BAcTKROLOGICAL REVIEWS, June 1977, p. 373-390 Vol. 41, No.

2
Copyright ©) 1977 American Society for Microbiology Printed in U.S.A.

Fusobacterium necrophorum: Its Characteristics and Role as

an Animal Pathogen
BARBARA F. LANGWORTH
American Cyanamid Company, Princeton, New Jersey 08540
INTRODUCTION.............................................................. 373
CLASSIFICATION ......... 374
DESCRIPTION OF F. NECR OPHORUM. 376
Cellular Morphology ................................... 376
Lipopolysaccharide ................................... 376

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Toxins ................................... 377
Biochemical Properties ........ ........................... 378
Serological Properties .................................... 378
Antibiotic Sensitivities ........ ............................ 381
Isolation and Cultivation .................................... 381
ANIMAL DISEASES AND CHEMOTHERAPY .................................. 382
Natural Infections .................................... 382
Calf diphtheria .................................... 382
Liver abscesses .................................... 383
Foot rot .......................................... 383
Other diseases .................................... 384
Experimental Infections ......... ........................... 385
IMMUNITY................................................................... 386
CONCLUDING REMARKS .................................... 387
LITERATURE CITED .................................... 387
INTRODUCTION pendently capable of causing disease. In natu-
Fusobacterium necrophorum is a gram-nega- ral infections with F. necrophorum other orga-
tive, nonsporeforming, nonmotile, strictly an- nisms are frequently isolated, but true syner-
aerobic, pleomorphic bacterium in the family gism has been described in only a few instances
Bacteroidaceae. Recognized as an animal path- (8, 74-76).
ogen since the late 1800s, F. necrophorum was The inability to subculture the organism
mainly described as occurring in liver abscesses hampered early investigators, and descriptions
in cattle, foot rot in many domestic animals, in the literature were mostly based on the fusi-
calf diphtheria (unrelated to the disease in hu- form morphology observed under the micro-
mans), and necrotic lesions in the oral cavity scope (see reference 57).
(see reference 82). F. necrophorum was also The study of anaerobic bacteria has been
known to survive in the soil of pastures, which greatly facilitated by the advent of the cultural
was an enigma, considering that it was a non- techniques of Hungate (47), who first developed
spore-forming anaerobe (35). a method for handling and cultivating anaero-
More recently, F. necrophorum has been iso- bic organisms by placing them under a stream
lated from the normal flora in the oral cavity, of deoxygenated carbon dioxide. His "roll-tube"
gastrointestinal tract, and genitourinary tract technique used a carbon dioxide-filled test tube
of humans and animals (85). When involved in coated with agar, the bacteria either being in
disease, it may cause localized necrotic lesions the agar or streaked on the surface. The sealed
and abscesses or be carried by the bloodstream tube was then placed in a standard incubator.
to internal tissues, causing necrosis and ab- This provided a closed anaerobic system under
scess formation (105). which the growth of colonies could be observed
Although the incidence in human disease is without their exposure to air. Holdeman and
not yet well established, F. necrophorum has Moore (45) further refined these techniques.
been isolated and cultivated from abscesses, They recognized the importance of collecting
blood, body fluids, and oral infections (3). specimens in containers devoid of oxygen and
F. necrophorum was originally thought to be processing the specimens as rapidly as possible.
a secondary invader requiring a previous infec- Holdeman and Moore (46) developed procedures
tion, wound, or other predisposing factor to for prereduced, anaerobically sterilized media
gain entry into the host. It has now been shown which, when used under a stream of deoxygen-
that pure cultures of F. necrophorum are inde- ated carbon dioxide, supported the growth of
373
374 LANGWORTH
most anaerobes. These techniques led to the
reevaluation of the taxonomy of anaerobic bac-
teria.
Descriptions of bacteria conforming to the
current definition of F. necrophorum have ap-
peared under the following names, of which
Spherophorus necrophorus is perhaps the most
common (the name Spherophorus is now con-
sidered invalid since it was initially used to
define two species of lichen [82]): Actinomyces
necrophorus, A. pseudonecrophorus, Bacillus
fundibuliforms, 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 family
Bacteriodaceae (gram-negative, anaerobic,
nonsporeforming rods) into four genera on the
basis of morphological criteria. Bacteroides
were simple rod-shaped cells with rounded
ends, Fusobacterium had pointed ends, Dialis-
ter had a diameter of less than 0.15 Mm, and
Spherophorus were pleomorphic rods. The Bac-
teroidaceae have now been divided into three
main genera based on the amount and type of
volatile fatty acids produced. Fusobacterium
are defined as producing major amounts of bu-
tyric acid, Leptotrichia as producing major
amounts of lactic acid, and Bacteroides as pro-
ducing a mixture of acids. F. necrophorum is
further differentiated from other species of Fu-
sobacterium by both the formation of indole
and the production of propionic acid from lac-
tate (14). Previously, F. necrophorum strains of
animal origin and strains ofhuman origin were
thought to be different species (5), but results
based on gas chromatographic analyses have
not substantiated this division (46).
The 16 species of Fusobacterium listed in the
eighth edition of Bergey's Manual (14) include
seven species that are encountered in clinical
specimens, F. gonidaformis, F. mortiferum, F.
naviforme, F. necrophorum, F. nucleatum, F.
russii, and F. varium, and nine other species
that are infrequently isolated from infectious
material (3, 14, 85). The species may be differ-
entiated on the basis of sugar fermentations
and biochemical reactions.
With contemporary techniques for isolation,
cultivation, and identification of anaerobes, it
BACTERIOL. REV.
seems appropriate to examine the literature on
the organisms that are now considered F. nec-
rophorum in order to collate the many diversi-
fied data and correlate various observations.
CLASSIFICATION
In the past, isolation and cultivation of an-
aerobic bacteria were hampered because of
their extreme susceptibility to oxygen. Cul-
tures were difficult to preserve, and therefore
little exchange of organisms between different
laboratories occurred. As a result, most descrip-
tions of anaerobes were based only on origin of
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the culture and morphology. Newly isolated
and named fusiforms were frequently the same
organisms as described previously, but because
they were reported by different investigators
who did not have access to similar strains for
comparison studies, they were mislabeled or
given a new name.
The first published description of F. necro-
phorum was by Loeffler who, in 1884, discussed
the 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 to
isolate the organism on a calf serum medium
but could not further subculture the bacteria.
In 1891 Bang isolated a fusiform bacillus from
abscessed livers of cattle, and Flugge, in a de-
scription of microorganisms in 1886, named the
organism Bacillus necrophorus (see reference
62). In 1905 Mohler and Washburn (see refer-
ence 26) thought that foot rot in cattle was also
caused by this "necrotic bacillus." Other de-
scriptions of fusiform bacilli reported in the
early literature include an account of orga-
nisms isolated from necrotic lesions of the
throat of humans by Vincent in 1896 and from
diseased appendixes by Veillon and Zuber in
1898. The organisms described were typical of
what is now called F. fusiforme (see reference
14).
