sodium hydroxide. Such characteris-tics indicated that the material was presum-ably LPS. The toxin was found to have a highmolecular weight as determined by gel filtra-

tion. Diethylaminoethyl-cellulose chromatog-raphy revealed two fractions. The partially pur-ified LPS fraction was resistant to ribonucle-ase, deoxyribonuclease, and Pronase, which in-dicated it was not a protein or nucleic acid. Themedian lethal dose (LD5,) in mice was found tobe 16.8 mg/kg ofbody weight. It was consideredto be less active than a comparable Salmonellatyphimurium LPS fraction.

Garcia et al. (32) also isolated an LPS frac-tion from F. necrophorum. They obtained anLD5n of 584 jig (29.2 mg/kg) compared to anLDw of 555 pg for E. coli. Electron microscopicstudies of the LPS fraction showed a matrix ofbranched, trilaminar ribbons. A lipid A ex-tract, solubilized with bovine serum, was foundto be highly toxic for 11-day-old chicken em-bryos. The investigators suggested that F. nec-rophorum had a classical gram-negative endo-toxin in which lipid A was the major toxiccomponent.The location of toxins in cell fractions of bo-

vine strains of F. necrophorum was studied byGarcia et al. (31). Using sonically treated cells,they determined toxic activity in the cell walland cytoplasmic fractions. The cell wall-associ-ated toxin was found to be heat stabile and wasconsidered to be an LPS. The cytoplasmic toxinwas found to be heat labile, hemolytic, and non-dialyzable. They suggested the cytoplasmic fac-tor may be either a high-molecular-weight pro-tein itself or bound to a protein.

Roberts (74) has described a leucocidal exo-toxin that was shown to destroy leukocytes mi-grating from blood vessels in the dermis of rab-bits, sheep, and guinea pigs. The exotoxin wasfound to consist of non-dialyzable macromole-cules, which were slowly inactivated by heatingat 1000C but not at 570C. Roberts demonstratedthat the leucocidin was not the same materialas the hemolysin by reacting the leucocidinwith antihemolytic antiserum. The antiseruminactivated the hemolysin but not the leucoci-din.The work of Garcia et al. (33) has shown that

a toxoid prepared from the cytoplasmic fractionofF. necrophorum is effective in producing im-munity to subsequent challenge with infectivedoses ofthe organism. This would indicate thatat least one factor responsible for the pathogen-icity ofF. necrophorum is located, or produced,intracellularly. From Roberts' (74) investiga-tions there is evidence of two separate exotox-ins, a leucocidin and a hemolysin.The leucocidin may be a significant compo-

nent of F. necrophorum as evidenced by Rob-erts' (74, 75) experiments. It has been notedthat swine, guinea pigs, and, to a lesser extent,

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sheep appear to be more resistant to F. necro-

phorum infections than other animals. Robertsinjected rabbits subcutaneously with F. necro-

phorum and observed the death of polymorpho-nuclear leucocytes and growth of the organismin tissue sections taken at intervals over sev-

eral hours. When he examined guinea pigs un-

der the same conditions, the leukocytes showedmuch less damage and there was little prolifer-ation of the bacteria. He obtained a similarreaction with the filtrate of cells grown in cul-ture, which demonstrated that the effects werenot dependent on the proliferation of the bac-terium in the animal tissue. It would certainlybe of interest to examine the relationship be-tween the leucocidin and the cytoplasmic toxoidof Garcia et al. (33) and to examine the appar-ent resistance of guinea pig leukocytes to both.Some investigators recognize two types ofF.

necrophorum, A and B. These two types are

differentiated by the ability to hemagglutinateerythrocytes. Type A will agglutinate chicken,human, and pigeon cells, whereas type B willnot. Type A strains are considered to be more

virulent for laboratory animals than type B(85).

J. N. Berg (personal communication) has in-dicated that type A strains belong to twogroups. One group is extremely virulent formice and produces a potent exotoxin (possiblythe leucocidin) and a potent endotoxin. Theother group of type A is much less virulent formice. It also produces a potent exotoxin but haslittle or no endotoxin activity.

Biochemical PropertiesF. necrophorum is indole positive, catalse

negative, methyl red negative, and Voges-Pros-kauer negative, reduces methylene blue, andforms hydrogen sulfide. Casein Is digest, anda soft clot is formed in litmus milk which maybe digested. Nitrates are not reduced, eseulin is

not hydrolyzed, and gelatin liquification is var-

iable. Threonine and lactate are broken downwith the production of propionic acid and gas.In peptone-yeast-glucose broth, major amountsof butyric acid and lesser amounts of acetic,formic, and fumaric acids are produced (14,105). Sugar fermentation reactions are variableand often weak. Most authors have reportedthe fermentation of glucose and variable fer-mentation of fructose. Wilson and Miles (105)also note variable reactions in maltose and le-vulose, and Werner (97) states that hexoses are

fermented (see Table 2).F. necrophorum can be differentiated from

other species of Fusobacterium by both thepresence of indole and the production of pro-pionic acid from lactate.