In 1923 Knorr suggested the genus Fusobac-
terium for all gram-negative, anaerobic fusi-
form bacteria.
In 1955 Wilson and Miles (105) did not think
there was sufficient information to subdivide
the gram-negative, nonsporeforming, anaero-
bic bacteria and lumped them together under
the species F. fusiformis (Table 1).
Morphology was still the main criterion for
separation in 1957 in the 7th edition of Bergey's
Manual (11). The family Bacteroidaceae was
divided into three genera: the Bacteroides de-
fined as rods, Fusobacterium defined as pleo-
morphic filamentous bacteria with pointed
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
TABLs 1. Nomenclature of anaerobic, gram-negative, nonsporeforming, rod-shaped bacteria'
Reference
Prevot, 1938 (72) Family
Genus
Wilson and Miles, 1955 (106)
Breed, Murray, and Smith, 1957 (11) Family
Genus
Prevot, Turpin, and Kaiser, 1967 (74) Family
Genus
Buchanan and Gibbons, 1974 (14) Family
Genus
0 Modified from Aalbaek.
ends, and Spherophorus defined as pleo-
morphic filamentous bacteria with rounded
ends.
Several species of Sphaerophorus 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 from
liver abscesses in cattle, pigs, and lambs (18).
Since no significant biochemical differences
among the strains were observed and inocula-
tion into rabbits indicated varying degrees of
pathogenicity, they suggested that all the iso-
lates were one species. Prevot and Kirchheimer
(74) were in disagreement with Dack et al. as a
result of serological studies of the two species
and decided that even though a common anti-
gen was shared, the isolates were indubitably
two distinct species.
In 1944 Boe and Thjotta (10) examined the
relationship between Leptotrichia and Fuso-
bacterium, which they thought had been con-
fused by previous authors due to the similar
morphological characteristics. They deter-
mined that the strong fermentation of carbohy-
drates and lack of indole production by the
Leptotrichia compared to weak carbohydrate
fermentation and strong indole production by
the Fusobacterium were valid differentiating
characteristics. Serological studies, however,
demonstrated that the Fusobacterium and Lep-
totrichia were related.
In 1945 Lahelle and Thjotta (57) published a
study comparing the Fusobacterium, which
was defined as being of human origin, with
Actinomyces necrophorus (which they sug-
gested be renamed Necrobacterium), which
was an animal pathogen. Based on the results
375
Ristellaceae Spherophoraceae
Ristella Zuberella Fusiformis Fusocillus
I Sphaerophorus Sphaerocillus
Fusiform group
Bacteroidaceae
Bacteroides Sphaerophorus Fusobacterium
Ristellaceae Spherophoraceae
Ristella Capsularis Zuberella Sphaerophorus Sphaerocillus
Bacteroidaceae
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Bacteroides Fusobacterium Leptotrichia
of serological studies, Lahelle and Thjotta de-
termined that the human and animal isolates
were closely related.
The separation of strains based on a host
preference was again examined in 1954 by Be-
erens (5), who supported the distinction based
on 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 (human
isolates) agglutinated chicken or sheep erythro-
cytes, and only two agglutinated human eryth-
rocytes.
In a later paper Beerens et al. (6) included a
study ofS. pseudonecrophorus and ascertained
that each of three putative species was actually
a different phase of one species. The character-
istics for each were as follows: S. necrophorus
produced a hemagglutinin and a hemolysin and
was pathogenic for mice; S. funduliformis did
not produce a hemagglutinin, produced a he-
molysin, and was nonpathogenic for mice; and
S. 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, based
on observations of changes in these properties
during subcultures of strains over several
years, which to them suggested mutation from
the A to B to C phase.
The use of gas chromatography for the sepa-
ration of volatile fatty acids was described in
1952 by James and Martin (49) and recom-
mended for the identification of microorga-
nisms (on Mars) by Oyama in 1963 (67), after
which followed many papers analyzing the
fatty acid and metabolic products of various
bacteria.
Werner et al. (101) examined 27 strains of
fusiform bacteria and were able to separate
376 LANGWORTH
Fusobacterium from Leptotrichia based on the
production of butyric acid. They were of the
opinion that fermentations of various sugars
were not adequate to differentiate the species of
Fusobacterium. They also determined that
morphologically similar clinical isolates ofBac-
teroidaceae that produced large amounts of bu-
tyric acid had more biochemical characteristics
in common with strains considered Fusobacte-
rium than with strains labeled Bacteroides or
Leptotrichia. In a subsequent paper, Werner
(98) noted the butyric acid ratio among F. nec-
rophorum, F. varium, and F. mortiferum
ranged 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 the
separation of Fusobacterium and Bacteroides
could be based solely on the formation of isoval-
eric and isobutyric acids by the Bacteroides
strains. Fritsche (27) studied strains of Bacte-
roides that produced butyric acid and observed
that the amount formed was much less than
that produced by Fusobacterium. Further in-
vestigations (29, 100) led to the conclusion that
production of major amounts of isovaleric and
isobutyric acids should take precedence in the
classification of Bacteroides, and that Fusobac-
terium could be distinguished by the production
of major amounts of butyric acid.
The taxonomic position of species within the
genus Fusobacterium is still dubious. Ameri-
can authors follow the biochemical distinctions
from Bergey's Manual, whereas many Euro-
pean authors favor Prevot's more morphologi-
cally based nomenclature. Slight discrepancies
in sugar fermentations as published by differ-
ent authors may result, not from strain varia-
tion, but from insufficient control of the test
media as to pH, etc. For example, Simon (80)
discovered the optimal initial pH ofhis medium
for maximum acid production of F. necropho-
rum was 7.7, whereas the initial pH of most
media commonly used was much lower.
Deoxyribonucleic acid homology studies have
been adjunctive in determining the relation-
ships of many organisms and may certainly
have application here. Even though Prevot et
al. (73) list guanine-plus-cytosine ratios, the
relatedness of similar deoxyribonucleic acids
has 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 phage
activity with F. necrophorum, a phage specific
for F. varium and two phages specific for B.
fragilis have been isolated (65).
BACTERIOL. REV.
DESCRIPTION OF F. NECROPHORUM
Cellular Morphology
F. necrophorum is a gram-negative, nonspore-
forming, pleomorphic bacillus from 0.5 to 1.75
gtm in diameter, ranging from small, almost
coccoid bodies to filaments greater than 100 pm
long, with parallel sides and blunt or tapering
ends. The morphology will be affected by the
type 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 older
cultures and when growth is on agar. Swelling
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along the filaments or at one end has also been
observed. With standard stains, e.g., carbol
fuchsin, irregular staining or beading may be
seen (105). Older cultures appear to stain more
irregularly, and Beveridge (9) attributed the
loss of staining ability to aging and degenera-
tion of the cells.
L-forms ofF. necrophorum were first isolated
by Klieneberger-Nobel (55) and Dienes (19).
The former was of the opinion that the forms
obtained were symbionic or virus-like bodies
since she could propagate them without rever-
sion to bacilli. Smith et al. (86) carefully stud-
ied the filaments under electron microscopy
and observed that, in addition to division by
binary fission, the bacteria swelled, forming
round bodies from which delicate filaments
arose and segmented to yield L-forms. On sub-
culturing, they observed reversion back to the
pleomorphic filaments. It was noted that L-
forms could be isolated more easily if penicillin
was added to the medium.
Ernst reported seeing branched forms of F.
necrophorum in 1902 (20). This reference is
cited by many investigators who state that
branching was never observed by them and
that 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 was
seen too infrequently to be a significant charac-
teristic.
Lipopolysaccharide
Biochemical studies of cell wall lipopolysac-
charide (LPS) components by Hofstad et al. (43,
44) resulted in the identification of heptose and
2-keto-deoxyoctonate (KDO) from water and
phenol extracts. Further investigating the LPS
composition, Hofstad (42) was able to determine
the additional presence of galactose, glucose,
and glucosamine. In both investigations KDO
was not found in Bacteroides strains. On the
other hand Sonnenwirth et al. (87) were able to
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
detect KDO in the four strains of Bacteroides
tested. Possible procedural differences have
been suggested to explain this discrepancy.
(KDO and heptose are also important compo-
nents of the LPS core of Salmonella and Esche-
richia coli [59].)
Further characterizing the LPS fraction,
Garcia et al. (32) determined that it contained
23.5% hexose, 6.4% heptose, 8.8% hexosamines,
0.8% KDO, 2.4% protein, and no nucleic acids.
Toxins
Strains of F. necrophorum have been shown
to possess a classical cell wall LPS endotoxin.
Investigators have also demonstrated the pres-
ence of exotoxins: hemolysin, leucocidin, and a
cytoplasmic toxin. However, whether each exo-
toxin is a separate component has yet to be
determined.
Orcutt (66) demonstrated the presence of an
exotoxin in bovine isolates of F. necrophorum.
Intraperitoneal or intravenous injection of cul-
ture filtrates into mice and rabbits produced
illness 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 sheep
produced diarrhea and anorexia. No response
was observed in guinea pigs by either route of
administration. Endotoxin activity of the
strains was demonstrated in rabbits and guinea
pigs using a carbolized or formalinized prepara-
tion. Intradermal inoculation produced necrosis
of the superficial and deep layers of the skin.
Dack et al. (18) also produced abscesses in
rabbits using the supernatant from cultures of
bovine, human, and monkey strains. They
noted that a more severe response resulted
from the bovine strain preparations.
Hofstad and Kristofferson (43) isolated the
LPS fraction of F. necrophorum from labora-
tory strains. When injected intradermally, in-
oculation of preparations into rabbits did not
result in a pronounced Schwartzman reaction.
Warner et al. (95), however, using a bovine
isolate of F. necrophorum, were able to elicit a
Schwar zman reaction in rabbits. They pre-
pared a cell wall fraction and a soluble fraction.
Both were toxic for mice. The soluble fraction
was heat stabile and insensitive to treatment
with 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 high
molecular weight as determined by gel filtra-
377
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. The
median lethal dose (LD5,) in mice was found to
be 16.8 mg/kg of body weight. It was considered
to be less active than a comparable Salmonella
typhimurium LPS fraction.
Garcia et al. (32) also isolated an LPS frac-
tion from F. necrophorum. They obtained an
LD5n of 584 jig (29.2 mg/kg) compared to an
LDw of 555 pg for E. coli. Electron microscopic
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studies of the LPS fraction showed a matrix of
branched, trilaminar ribbons. A lipid A ex-
tract, solubilized with bovine serum, was found
to 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 toxic
component.
The location of toxins in cell fractions of bo-
vine strains of F. necrophorum was studied by
Garcia et al. (31). Using sonically treated cells,
they determined toxic activity in the cell wall
and cytoplasmic fractions. The cell wall-associ-
ated toxin was found to be heat stabile and was
considered to be an LPS. The cytoplasmic toxin
was 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 was
found to consist of non-dialyzable macromole-
cules, which were slowly inactivated by heating
at 1000C but not at 570C. Roberts demonstrated
that the leucocidin was not the same material
as the hemolysin by reacting the leucocidin
with antihemolytic antiserum. The antiserum
inactivated the hemolysin but not the leucoci-
din.
The work of Garcia et al. (33) has shown that
a toxoid prepared from the cytoplasmic fraction
of F. necrophorum is effective in producing im-
munity to subsequent challenge with infective
doses ofthe organism. This would indicate that
at least one factor responsible for the pathogen-
icity of F. 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 noted
that swine, guinea pigs, and, to a lesser extent,
378 LANGWORTH BACTERIOL. REV.
sheep appear to be more resistant to F. necro- Porschen and 80tntag (69) have demon-
phorum infections than other animals. Roberts strated the production of extracellular deoxyri-
injected rabbits subcutaneously with F. necro- bonuclease in strains of Fusobacterium isolated
phorum and observed the death of polymorpho- from clinical material, Porschen and Spaulding
nuclear leucocytes and growth of the organism (70) were not able to demonstrate phosphatase
in tissue sections taken at intervals over sev- activity in a clinical isolate of F. necrophorum
eral hours. When he examined guinea pigs un- but found a strong positive reaction with F.
der the same conditions, the leukocytes showed mortiferum and weak reactiots with a few
much less damage and there was little prolifer- other species.
ation of the bacteria. He obtained a similar Wahren et al. (93) showed that . ntecropho-
reaction with the filtrate of cells grown in cul- rum possessed proteolytk enzymes, which were
ture, which demonstrated that the effects were bound to the cell wall, They also observed that
not dependent on the proliferation of the bac- the proteolytic activity (inferring production)
terium in the animal tissue. It would certainly was related to the amount of glucose in the

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be of interest to examine the relationship be- medium, decreasinig when high concentrations
tween the leucocidin and the cytoplasmic toxoid were added. The addition of sucrose had no
of Garcia et al. (33) and to examine the appar- such eftect. Tho possibility that more than one
ent resistance of guinea pig leukocytes to both. proteolytic enzyme was being synthesized was
Some investigators recognize two types of F. suggested to explain this observation.