Porschen and 80tntag (69) have demon-strated the production of extracellular deoxyri-bonuclease in strains ofFusobacterium isolatedfrom clinical material, Porschen and Spaulding(70) were not able to demonstrate phosphataseactivity in a clinical isolate ofF. necrophorumbut found a strong positive reaction with F.mortiferum and weak reactiots with a fewother species.Wahren et al. (93) showed that . ntecropho-

rum possessed proteolytk enzymes, which werebound to the cell wall, They also observed thatthe proteolytic activity (inferring production)was related to the amount of glucose in themedium, decreasinig when high concentrationswere added. The addition of sucrose had nosuch eftect. Tho possibility that more than oneproteolytic enzyme was being synthesized wassuggested to explain this observation.Wahren (92) found P, necrophorum capable

of storing large anounts of polyglucose (mostlikely as glycogen), which increased as cellgrowth decreased. She also noted that theamount of polyglucose was doubled when thecells were grown at 37 to39XC compared withwhen cells were grown at 33TC. Polyglucosecould reach as much as 60% of the dry cellweight.Werner (99) determined that strains of F.

hecrophorum, PF. mortiferum, Leptotrichia,and Bacteroides were lacking in lysine decar-boxylase. F. varium was shown to have lysinedecarboxylase activity.

Pritsche and J3oehmer (29) analyzed lipidsextracted from whole cells and determined thatunlike Bacteroides, Fusobacterium did not pos-sess sphingosine bases or branched pentadecan-oic acid. It was further demonstrated (28) thatFusobacterium did not synthesize 3-hydroxyfatty acids as did Bacteroides, and it was sug-gested that this difference may be useful forseparating the two genera.

Serological PropertiesThe investigation of serological reactions

among Fusobacterium was initiated mainly forthe study of taxonomic relationships.

Orcutt (66) examined the agglutination reac-tions ofnine strains ofF. necrophorum isolatedfrom cows. She observed at least seven differentcross-agglutination patterns among thestrains. Antiserum prepared with one of thecultures appeared to possess agglutinins foreach of the other strains. No biochemical differ-ences (sugar fermentations, indole production,catalase production, etc.) were observed amongthe strains, and all nine strains were consid-ered to be one species. Animal pathogens weredesignated Sphaerophorus necrophorus, and

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F. NECROPHORUM AS AN ANIMAL PATHOGEN 379

TALz 2. Biochemical reactions ofFusobacterium, B. fragilis, and L. buccalisa

Phopha Shino-Lysine DNAOrgnis Indoleb Glucose, Maltose" Fructose" Esculin" Lipase" DNasec Phsed Sphingo decar- G+Ctased lipidse boxylasef (mol%)o

Fgonidaormis + + - - - - + V 0 0 0. mrtiferurn - + + + + - + + - - 26-28

F. napiforme + - - - - - + W 0 0 0F.nerophorum + W - V - + + - - - 31-34F. nueleatum + W - W - - + - 0 0 27-28F. rusi - - - - - - + W 0 0 30F. Parium + W - W - - + - - + 29B.frqgilw - + + + + - + + + _ 43L. buccalis - + + + + - 0 0 0 - 31-34

vSymbols: M,Most strains positive; -, most strains negative; W, weak reaction; V, variable reactions; 0, not reported.From Holdenan and Moore (46) and Smith (86).From PorPchen and Sonntag (70). DNase, Deoxyribonuclease.

" rom Porschen and Spaulding (71).From Fritsche and Thelen (30).

'From Werner (100).'From Buchanan and Gibbons (14) and Prevot et al. (74). DNA, Deoxyribonucleic acid; G+C, guanine plus cytosine.

anwobic fusiforms isolated from humans werealld S funduliforrzs- Ai 1939 Prevot AdKichheimer ( w),uing a trichioroacetic acidegrwoton prqcedure, demonptratd the pres-onee of a comupon Antigen in isolates of S.nerphus and S. funduliformis. The anti-gen was considered to be an LPS since it waseXtractable in trichloroacetic acid, was non-di-yzable, and could be precipitated with abso-

lute alcohol or acetone. However, they main-tained that these strains still represented twodifferent species. To discover antigenic proper-ties useful for diagnostic procedures, Wernerand Sebald (103) reviewed the literature con-cerning serological studies of gram-negative,nonsporeforming, anaerobic bacilli since 1927.They determined that previous studies were toovaried, and no significant conclusions could bemade. Using stock cultures from several differ-ent laboratories, they examined many strainsofFusobacterium and Bacteroides for antigenicreactions as measured by agglutination andprecipitin tests. Antisera were produced in rab-bits by either subcutaneous or intraveneous in-jection of either bacterial suspensions or super-natant fractions from sonically treated cells.They determined that both heat-labile andheat-stabile antigens were present and ob-served heterologous agglutination reactionsamong the strains ofFusobacterium. Their re-sults indicated that most agglutinogens weretype specific. No cross-reactions occurredamong any of the Fusobacterium and Bacte-roides strains tested.

Caselitz et al. (16) compared 10 strains ofF.necrophorum isolated from human clinicalspecimens and could not detect a common anti-gen. Five of the strains showed some cross-reactivity. Wattre et al. (96), however, havedemonstrated, using gel diffusion and immuno-

electrophoretic techniques, that three strains ofF. necrophorum have common antigens. Linesof precipitation indicated that at least four dis-tinct antigens were present for one of thestrains. Two of these were shared by the secondstrain and three by the third.Werner (98) conducted a serological study to

justify the separation of three species of Fuso-bacterium. He observed that F. necrophorumstrains autoagglutinated in phosphate-bufferedsaline but was able to identify strain-specificand common antigens between F. varium andF. mortiferum. Gel diffusion procedures withextracts of F. necrophorum revealed specificantigens that were heat stabile and commonantigens that were heat labile.Garcia et al. (35) prepared antiserum to

whole cells ofF. necrophorum of bovine origin.When tested using a gel diffusion technique,the antiserum produced five precipitin bandswith a cytoplasmic cell fraction preparation ofantigen, three bands with an intact cell frac-tion, and one band with a crude cell wall frac-tion. Immunofluorescent studies with antiseraprepared from these same isolates showed spe-cific reactions with F. necrophorum cells inbovine tissues (liver abscesses, small intestine,gall bladder, mesentery lymph node, and ru-men contents of diseased and healthy cattle).The fluorescent antibodies were tested againstmore than 20 different bacterial species andreacted specifically only with other F. necro-phorum isolates.