necrophorum, A and B. These two types are Wahren (92) found P, necrophorum capable
differentiated by the ability to hemagglutinate of storing large anounts of polyglucose (most
erythrocytes. Type A will agglutinate chicken, likely as glycogen), which increased as cell
human, and pigeon cells, whereas type B will growth decreased. She also noted that the
not. Type A strains are considered to be more amount of polyglucose was doubled when the
virulent for laboratory animals than type B cells were grown at 37 39XC to compared with
(85). when cells were grown at 33TC. Polyglucose
J. N. Berg (personal communication) has in- could reach as much as 60% of the dry cell
dicated that type A strains belong to two weight.
groups. One group is extremely virulent for Werner (99) determined that strains of F.
mice and produces a potent exotoxin (possibly hecrophorum, PF. mortiferum, Leptotrichia,
the leucocidin) and a potent endotoxin. The and Bacteroides were lacking in lysine decar-
other group of type A is much less virulent for boxylase. F. varium was shown to have lysine
mice. It also produces a potent exotoxin but has decarboxylase activity.
little or no endotoxin activity. Pritsche and J3oehmer (29) analyzed lipids
extracted from whole cells and determined that
Biochemical Properties unlike Bacteroides, Fusobacterium did not pos-
F. necrophorum is indole positive, catalse sess sphingosine bases or branched pentadecan-
negative, methyl red negative, and Voges-Pros- oic acid. It was further demonstrated (28) that
kauer negative, reduces methylene blue, and Fusobacterium did not synthesize 3-hydroxy
forms hydrogen sulfide. Casein Is digest, and fatty acids as did Bacteroides, and it was sug-
a soft clot is formed in litmus milk which may gested that this difference may be useful for
be digested. Nitrates are not reduced, eseulin is separating the two genera.
not hydrolyzed, and gelatin liquification is var-
iable. Threonine and lactate are broken down Serological Properties
with the production of propionic acid and gas. The investigation of serological reactions
In peptone-yeast-glucose broth, major amounts among Fusobacterium was initiated mainly for
of butyric acid and lesser amounts of acetic, the study of taxonomic relationships.
formic, and fumaric acids are produced (14, Orcutt (66) examined the agglutination reac-
105). Sugar fermentation reactions are variable tions of nine strains of F. necrophorum isolated
and often weak. Most authors have reported from cows. She observed at least seven different
the fermentation of glucose and variable fer- cross-agglutination patterns among the
mentation of fructose. Wilson and Miles (105) strains. Antiserum prepared with one of the
also note variable reactions in maltose and le- cultures appeared to possess agglutinins for
vulose, and Werner (97) states that hexoses are each of the other strains. No biochemical differ-
fermented (see Table 2). ences (sugar fermentations, indole production,
F. necrophorum can be differentiated from catalase production, etc.) were observed among
other species of Fusobacterium by both the the strains, and all nine strains were consid-
presence of indole and the production of pro- ered to be one species. Animal pathogens were
pionic acid from lactate. designated Sphaerophorus necrophorus, and
VOL, 41, i977 F. NECROPHORUM AS AN ANIMAL PATHOGEN 379
TALz 2. Biochemical reactions of Fusobacterium, B. fragilis, and L. buccalisa
Phopha Shino-Lysine G+C
DNA
Orgnis Indoleb Glucose, Maltose" Fructose" Esculin" Lipase" DNasec tased Sphingo
Phsed decar-
lipidse boxylasef (mol%)o
Fgonidaormis + + - - - - + V 0 0 0
. mrtiferurn - + + + + - + + - - 26-28
F. napiforme + - - - - - + W 0 0 0
F. nerophorum + W - V - + + - - - 31-34
F. nueleatum + W - W - - + - 0 0 27-28
F. rusi - - - - - - + W 0 0 30
F. Parium + W - W - - + - - + 29
B.frqgilw - + + + + - + + + _ 43
L. 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).

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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 were
alld S funduliforrzs- Ai 1939 Prevot Ad
Kichheimer ( w),uing a trichioroacetic acid
egrwoton 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 was
eXtractable 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 two
different species. To discover antigenic proper-
ties useful for diagnostic procedures, Werner
and Sebald (103) reviewed the literature con-
cerning serological studies of gram-negative,
nonsporeforming, anaerobic bacilli since 1927.
They determined that previous studies were too
varied, and no significant conclusions could be
made. Using stock cultures from several differ-
ent laboratories, they examined many strains
ofFusobacterium and Bacteroides for antigenic
reactions as measured by agglutination and
precipitin 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 and
heat-stabile antigens were present and ob-
served heterologous agglutination reactions
among the strains of Fusobacterium. Their re-
sults indicated that most agglutinogens were
type specific. No cross-reactions occurred
among any of the Fusobacterium and Bacte-
roides strains tested.
Caselitz et al. (16) compared 10 strains of F.
necrophorum isolated from human clinical
specimens and could not detect a common anti-
gen. Five of the strains showed some cross-
reactivity. Wattre et al. (96), however, have
demonstrated, using gel diffusion and immuno-
electrophoretic techniques, that three strains of
F. necrophorum have common antigens. Lines
of precipitation indicated that at least four dis-
tinct antigens were present for one of the
strains. Two of these were shared by the second
strain 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. necrophorum
strains autoagglutinated in phosphate-buffered
saline but was able to identify strain-specific
and common antigens between F. varium and
F. mortiferum. Gel diffusion procedures with
extracts of F. necrophorum revealed specific
antigens that were heat stabile and common
antigens that were heat labile.
Garcia et al. (35) prepared antiserum to
whole cells of F. necrophorum of bovine origin.
When tested using a gel diffusion technique,
the antiserum produced five precipitin bands
with a cytoplasmic cell fraction preparation of
antigen, three bands with an intact cell frac-
tion, and one band with a crude cell wall frac-
tion. Immunofluorescent studies with antisera
prepared from these same isolates showed spe-
cific reactions with F. necrophorum cells in
bovine tissues (liver abscesses, small intestine,
gall bladder, mesentery lymph node, and ru-
men contents of diseased and healthy cattle).
The fluorescent antibodies were tested against
more than 20 different bacterial species and
reacted specifically only with other F. necro-
phorum isolates.
Fales and Teresa (22) also examined the sero-
logical relationship of F. necrophorum strains
using immunofluorescent procedures. They too
observed little cross-reactivity with other spe-
cies of bacteria. However, reactions with 17
bovine liver isolates of F. necrophorum showed
a greater degree of serological diversity. Com-
380 LANGWORTH
mon antigens were found among 10 of the iso-
lates, and the cross-reaction pattern for each
strain was slightly different. None of the
strains 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, some
of which are common to more than one strain.