Fales and Teresa (22) also examined the sero-logical relationship of F. necrophorum strainsusing immunofluorescent procedures. They tooobserved little cross-reactivity with other spe-cies of bacteria. However, reactions with 17bovine liver isolates ofF. necrophorum showeda greater degree of serological diversity. Com-

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mon antigens were found among 10 of the iso-lates, and the cross-reaction pattern for eachstrain was slightly different. None of thestrains reacted with other isolates ofFusobacte-rium spp. from stock culture collections.

Serological studies of F. necrophorum indi-cate the presence of a variety of antigens, someof which are common to more than one strain.Feldman et al. (25) determined four distinctantigenic groups among 14 strains ofF. necro-phorum isolated from bovine liver abscesses.Since neither flagella nor capsules are present,it appears probable that we are dealing with asomatic type of antigen.

Since investigators tested few strains in com-mon it is difficult to establish the antigenicrelationships among the many strains of F.necrophorum from an examination of the liter-ature. However, the observation that no cross-reactivity occurs between Fusobacterium andeither Bacteroides orLeptotrichia appears to beconsistent. What may exist is a situation simi-lar to the many and varied O-antigen determi-nants of the Salmonella genus. The specificityof serotypes in the Salmonella group provides amarker useful in etiological studies where theparticular strain is to be selectively identified.It has also been realized that certain serotypesare more frequently associated with specifichosts, e.g., S. choleraesius with swine, S.dublin with cattle, etc.One study investigating the presence of dif-

ferent antigens in Bacteroides has been re-cently published. Beerens et al. (7) have pre-pared antisera and classified 131 strains ofBac-teroides in the Pasteur Institute culture collec-tion. They have determined six major groupsand several minor subgroups. In a later studyRomond et al. (78) examined 58 strains ofBac-teroides from clinical specimens to determine ifa relationship existed between pathogenicityand serotype. They were not able to make adefinitive conclusion. However, they did nottest strains considered to be nonpathogenic.Such a classification may be extremely usefulin the study ofFusobacterium not only for taxo-nomic purposes, but practically for the identifi-cation of strains isolated from various sources.It would be prudent to investigate the serologi-cal types characterizing strains ofF. necropho-rum isolated from normal flora and from clini-cal specimens. Strains from different species ofanimals should also be compared.

Fluorescent antibody techniques have beenused in attempting to diagnose F. necrophoruminfections. Griffin (37) observed that antibodyprepared against S. necrophorus and S. fundu-liformis (both now considered F. necrophorum)did not cross-react. Garcia et al. (35) also testedvarious fluorescent antibodies prepared from

bovine liver isolates of F. necrophorum. Theyobtained reactions with F. necrophorumstrains isolated from bovine foot infections anda bovine liver abscess. No cross-reactions wereseen with other Fusobacterium spp. isolates.The observation of specific fluorescence in bothhealthy and diseased tissue from cattle sug-gests that for a diagnostic procedure refinementof this technique is necessary.

Fales and Teresa (22) prepared fluorescentantisera from several bovine on one humanisolate of F. necrophorum. The bovine strainsdid not cross-react with the human strain orwith various Spherophorus species that arenow considered to be F. necrophorum. Theysuggested the use of polyvalent antiserumwhen using this technique for determining thepresence of F. necrophorum in clinical speci-mens. The necessity for polyvalent antiserumwas appreciated by Stauffer et al. (89), whoexamined human clinical specimens for a reac-tion to Bacteroides antigens. The antisera werespecific for the various groups of Bacteroida-ceae involved in the study but still lacked activ-ity for some ofthe Bacteroides isolated from theclinical material.To differentiate S. necrophorus from S. fun-

duliformis, Beerens (5) examined 4 animal iso-lates and 14 human isolates for their ability toagglutinate erythrocytes obtained from variousanimal species. Two of the human strains (S.funduliformis) agglutinated human andguinea pig erythrocytes and one strain agglu-tinated rabbit erythrocytes. All four of the ani-mal bacterial isolates (S. necrophorus) agglu-tinated chicken, sheep, and human erythro-cytes; three strains also agglutinated bovine,rabbit, and guinea pig cells.Simon (80) tested 20 strains of F. necropho-

rum isolated from bovine liver abscesses. Hefound that all 20 strains agglutinated human,rabbit, and guinea pig erythrocytes, only twostrains agglutinated chicken erythrocytes, andnone agglutinated bovine or sheep erythro-cytes. The agglutination could be completelyinhibited by the addition of F. necrophorumantiserum. Many other bacterial species testedagglutinated rabbit erythrocytes and a few alsoagglutinated guinea pig and human erythro-cytes, but this could be eliminated by F. necro-phorum antisera, which inhibited hemaggluti-nation caused by F. necrophorum strains butnot hemagglutination caused by other strains.Warner et al. (94), in attempting to develop a

hemagglutination test for antibody determina-tion, observed that rabbit erythrocytes weremore active against different strains ofF. nec-rophorum after being sensitized with polyva-lent antigens.The occurrence of a specific hemagglutinin