Feldman et al. (25) determined four distinct
antigenic groups among 14 strains of F. necro-
phorum isolated from bovine liver abscesses.
Since neither flagella nor capsules are present,
it appears probable that we are dealing with a
somatic type of antigen.
Since investigators tested few strains in com-
mon it is difficult to establish the antigenic
relationships among the many strains of F.
necrophorum from an examination of the liter-
ature. However, the observation that no cross-
reactivity occurs between Fusobacterium and
either Bacteroides orLeptotrichia appears to be
consistent. What may exist is a situation simi-
lar to the many and varied O-antigen determi-
nants of the Salmonella genus. The specificity
of serotypes in the Salmonella group provides a
marker useful in etiological studies where the
particular strain is to be selectively identified.
It has also been realized that certain serotypes
are more frequently associated with specific
hosts, 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 groups
and several minor subgroups. In a later study
Romond et al. (78) examined 58 strains of Bac-
teroides from clinical specimens to determine if
a relationship existed between pathogenicity
and serotype. They were not able to make a
definitive conclusion. However, they did not
test strains considered to be nonpathogenic.
Such a classification may be extremely useful
in 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 of
animals should also be compared.
Fluorescent antibody techniques have been
used in attempting to diagnose F. necrophorum
infections. Griffin (37) observed that antibody
prepared against S. necrophorus and S. fundu-
liformis (both now considered F. necrophorum)
did not cross-react. Garcia et al. (35) also tested
various fluorescent antibodies prepared from
BACTERIOL. REV.
bovine liver isolates of F. necrophorum. They
obtained reactions with F. necrophorum
strains isolated from bovine foot infections and
a bovine liver abscess. No cross-reactions were
seen with other Fusobacterium spp. isolates.
The observation of specific fluorescence in both
healthy and diseased tissue from cattle sug-
gests that for a diagnostic procedure refinement
of this technique is necessary.
Fales and Teresa (22) prepared fluorescent
antisera from several bovine on one human
isolate of F. necrophorum. The bovine strains
did not cross-react with the human strain or
with various Spherophorus species that are
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now considered to be F. necrophorum. They
suggested the use of polyvalent antiserum
when using this technique for determining the
presence of F. necrophorum in clinical speci-
mens. The necessity for polyvalent antiserum
was appreciated by Stauffer et al. (89), who
examined human clinical specimens for a reac-
tion to Bacteroides antigens. The antisera were
specific for the various groups of Bacteroida-
ceae involved in the study but still lacked activ-
ity for some ofthe Bacteroides isolated from the
clinical material.
To differentiate S. necrophorus from S. fun-
duliformis, Beerens (5) examined 4 animal iso-
lates and 14 human isolates for their ability to
agglutinate erythrocytes obtained from various
animal species. Two of the human strains (S.
fund uliformis) agglutinated human and
guinea 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. He
found that all 20 strains agglutinated human,
rabbit, and guinea pig erythrocytes, only two
strains agglutinated chicken erythrocytes, and
none agglutinated bovine or sheep erythro-
cytes. The agglutination could be completely
inhibited by the addition of F. necrophorum
antiserum. Many other bacterial species tested
agglutinated rabbit erythrocytes and a few also
agglutinated 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 but
not hemagglutination caused by other strains.
Warner et al. (94), in attempting to develop a
hemagglutination test for antibody determina-
tion, observed that rabbit erythrocytes were
more active against different strains of F. nec-
rophorum after being sensitized with polyva-
lent antigens.
The occurrence of a specific hemagglutinin
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
for chicken erythrocytes appears to be directly
correlated with pathogenicity of F. necropho-
rum (J. N. Berg, personal communication), and
strains have been separated into two types
based on this characteristic (85). The virulent
(for mice) type A is isolated more frequently
from animals, and the less virulent type B is
isolated more frequently from humans. If path-
ogenicity is related to the cytoplasmic factor
discussed by Garcia et al. (33), it would be of
value to determine if a toxoid prepared from a
cytoplasmic fraction of a type B strain conferred
immunity to challenge with a type A strain.
Beerens et al. (6) noted that in vitro the loss
of hemagglutination activity paralleled the loss
of pathogenicity for mice in the several strains
of F. necrophorum examined. It was not deter-
mined whether the two effects were coinciden-
tal or whether they were the consequence of a
single component.
The precipitin test for antibody determina-
tion presents problems in the case of F. 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 more
sensitive than a precipitin test. Formalinized
rabbit erythrocytes were sensitized with anti-
genic preparations of F. necrophorum, which
were 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. If
indeed there are several serological types of F.
necrophorum, it would be mandatory to use a
polyvalent antigenic preparation. Until the ser-
ology is resolved, it would be prudent to iden-
tify F. necrophorum in clinical specimens by
isolation where an infection is suspected and
when examination for circulating antibodies is
negative.
These results support previous observations
that many specific serotypes exist among the
Bacteroidaceae. In addition, these observations
suggest a need for a re-evaluation ofthe species
F. necrophorum. Although the species may be
biochemically 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 existence
and commonly encountered.
Antibiotic Sensitivities
Chemotherapy of anaerobic infections is a
subject of much current research since their
role in human infections has become more evi-
381
dent. Lincomycin, clindamycin, minocycline,
metronidazole, and, to a lesser extent, penicil-
lin and carbenicillin have been shown to have
in vivo activity against many gram-negative,
nonsporeforming anaerobes in both human and
animal 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 be
useful against F. necrophorum infections in
animals. But since liver abscesses and foot rot
remain significant diseases, these antibiotics
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are 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 the
LPS, 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. They
have also suggested that the presence of L-
forms ofF. necrophorum may be responsible for
persistent infection after penicillin therapy. On
the other hand, Klieneberger-Nobel (55) and
Dienes (19) were not able to demonstrate patho-
genicity for laboratory animals with their L-
form isolates.
Isolation and Cultivation
F. necrophorum will grow only under anaer-
obic conditions. An atmosphere of 5 to 10%
carbon dioxide enhances growth. The optimal
temperature is 370C, with a range of 30 to 400C.
The addition of yeast, blood, or serum to the
medium, plus a reducing agent such as cysteine,
encourages growth. On blood agar, alpha- and
beta-hemolysis may be observed. We normally
see alpha-hemolysis on fresh media, but have
noticed that on older blood agar plates beta-
hemolysis may occur.
Lahelle (see reference 86) first recommended
the addition of 0.01% brilliant green and 0.02%
crystal violet to human blood agar for the selec-
tive isolation of Fusobacterium. Fales and Ter-
esa (21) have developed a highly selective agar
for the isolation of F. necrophorum from liver
abscesses in cattle. The medium consists of an
egg yolk agar base supplemented with crystal
violet and phenethyl alcohol, the latter to in-
hibit gram-negative facultative anaerobes.