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for chicken erythrocytes appears to be directlycorrelated with pathogenicity of F. necropho-rum (J. N. Berg, personal communication), andstrains have been separated into two typesbased on this characteristic (85). The virulent(for mice) type A is isolated more frequentlyfrom animals, and the less virulent type B isisolated more frequently from humans. If path-ogenicity is related to the cytoplasmic factordiscussed by Garcia et al. (33), it would be ofvalue to determine if a toxoid prepared from acytoplasmic fraction ofa type B strain conferredimmunity to challenge with a type A strain.Beerens et al. (6) noted that in vitro the loss

ofhemagglutination activity paralleled the lossof pathogenicity for mice in the several strainsofF. necrophorum examined. It was not deter-mined whether the two effects were coinciden-tal or whether they were the consequence of asingle component.The precipitin test for antibody determina-

tion presents problems in the case ofF. necro-phorum, since many strains autoagglutinate.Warner et al. (95) have described a hemaggluti-nation test that eliminated the problem of au-toagglutination, and which they consider moresensitive than a precipitin test. Formalinizedrabbit erythrocytes were sensitized with anti-genic preparations of F. necrophorum, whichwere then reacted with the serum of test ani-mals. If the antibodies were present, the eryth-rocytes clumped.The crux of these tests is the ability of the

prepared antigen to react with the antisera. Ifindeed there are several serological types ofF.necrophorum, it would be mandatory to use apolyvalent antigenic preparation. Until the ser-ology is resolved, it would be prudent to iden-tify F. necrophorum in clinical specimens byisolation where an infection is suspected andwhen examination for circulating antibodies isnegative.These results support previous observations

that many specific serotypes exist among theBacteroidaceae. In addition, these observationssuggest a need for a re-evaluation ofthe speciesF. necrophorum. Although the species may bebiochemically identical, the serological diver-sity may warrant further divisions. The pro-spective value of serological techniques as diag-nostic tools will require a more precise identifi-cation of the number of serotypes in existenceand commonly encountered.

Antibiotic Sensitivities

Chemotherapy of anaerobic infections is asubject of much current research since theirrole in human infections has become more evi-

dent. Lincomycin, clindamycin, minocycline,metronidazole, and, to a lesser extent, penicil-lin and carbenicillin have been shown to havein vivo activity against many gram-negative,nonsporeforming anaerobes in both human andanimal infections (88). Unfortunately, clinda-mycin has been shown to cause ulcerative coli-tis in humans (90), and metronidazole is a sus-pect carcinogen (17).

Chlortetracycline and tylosin appear to beuseful against F. necrophorum infections inanimals. But since liver abscesses and foot rotremain significant diseases, these antibioticsare far from satisfactory.The atypical in vitro activity of penicillin

againstF. necrophorum is interesting consider-ing that the organism is gram negative.Whether the sensitivity is a result of an in-creased permeability of the cell, charge on theLPS, or other factors has not been investigated.Hackman and Wilkins (39, 40) have demon-strated the in vivo protection of F. necropho-rum to the action of penicillin by beta-lacta-mase-producing strains of Bacteroides. Theyhave also suggested that the presence of L-forms ofF. necrophorum may be responsible forpersistent infection after penicillin therapy. Onthe other hand, Klieneberger-Nobel (55) andDienes (19) were not able to demonstrate patho-genicity for laboratory animals with their L-form isolates.

Isolation and CultivationF. necrophorum will grow only under anaer-

obic conditions. An atmosphere of 5 to 10%carbon dioxide enhances growth. The optimaltemperature is 370C, with a range of 30 to 400C.The addition of yeast, blood, or serum to themedium, plus a reducing agent such as cysteine,encourages growth. On blood agar, alpha- andbeta-hemolysis may be observed. We normallysee alpha-hemolysis on fresh media, but havenoticed that on older blood agar plates beta-hemolysis may occur.

Lahelle (see reference 86) first recommendedthe addition of 0.01% brilliant green and 0.02%crystal violet to human blood agar for the selec-tive isolation ofFusobacterium. Fales and Ter-esa (21) have developed a highly selective agarfor the isolation of F. necrophorum from liverabscesses in cattle. The medium consists of anegg yolk agar base supplemented with crystalviolet and phenethyl alcohol, the latter to in-hibit gram-negative facultative anaerobes.After incubation under carbon dioxide, coloniesare blue, surrounded by an opaque zone and aclear zone. Possible explanations for the occur-rence of these zones were discussed, but themechanism could not be determined. Others

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have suggested (see reference 3) the inclusion ofantibiotics such as erythromycin, vancomycin,kanamycin, and bacitracin in the agar mediumto inhibit facultative anaerobes.F. necrophorum may be isolated, as most

other anaerobes, on one of several commercialagars (Trypticase soy, Schaedler, brucella,brain heart infusion) with 5% sheep bloodadded and incubated at 370C in an anaerobicatmosphere such as a GasPak (BBL). On sheepblood agar, colonies are round and grey, withan entire edge. Size will vary from 1 to 3 mmdepending on the base agar used. Once iso-lated, F. necrophorum will grow rapidly influid thioglycolate or even better in any one ofseveral prereduced broths (peptone yeast,chopped meat, brain heart infusion) inoculatedunder deoxygenated carbon dioxide. Growth isfluffy, slowly sedimenting to the bottom of thetube, and gas is produced. The fetid odor ofbutyric acid is characteristic.Garcia and McKay (34) have grown large

batch cultures in a chemostat using a modifiedCasitone broth adjusted to pH 7.3 under carbondioxide and nitrogen. Simon (80) reported thatoptimal growth occurred in his medium 159 atpH 6.84, but that the optimal pH for acid pro-duction was at pH 7.7.

Cultures may be preserved by lyophilizationin skim milk or freezing and preserving at-60°C in calf serum (21). Viability and highvirulence for mice has been maintained in ourlaboratory (American Cyanamid Co., Prince-ton, N.J.) by subculturing monthly at roomtemperature using a commercially prepared,prereduced chopped-meat carbohydrate broth(Scott Labs, Fiskeville, R.I.) inoculated underdeoxygenated carbon dioxide.

ANIMAL DISEASES ANDCHEMOTHERAPYNatural Infections

F. necrophorum has been reported to be in-volved in diseases of cattle, sheep, goats, rab-bits, and wild animals (see references 9, 81).Such references must be treated cautiously,since many of the organisms mentioned arenow considered Bacteroides, Leptotrichia, orother genera; the etiological role of the Fuso-bacterium isolated in relation to the diseasedescribed was not established, or the descrip-tion of the organism (especially in older litera-ture) is insufficient and the cultures are nolonger extant to make a valid identification.For example, swine atrophic rhinitis wasthought to be caused by L-forms ofF. necropho-rum (60, 61), and these observations were being

cited even after a subsequent publication by thesame authors (15) suggested the etiologicalagent was a mycoplasma. The primary cause inthe United States in now considered to be Bor-detella bronchiseptica (79). Carter and McKay(15) suggested that the fusiform organisms ob-served in stained smears were most likely natu-ral inhabitants of the oral and nasal cavities.The type of disease associated with animals

is typified by necrosis of the tissues involved,abscess formation, and usually a characteristicputrid odor. Bacteremia can be present, theorganism may cause lesions, and F. necropho-rum has been isolated in pure culture fromalmost all organs including the brain (105).

Calf diphtheria, liver abscesses, and foot rotare the three manifestations ofF. necrophorumdiseases that have the most severe economicimpact. Losses due to calf diphtheria are diffi-cult to estimate, but losses of 17 million dollarsa year result from condemned beef livers andlosses due to foot rot are approximately onemillion dollars (52).

Calf diphtheria. The involvement of F. nec-rophorum in this disease was first recognized in1884 by Loeffler (58), who conducted a study todetermine the etiological agent ofdiphtheria byinvestigating clinical material from humans,doves, and calves. Loeffler was able to deter-mine that a different bacterium was responsi-ble in each host, although superficial similari-ties were evident in the clinical symptoms. Theterm calf diphtheria is a general description fora noncontagious necrotizing type of infectionwhich may involve the oral mucosa, tongue,pharangeal, and laryngeal areas (79). Invasionof F. necrophorum is thought to require sometype of damage to the oral cavity before theorganism can proliferate in the mucosa andsubmucosa to the underlying muscle tissue.The disease occurs more frequently in youngcalves (up to 2 years old) than in older cattle.Fatality may be a result of the larynx becomingoccluded with caseous material, inhibiting res-piration. Pieces of infectious material may beaspirated to the lungs, where abscesses orpneumonia may result. Except for lung in-volvement the infection is localized in the oralcavity. F. necrophorum can be seen in clinicalmaterial, but so far efforts to reproduce thedisease by introducing F. necrophorum cul-tures or infected tissue onto the larynx havebeen unsuccessful (52, 79).

Successful therapy with sulfa drugs was re-ported by Farquharson (23, 24), and the adventof antibiotics such as the tetracyclines hasgreatly reduced fatalities.Gay et al. (36) have reported a severe out-

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break of an oral disease in Canada under thename agranulocytopenic syndrome. The dis-ease is described as an oral necrosis typical ofan F. necrophorum infection, and fusiform bac-teria were profusely evident in the lesions. Thereason for a low granulocyte count in thesecalves is not discussed. No specific antibiotictreatment was mentioned, and mortalityranged from 30 to 100%.

Liver abscesses. The condemnation of ab-scessed livers in cattle presents a serious eco-nomic problem, hence the interest in the dis-ease and the etiological considerations. Liverabscesses do not appear to be a problem in othermeat animals, but there is a paucity of data onthe subject.

Liver abscesses in cattle attributed to a bac-terial infection were described in the late 1880s.The etiological agent was disputable becauseCorynebacterium pyogenes, F. necrophorum,streptococci, staphylococci, and E. coli were allreported to have been isolated from lesions. Nodoubt the inability to cultivate many anaerobesprevented some investigators from isolating F.necrophorum, although several described typi-cal F. necrophorum morphology in stainedsmears.Feldman et al. (25) were able to isolate F.

necrophorum from 44 of 50 liver abscesses andfound antibodies to F. necrophorum in serumsamples from 29 of 30 cattle in an area with ahistory of abscesses. Smith (84) proposed thatthere was a relationship between rumen ulcersand liver abscesses of cattle raised in feedlots.Forty-two percent of the cattle examined hadruminal lesions and liver abscesses, whereasonly 9% had liver abscesses without rumen le-sions. Gram-negative filamentous bacteriawere seen in both liver abscesses and ruminallesions. It was hypothesized that F. necropho-rum entered the liver via the portal circulation.Madin (62) was able to isolate F. necrophorumfrom 89%o of abscessed livers, but not from nor-mal livers, and did not consistently find otherorganisms. He also suggested that the portalcirculation was the mode of entry. Robinson etal. (77) examined range animals and feedlotanimals and concluded that feedlot conditionshad no relation to the occurrence of liver ab-scesses. They were not able to reproduce liverlesions by an oral or intravenous inoculation ofF. necrophorum, although the organism couldbe recovered from the rumen. (The origin of theF. necrophorum strain was not indicated.) Jen-sen and Mackey (52), on the other hand, notedthat the change from range feeding to a high-concentrate diet when in feedlots led to an in-creased acid production by the rumen flora.