After incubation under carbon dioxide, colonies
are blue, surrounded by an opaque zone and a
clear zone. Possible explanations for the occur-
rence of these zones were discussed, but the
mechanism could not be determined. Others
382 LANGWORTH
have suggested (see reference 3) the inclusion of
antibiotics such as erythromycin, vancomycin,
kanamycin, and bacitracin in the agar medium
to inhibit facultative anaerobes.
F. necrophorum may be isolated, as most
other anaerobes, on one of several commercial
agars (Trypticase soy, Schaedler, brucella,
brain heart infusion) with 5% sheep blood
added and incubated at 370C in an anaerobic
atmosphere such as a GasPak (BBL). On sheep
blood agar, colonies are round and grey, with
an entire edge. Size will vary from 1 to 3 mm
depending on the base agar used. Once iso-
lated, F. necrophorum will grow rapidly in
fluid thioglycolate or even better in any one of
several prereduced broths (peptone yeast,
chopped meat, brain heart infusion) inoculated
under deoxygenated carbon dioxide. Growth is
fluffy, slowly sedimenting to the bottom of the
tube, and gas is produced. The fetid odor of
butyric acid is characteristic.
Garcia and McKay (34) have grown large
batch cultures in a chemostat using a modified
Casitone broth adjusted to pH 7.3 under carbon
dioxide and nitrogen. Simon (80) reported that
optimal growth occurred in his medium 159 at
pH 6.84, but that the optimal pH for acid pro-
duction was at pH 7.7.
Cultures may be preserved by lyophilization
in skim milk or freezing and preserving at
-60°C in calf serum (21). Viability and high
virulence for mice has been maintained in our
laboratory (American Cyanamid Co., Prince-
ton, N.J.) by subculturing monthly at room
temperature using a commercially prepared,
prereduced chopped-meat carbohydrate broth
(Scott Labs, Fiskeville, R.I.) inoculated under
deoxygenated carbon dioxide.
ANIMAL DISEASES AND
CHEMOTHERAPY
Natural 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 are
now considered Bacteroides, Leptotrichia, or
other genera; the etiological role of the Fuso-
bacterium isolated in relation to the disease
described was not established, or the descrip-
tion of the organism (especially in older litera-
ture) is insufficient and the cultures are no
longer extant to make a valid identification.
For example, swine atrophic rhinitis was
thought to be caused by L-forms of F. necropho-
rum (60, 61), and these observations were being
BACTERIOL. REV.
cited even after a subsequent publication by the
same authors (15) suggested the etiological
agent was a mycoplasma. The primary cause in
the 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 characteristic
putrid odor. Bacteremia can be present, the
organism may cause lesions, and F. necropho-
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rum has been isolated in pure culture from
almost all organs including the brain (105).
Calf diphtheria, liver abscesses, and foot rot
are the three manifestations of F. necrophorum
diseases that have the most severe economic
impact. Losses due to calf diphtheria are diffi-
cult to estimate, but losses of 17 million dollars
a year result from condemned beef livers and
losses due to foot rot are approximately one
million dollars (52).
Calf diphtheria. The involvement of F. nec-
rophorum in this disease was first recognized in
1884 by Loeffler (58), who conducted a study to
determine the etiological agent of diphtheria by
investigating 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. The
term calf diphtheria is a general description for
a noncontagious necrotizing type of infection
which may involve the oral mucosa, tongue,
pharangeal, and laryngeal areas (79). Invasion
of F. necrophorum is thought to require some
type of damage to the oral cavity before the
organism can proliferate in the mucosa and
submucosa to the underlying muscle tissue.
The disease occurs more frequently in young
calves (up to 2 years old) than in older cattle.
Fatality may be a result of the larynx becoming
occluded with caseous material, inhibiting res-
piration. Pieces of infectious material may be
aspirated to the lungs, where abscesses or
pneumonia may result. Except for lung in-
volvement the infection is localized in the oral
cavity. F. necrophorum can be seen in clinical
material, but so far efforts to reproduce the
disease by introducing F. necrophorum cul-
tures or infected tissue onto the larynx have
been unsuccessful (52, 79).
Successful therapy with sulfa drugs was re-
ported by Farquharson (23, 24), and the advent
of antibiotics such as the tetracyclines has
greatly reduced fatalities.
Gay et al. (36) have reported a severe out-
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
break of an oral disease in Canada under the
name agranulocytopenic syndrome. The dis-
ease is described as an oral necrosis typical of
an F. necrophorum infection, and fusiform bac-
teria were profusely evident in the lesions. The
reason for a low granulocyte count in these
calves is not discussed. No specific antibiotic
treatment was mentioned, and mortality
ranged 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. Liver
abscesses do not appear to be a problem in other
meat animals, but there is a paucity of data on
the subject.
Liver abscesses in cattle attributed to a bac-
terial infection were described in the late 1880s.
The etiological agent was disputable because
Corynebacterium pyogenes, F. necrophorum,
streptococci, staphylococci, and E. coli were all
reported to have been isolated from lesions. No
doubt the inability to cultivate many anaerobes
prevented some investigators from isolating F.
necrophorum, although several described typi-
cal F. necrophorum morphology in stained
smears.
Feldman et al. (25) were able to isolate F.
necrophorum from 44 of 50 liver abscesses and
found antibodies to F. necrophorum in serum
samples from 29 of 30 cattle in an area with a
history of abscesses. Smith (84) proposed that
there was a relationship between rumen ulcers
and liver abscesses of cattle raised in feedlots.
Forty-two percent of the cattle examined had
ruminal lesions and liver abscesses, whereas
only 9% had liver abscesses without rumen le-
sions. Gram-negative filamentous bacteria
were seen in both liver abscesses and ruminal
lesions. It was hypothesized that F. necropho-
rum entered the liver via the portal circulation.
Madin (62) was able to isolate F. necrophorum
from 89%o of abscessed livers, but not from nor-
mal livers, and did not consistently find other
organisms. He also suggested that the portal
circulation was the mode of entry. Robinson et
al. (77) examined range animals and feedlot
animals and concluded that feedlot conditions
had no relation to the occurrence of liver ab-
scesses. They were not able to reproduce liver
lesions by an oral or intravenous inoculation of
F. necrophorum, although the organism could
be recovered from the rumen. (The origin of the
F. necrophorum strain was not indicated.) Jen-
sen and Mackey (52), on the other hand, noted
that the change from range feeding to a high-
concentrate diet when in feedlots led to an in-
creased acid production by the rumen flora.
383
Other conditions, such as the presence of metal
objects (nails, wire, etc.) may lead to irritation
and ulceration of the rumen (50). F. necropho-
rum may then invade the mucosa, and often
the underlying musculature, and thereby gain
entry to the circulation.
Jensen et al. (51) tried to produce liver ab-
scesses with a bovine isolate of F. necrophorum
by giving intraportal inoculations to cattle,
sheep, and swine. Liver abscesses resulted in
cattle and sheep but not in swine. Attempts by
Simon and Stovell (83) to isolate F. necropho-
rum from liver abscesses in cattle resulted in
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organisms from 97% of the samples, of which
67% were pure cultures ofF. necrophorum, and
Hussein and Shigidi (48) recovered F. necro-
phorum from 84% of bovine liver abscesses, 57%
of which were in pure culture. They also ob-
served staphylococci and diphtheroids in some
of the abscesses.
The isolation of other organisms from liver
abscesses is not consistent, and what role, if
any, such organisms may have in the produc-
tion of liver lesions has not yet been deter-
mined.
Chemotherapy may reduce the incidence of
liver abscesses, but a definitive regimen for
prevention has not been established.
Matsushima et al. (64) examined the livers of
animals fed chlortetracycline for the control of
calf scours. Animals that had received chloro-
tetracycline for 12 weeks after birth had normal
livers where previous experience had shown
75% of livers to be scarred or abscessed. How-
ever, Johnson et al. (54) noted that at slaughter
20 of 684 livers from cattle fed chlortetracycline
contained abscesses, and 23 of 681 control cattle
had abscesses. Brown et al. (13) tested tylosin
in feedlot cattle receiving a high-concentrate
ration and found an 81% reduction in liver ab-
scesses; in another study (12) using chlortetra-
cycline and tylosin only a 13% reduction was
observed.
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 infections
also may be involved. The disease may be mild
to severely debilitating, even requiring sur-
gery. Occasionally the infection will spread and
involve internal organs (79).
Damp soil and injury to the foot appear to be
predisposing factors for the development of foot
rot. Garcia et al. (35) used the fluorescent anti-
body technique to examine the survival of F.
necrophorum in the soil. They could still see a
reaction after inoculated soil (with an 80% wa-
ter-holding capacity) was maintained under an-
384 LANGWORTH
aerobic conditions for 11 months at 4VC and 8
months at 370C. Even under aerobic conditions
at 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 and
it was generally accepted as the etiological
agent, attempts to reproduce the disease were
not too successful. Flint and Jensen (26) in-
jected F. necrophorum isolated from bovine
liver abscesses into the left common digital ar-
tery of experimental cattle. The resultant infec-
tions were compared with natural infections of
diseased feet. The pathology of the experimen-
tal animals showed infection of bones, joints,
skin, and tendons, which was different from the
skin and joint inflammation and necrosis of
interdigital tissue observed in the naturally
infected hooves.
Johnson et al. (53) isolated gram-negative
anaerobes including F. necrophorum from only
12 out of 34 cases of foot rot and also isolated
various other organisms whose contribution to
the disease was not established. Gupta et al.
(38) also isolated many types of bacteria from
cattle with foot rot. In attempts to reproduce
the disease, pure cultures and combinations
were 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-negative
rods, filamentous forms, gram-positive cocci,
and diphtheroides (including C. pyogenes) from
about 500 cases of ovine interdigital dermatitis.
They were able to reproduce the disease by
placing F. necrophorum-soaked pads on scari-
fied interdigital skin. F. necrophorum could
not be isolated from sheep that had no history
of foot rot.
Roberts (74, 75) considered F. necrophorum
the primary pathogen in ovine foot abscesses
but also frequently isolated E. coli and C. py-
ogenes. In an elegant series of experiments, he
determined 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 the
growth and invasiveness of F. necrophorum.
Parsonson et al. (68) had suggested that the
production of catalase by diphtheroides lowered
the oxygen tension of the tissues, permitting F.
necrophorum to grow. Roberts (74, 75), how-
ever, used typically catalase-negative strains of
BACTERIOL. REV.
C. pyogenes. By definition, ovine interdigital
dermatitis is caused by F. necrophorum and C.
pyogenes in association. The presence of ovine
interdigital dermatitis is indicated by red and
swollen interdigital skin covered by a film of
necrotic 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 other
organisms from all eight cases examined. In
experimental infections with pure and mixed
cultures, they obtained the most severe lesions
with a dual inoculum of F. necrophorum and B.
melaninogenicus; both were recovered from the
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lesions. Both organisms were also recovered
from the animals infected with F. necrophorum
only. Intradermal inoculation of F. necropho-
rum plus C. pyogenes into nonscarified interdi-
gital skin produced only superficial dermatitis
and was not typical of foot rot. Neither C. py-
ogenes nor B. melaninogenicus alone produced
an infection.
It may be concluded that the concurrent in-
fection by F. necrophorum plus one of several
different types of bacteria produces various
types of foot infections. Foot rot as observed in
sheep, cattle, goats, and deer may begin as an
F. necrophorum infection of the dermis. In cat-
tle the occurrence of Bacteroides nodosus
(which is sometimes considered an essential
additional 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 of B.
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 further
investigation (79).
Prevention of foot rot is preferable to having
to treat the disease. Most investigators agree
that damage to the foot by stones, sticks, stub-
ble, etc., should be avoided and that feet should
be cared for, especially in the damp weather.
Chemotherapy is usually confined to a foot dip
in copper sulfate or chlortetracycline solution.
Johnson et al. (54), studying the effect of chlor-
tetracycline-supplemented feed on the perform-
ance of feedlot cattle, observed that only 2 of 684
animals on medicated feed developed foot rot
compared 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 of
swine, which usually occurs in young animals
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
and requires traumatic damage to the nose;
neonatal bacteremias, usually contracted
through the umbilical cord in calves and lambs;
and oral infections in many animals (84). None
of 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. As
the bacteria proliferate an exotoxin is produced
which arrests and destroys the invading leuco-
cytes, thus inhibiting phagocytosis and permit-
ting the bacteria to grow. Necrosis of the local
tissue 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 around
the abscess. It has been observed in liver tissue
that the lesion may heal spontaneously as evi-
dence by scar formation. Although the infection
tends to remain localized, F. necrophorum may
enter the blood stream and be carried to other
areas of the body, where it may lead to other
foci of infection if predisposing factors are evi-
dent. There has been no mention of invasion of
the 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 whereby
the pathogen may be controlled, it is desirable
to reproduce the disease in the primary host or
other suitable animal. One also must prove
that 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 subcutaneous
infection of F. necrophorum strains isolated
from cows, and in 1940 Prevot (see reference
104) used an infection in rabbits to test the
efficacy of sulfa drugs. Different early investi-
gators had various success in producing lesions
in laboratory animals with isolates ofF. necro-
phorum (see reference 9).