Other conditions, such as the presence of metalobjects (nails, wire, etc.) may lead to irritationand ulceration of the rumen (50). F. necropho-rum may then invade the mucosa, and oftenthe underlying musculature, and thereby gainentry to the circulation.Jensen et al. (51) tried to produce liver ab-

scesses with a bovine isolate ofF. necrophorumby giving intraportal inoculations to cattle,sheep, and swine. Liver abscesses resulted incattle and sheep but not in swine. Attempts bySimon and Stovell (83) to isolate F. necropho-rum from liver abscesses in cattle resulted inorganisms from 97% of the samples, of which67% were pure cultures ofF. necrophorum, andHussein and Shigidi (48) recovered F. necro-phorum from 84% ofbovine liver abscesses, 57%of which were in pure culture. They also ob-served staphylococci and diphtheroids in someof the abscesses.The isolation of other organisms from liver

abscesses is not consistent, and what role, ifany, such organisms may have in the produc-tion of liver lesions has not yet been deter-mined.

Chemotherapy may reduce the incidence ofliver abscesses, but a definitive regimen forprevention has not been established.Matsushima et al. (64) examined the livers of

animals fed chlortetracycline for the control ofcalf scours. Animals that had received chloro-tetracycline for 12 weeks after birth had normallivers where previous experience had shown75% of livers to be scarred or abscessed. How-ever, Johnson et al. (54) noted that at slaughter20 of 684 livers from cattle fed chlortetracyclinecontained abscesses, and 23 of 681 control cattlehad abscesses. Brown et al. (13) tested tylosinin feedlot cattle receiving a high-concentrateration and found an 81% reduction in liver ab-scesses; in another study (12) using chlortetra-cycline and tylosin only a 13% reduction wasobserved.Foot rot. Foot rot is a disease of ungulates

characterized by necrotic tissue within and sur-rounding the hooves. F. necrophorum is impli-cated in most forms, although viral infectionsalso may be involved. The disease may be mildto severely debilitating, even requiring sur-gery. Occasionally the infection will spread andinvolve internal organs (79).Damp soil and injury to the foot appear to be

predisposing factors for the development of footrot. Garcia et al. (35) used the fluorescent anti-body technique to examine the survival of F.necrophorum in the soil. They could still see areaction after inoculated soil (with an 80% wa-ter-holding capacity) was maintained under an-

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aerobic conditions for 11 months at 4VC and 8months at 370C. Even under aerobic conditionsat the higher temperatures, reduction of fluo-resence did not occur until 18 weeks.Even though what appeared to be F. necro-

phorum was isolated from cases of foot rot andit was generally accepted as the etiologicalagent, attempts to reproduce the disease werenot too successful. Flint and Jensen (26) in-jected F. necrophorum isolated from bovineliver abscesses into the left common digital ar-tery of experimental cattle. The resultant infec-tions were compared with natural infections ofdiseased feet. The pathology of the experimen-tal animals showed infection of bones, joints,skin, and tendons, which was different from theskin and joint inflammation and necrosis ofinterdigital tissue observed in the naturallyinfected hooves.Johnson et al. (53) isolated gram-negative

anaerobes including F. necrophorum from only12 out of 34 cases of foot rot and also isolatedvarious other organisms whose contribution tothe disease was not established. Gupta et al.(38) also isolated many types of bacteria fromcattle with foot rot. In attempts to reproducethe disease, pure cultures and combinationswere used. Typical foot rot pathology was pro-duced with a combination of fusiform-like orga-nisms plus staphylococci. No infection was pro-duced with F. necrophorum alone (which was a"stock" culture).Parsonson et al. (68), working with ovine

interdigital dermatitis, isolated gram-negativerods, filamentous forms, gram-positive cocci,and diphtheroides (including C. pyogenes) fromabout 500 cases of ovine interdigital dermatitis.They were able to reproduce the disease byplacing F. necrophorum-soaked pads on scari-fied interdigital skin. F. necrophorum couldnot be isolated from sheep that had no historyof foot rot.Roberts (74, 75) considered F. necrophorum

the primary pathogen in ovine foot abscessesbut also frequently isolated E. coli and C. py-ogenes. In an elegant series of experiments, hedetermined the synergism between F. necro-phorum and C. pyogenes. F. necrophorum pro-duced a leucocidal exotoxin which allowed C.pyogenes to become established, and C. py-ogenes produced a filterable, heat-labile, non-dialyzable macromolecule which stimulated thegrowth and invasiveness of F. necrophorum.Parsonson et al. (68) had suggested that theproduction of catalase by diphtheroides loweredthe oxygen tension of the tissues, permitting F.necrophorum to grow. Roberts (74, 75), how-ever, used typically catalase-negative strains of

C. pyogenes. By definition, ovine interdigitaldermatitis is caused by F. necrophorum and C.pyogenes in association. The presence of ovineinterdigital dermatitis is indicated by red andswollen interdigital skin covered by a film ofnecrotic tissue (79).Berg and Loan (8), in clinically diagnosed

cases of foot rot in cattle, isolated F. necropho-rum and B. melaninogenicus as well as otherorganisms from all eight cases examined. Inexperimental infections with pure and mixedcultures, they obtained the most severe lesionswith a dual inoculum ofF. necrophorum and B.melaninogenicus; both were recovered from thelesions. Both organisms were also recoveredfrom the animals infected with F. necrophorumonly. Intradermal inoculation of F. necropho-rum plus C. pyogenes into nonscarified interdi-gital skin produced only superficial dermatitisand was not typical of foot rot. Neither C. py-ogenes nor B. melaninogenicus alone producedan infection.

It may be concluded that the concurrent in-fection by F. necrophorum plus one of severaldifferent types of bacteria produces varioustypes of foot infections. Foot rot as observed insheep, cattle, goats, and deer may begin as anF. necrophorum infection of the dermis. In cat-tle the occurrence of Bacteroides nodosus(which is sometimes considered an essentialadditional etiological agent) results in an infec-tion involving the hoof and underlying connec-tive tissues, eventually causing the hoof to be-come detached. A less severe form of the dis-ease is attributed to a less virulent strain ofB.nodosus (79).A foot rot of swine also occurs, especially

when the animals are kept on concrete. Pre-sumably this disease is caused by F. necropho-rum and a spirochete, but it requires furtherinvestigation (79).

Prevention of foot rot is preferable to havingto treat the disease. Most investigators agreethat damage to the foot by stones, sticks, stub-ble, etc., should be avoided and that feet shouldbe cared for, especially in the damp weather.Chemotherapy is usually confined to a foot dipin copper sulfate or chlortetracycline solution.Johnson et al. (54), studying the effect of chlor-tetracycline-supplemented feed on the perform-ance offeedlot cattle, observed that only 2 of 684animals on medicated feed developed foot rotcompared with 172 out of 681 cattle not receiv-ing medicated feed.Other diseases. Pathological conditions

where F. necrophorum may be a serious sec-ondary invader include: necrotic rhinitis ofswine, which usually occurs in young animals

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and requires traumatic damage to the nose;neonatal bacteremias, usually contractedthrough the umbilical cord in calves and lambs;and oral infections in many animals (84). Noneof these conditions have been extensively stud-ied.The pathogenesis ofF. necrophorum may in-

volve the following series of events. Initially,there is an interruption in the surface epithe-lium by which F. necrophorum gains entry. Asthe bacteria proliferate an exotoxin is producedwhich arrests and destroys the invading leuco-cytes, thus inhibiting phagocytosis and permit-ting the bacteria to grow. Necrosis of the localtissue ensues as toxins are produced and deoxy-ribonuclease may also be involved. Pus sur-rounds the area of necrotic tissue, and fibro-blasts form a layer of connective tissue aroundthe abscess. It has been observed in liver tissuethat the lesion may heal spontaneously as evi-dence by scar formation. Although the infectiontends to remain localized, F. necrophorum mayenter the blood stream and be carried to otherareas of the body, where it may lead to otherfoci of infection if predisposing factors are evi-dent. There has been no mention of invasion ofthe lymphatics in natural F. necrophorum in-fections.

Experimental Infections

To study and understand the role of a patho-gen in disease and to develop methods wherebythe pathogen may be controlled, it is desirableto reproduce the disease in the primary host orother suitable animal. One also must provethat the suspected pathogen is, indeed, the etio-logical agent of a particular disease.

Orcutt (66) was able to produce a lethal infec-tion in rabbits and mice with a subcutaneousinfection of F. necrophorum strains isolatedfrom cows, and in 1940 Prevot (see reference104) used an infection in rabbits to test theefficacy of sulfa drugs. Different early investi-gators had various success in producing lesionsin laboratory animals with isolates ofF. necro-phorum (see reference 9).

Flint and Jensen (26) inoculated a bovineliver abscess isolate ofF. necrophorum into theleft common digital artery of cattle in an effortto reproduce foot rot, but the resultant lesionswere not typical of the disease.Jensen et al. (51) were able to produce liver

abscesses in cattle and sheep by intraportalinoculation of F. necrophorum, but not inswine. No hypothesis for the recalcitrance ofswine was suggested.Although it may be preferable to investigate

disease processes in the host animal, it is notalways practical. Several investigators have re-cently developed laboratory animal models thatappear to be extremely useful for studying F.necrophorum. Wilkins and Smith (104) used anisolate from sheep foot rot to establish an infec-tion in mice. They determined the LD50 ofeither a subcutaneous or intraperitoneal routeof infection to be 106 cells. Mortality after 14days was 97%, with a mean survival time of10.2 days for the subcutaneous route and 88%mortality and a mean survival time of 5.5 daysfor the intraperitoneal route. Abscesses wereseen in liver, mesentery, pancreas, gonads, gutwall, and peritoneal membrane with the intra-peritoneal infection, and a local abscess in thethigh was observed with the subcutaneous in-fection.

In our laboratory the same strain has consist-ently produced 90 to 100% mortality when inoc-ulated intraperitoneally in mice. Necropsy re-vealed purulent abscesses mainly in the perito-neal cavity, liver, and kidney. Splenomegalywas also observed. The LD50 was 2.4 x 107 cells,and the mean survival time was 7.3 + 1.2 days.

Hill et al. (41) were able to produce liverabscesses in 70% of mice using a human isolateofF. necrophorum, but no deaths occurred. Thepercentage of lesions produced was enhancedby a mixed infection combining F. necropho-rum withB. melaninogenicus, B. fragilis, orF.nucleatum strains. Deaths were occasionallyobserved in mixtures using the latter strains.Kuck (56) also infected mice with human

strains of F. necrophorum. Infrequent mortal-ity was observed, but reproducible abscesseswere obtained with a subcutaneous inoculationat the base of the tail.Maestrone et al. (63) were able to produce

infections similar to those seen in cattle byinfecting mice intrahepatically, intrathoraci-cally, intraperitoneally, subcutaneously, or inthe plantar area of the footpad with severalfield strains of F. necrophorum from sheep orcattle.

Thoracic infection resulted in lung abscesseswith involvement of the pleural and pericardialcavities. Liver abscesses were observed withthe intrahepatic inoculation, and death oc-curred in 2 to 3 days after either route of inoc-ulation. Intraperitoneal inoculation causeddeath within 7 days, and autopsy revealed alarge amount of purulent exudate. Injection ofF. necrophorum into the footpad of mice re-sulted in a local necrotizing infection where thesoft tissues of the foot sloughed off. A reactionin the popliteal lymph nodes was also observed.These models have provided excellent in vivo

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systems for testing the effects of chemothera-peutic agents. Wilkins and Smith (104), withtheir model tested penicillin, tetracycline, clin-damycin, and lincomycin as typical antibioticsused against human clinical anaerobic infec-tions and found clindamycin to be the mosteffective and penicillin the least effective.Kuck (56), in her system, found minocycline

the most active in preventing abscesses. Clin-damycin was also found to be very active, butampicillin, cephalexin, and penicillin were notquite as effective.Maestrone et al. (63) found sulfadimethoxine

plus ormetoprin administered in the diet to bemost effective against each of their model F.necrophorum infections. AUREO S P 250(chlortetracycline, sulfamethazine, and penicil-lin, American Cyanamid Co.) and NEO-TERRA (oxytetracycline and neomycin, Pfizer,Inc.) were found to be more effective than TY-LAN-10 (tylosin and sulfamethazine, ElancoProducts Co.) against hepatic and thoracic in-fections. All four preparations were similarlyeffective when administered intraperitoneallyagainst the footpad infection. Since tylosin isknown to be poorly absorbed from the gut, oraltylosin treatment would not be indicated.

In the model developed in our laboratory,various chemotherapeutic agents were admin-istered either in the diet or intraperitoneally.Antibiotics effective in significantly reducingmortality were chlortetracycline, minocycline,clindamycin, penicillin, and lincomycin. Chlor-amphenicol was not found to be of therapeuticvalue. Other drugs, used in animal husbandry,that were able to suppress or eliminate lesionsand prevent mortality included carbadox, roni-dazole, dimetridazole, and ipronidazole. Tylo-sin and virginiamycin were only effective ifadministered intraperitoneally. Metronidazolewas found to be active by both routes of admin-istration.

IMMUNITY

Beveridge (9) attempted to protect rabbitsfrom an F. necrophorum infection by using a

formalinized preparation of cells and was notsuccessful in establishing immunity. He sug-gested that the pathogenicity of F. necro-phorum was attributable to the action of anendotoxin. At that time methods of protectionagainst endotoxins had not been developed.Jensen et al. (51) were not successful in pro-tecting sheep from developing liver abscessesby inoculation with culture filtrates ofF. necro-phorum. Other investigators also tried to de-velop immunological protection against F.

necrophorum infections in various animals, butresults were ineffectual (see reference 82).The possibility that antibodies formed

against natural exposure to F. necrophorumoffered little protection against infection wassuggested by Feldman et al. (25). They exam-ined the serum of laboratory and domestic ani-mals, both healthy and diseased of differingages, for the presence of antibodies to F. necro-phorum. In adult cattle with and without liverabscesses, agglutinins were found, but serumfrom calves usually had no agglutinins. Higheragglutination titers were also observed in se-rum from adult swine and sheep when thesewere compared with younger animals. Theredid not appear to be a relationship between thepresence of disease and the level of agglutina-tion achieved. Laboratory animals includingrats, dogs, and rabbits did not possess signifi-cant antibodies to F. necrophorum in serumsamples. These results suggest that exposure toF. necrophorum may lead to the formation ofantibodies, but these antibodies have little orno immunological significance.

Garcia et al. (33) have been able to protectcattle from developing liver abscesses by usinga toxoid prepared from the cytoplasmic fractionof sonically treated F. necrophorum cells. Atoxoid prepared from sonically treated wholecells did not significantly reduce the incidenceof liver abscesses. The amount of cytoplasmictoxoid administered appeared to be critical. Us-ing 5 to 10 cattle per group, they observed thatcontrol animals had an incidence of 35% liverabscesses or scars. A total dose of 10.5 mg ofcytoplasmic protein resulted in 30% ofthe cattlehaving liver damage, whereas 10% of the cattlereceiving a dose of 15.5 mg of protein showedevidence of liver damage. F. necrophorum wasisolated from the liver abscesses examined.Serum samples from animals taken during

the 6-month trial were examined by gel diffu-sion techniques for the presence of agglutinins.Thirty-five percent of the control animals dem-onstrated positive reactions compared with 74%of sera from animals receiving sonically treatedtoxoid, 70% of animals receiving the lower doseof cytoplasmic toxoid, and 95% of cattle receiv-ing the higher level of cytoplasmic toxoid.These observations may lead to the develop-ment of a vaccine useful in controlling F. necro-phorum infections.

It is apparent from the study of Dack et al.(18) that circulating antibodies to F. necropho-rum occur in adult animals, including humans,who have probably been exposed to this bacte-rium but have no overt signs of disease. Experi-mental evidence indicates that these antibodies

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offer little or no protection against infection.Calf diphtheria has been the only type of dis-ease involving F. necrophorum that appears tooccur more frequently in younger animals;many older animals are presumably immune.

Caselitz et al. (16) have studied the antibodytiters in three human patients with F. necro-phorum infections and observed an increase asthe disease progressed, whereas Feldman et al.(25) found little differences in the titers of cattlewith or without hepatic abscesses. They didsuggest that the lesions may have containedone or more antigenic types ofF. necrophorumwhich were not included i.. the test antigenpreparation.

CONCLUDING REMARKS

Means are now available for the isolation andidentification of certain strains ofF. necropho-rum. With the study of additional strains, fur-ther characterization of the cell wall antigens,the isolation and characterization of bacterio-phages specific for F. necrophorum, and thepreparation of antisera capable of sorting spe-cific antigenic types, the identification of thisgram-negative anaerobe should be possible inlaboratories concerned with anaerobic infec-tions.

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