Flint and Jensen (26) inoculated a bovine
liver abscess isolate ofF. necrophorum into the
left common digital artery of cattle in an effort
to reproduce foot rot, but the resultant lesions
were not typical of the disease.
Jensen et al. (51) were able to produce liver
abscesses in cattle and sheep by intraportal
inoculation of F. necrophorum, but not in
swine. No hypothesis for the recalcitrance of
swine was suggested.
Although it may be preferable to investigate
385
disease processes in the host animal, it is not
always practical. Several investigators have re-
cently developed laboratory animal models that
appear to be extremely useful for studying F.
necrophorum. Wilkins and Smith (104) used an
isolate from sheep foot rot to establish an infec-
tion in mice. They determined the LD50 of
either a subcutaneous or intraperitoneal route
of infection to be 106 cells. Mortality after 14
days was 97%, with a mean survival time of
10.2 days for the subcutaneous route and 88%
mortality and a mean survival time of 5.5 days
for the intraperitoneal route. Abscesses were
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seen in liver, mesentery, pancreas, gonads, gut
wall, and peritoneal membrane with the intra-
peritoneal infection, and a local abscess in the
thigh 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. Splenomegaly
was 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 liver
abscesses in 70% of mice using a human isolate
ofF. necrophorum, but no deaths occurred. The
percentage of lesions produced was enhanced
by a mixed infection combining F. necropho-
rum withB. melaninogenicus, B. fragilis, orF.
nucleatum strains. Deaths were occasionally
observed in mixtures using the latter strains.
Kuck (56) also infected mice with human
strains of F. necrophorum. Infrequent mortal-
ity was observed, but reproducible abscesses
were obtained with a subcutaneous inoculation
at the base of the tail.
Maestrone et al. (63) were able to produce
infections similar to those seen in cattle by
infecting mice intrahepatically, intrathoraci-
cally, intraperitoneally, subcutaneously, or in
the plantar area of the footpad with several
field strains of F. necrophorum from sheep or
cattle.
Thoracic infection resulted in lung abscesses
with involvement of the pleural and pericardial
cavities. Liver abscesses were observed with
the intrahepatic inoculation, and death oc-
curred in 2 to 3 days after either route of inoc-
ulation. Intraperitoneal inoculation caused
death within 7 days, and autopsy revealed a
large amount of purulent exudate. Injection of
F. necrophorum into the footpad of mice re-
sulted in a local necrotizing infection where the
soft tissues of the foot sloughed off. A reaction
in the popliteal lymph nodes was also observed.
These models have provided excellent in vivo
386 LANGWORTH
systems for testing the effects of chemothera-
peutic agents. Wilkins and Smith (104), with
their model tested penicillin, tetracycline, clin-
damycin, and lincomycin as typical antibiotics
used against human clinical anaerobic infec-
tions and found clindamycin to be the most
effective 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, but
ampicillin, cephalexin, and penicillin were not
quite as effective.
Maestrone et al. (63) found sulfadimethoxine
plus ormetoprin administered in the diet to be
most 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, Elanco
Products Co.) against hepatic and thoracic in-
fections. All four preparations were similarly
effective when administered intraperitoneally
against the footpad infection. Since tylosin is
known to be poorly absorbed from the gut, oral
tylosin 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 reducing
mortality were chlortetracycline, minocycline,
clindamycin, penicillin, and lincomycin. Chlor-
amphenicol was not found to be of therapeutic
value. Other drugs, used in animal husbandry,
that were able to suppress or eliminate lesions
and prevent mortality included carbadox, roni-
dazole, dimetridazole, and ipronidazole. Tylo-
sin and virginiamycin were only effective if
administered intraperitoneally. Metronidazole
was found to be active by both routes of admin-
istration.
IMMUNITY
Beveridge (9) attempted to protect rabbits
from an F. necrophorum infection by using a
formalinized preparation of cells and was not
successful in establishing immunity. He sug-
gested that the pathogenicity of F. necro-
phorum was attributable to the action of an
endotoxin. At that time methods of protection
against endotoxins had not been developed.
Jensen et al. (51) were not successful in pro-
tecting sheep from developing liver abscesses
by inoculation with culture filtrates ofF. necro-
phorum. Other investigators also tried to de-
velop immunological protection against F.
BACTISRIOL. REV.-
necrophorum infections in various animals, but
results were ineffectual (see reference 82).
The possibility that antibodies formed
against natural exposure to F. necrophorum
offered little protection against infection was
suggested by Feldman et al. (25). They exam-
ined the serum of laboratory and domestic ani-
mals, both healthy and diseased of differing
ages, for the presence of antibodies to F. necro-
phorum. In adult cattle with and without liver
abscesses, agglutinins were found, but serum
from calves usually had no agglutinins. Higher
agglutination titers were also observed in se-
rum from adult swine and sheep when these
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were compared with younger animals. There
did not appear to be a relationship between the
presence of disease and the level of agglutina-
tion achieved. Laboratory animals including
rats, dogs, and rabbits did not possess signifi-
cant antibodies to F. necrophorum in serum
samples. These results suggest that exposure to
F. necrophorum may lead to the formation of
antibodies, but these antibodies have little or
no immunological significance.
Garcia et al. (33) have been able to protect
cattle from developing liver abscesses by using
a toxoid prepared from the cytoplasmic fraction
of sonically treated F. necrophorum cells. A
toxoid prepared from sonically treated whole
cells did not significantly reduce the incidence
of liver abscesses. The amount of cytoplasmic
toxoid administered appeared to be critical. Us-
ing 5 to 10 cattle per group, they observed that
control animals had an incidence of 35% liver
abscesses or scars. A total dose of 10.5 mg of
cytoplasmic protein resulted in 30% ofthe cattle
having liver damage, whereas 10% of the cattle
receiving a dose of 15.5 mg of protein showed
evidence of liver damage. F. necrophorum was
isolated 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 treated
toxoid, 70% of animals receiving the lower dose
of 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
VOL. 41, 1977 F. NECROPHORUM AS AN ANIMAL PATHOGEN
offer little or no protection against infection.
Calf diphtheria has been the only type of dis-
ease involving F. necrophorum that appears to
occur more frequently in younger animals;
many older animals are presumably immune.
Caselitz et al. (16) have studied the antibody
titers in three human patients with F. necro-
phorum infections and observed an increase as
the disease progressed, whereas Feldman et al.
(25) found little differences in the titers of cattle
with or without hepatic abscesses. They did
suggest that the lesions may have contained
one or more antigenic types of F. necrophorum
which were not included i.. the test antigen
preparation.
CONCLUDING REMARKS
Means are now available for the isolation and
identification of certain strains of F. 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 the
preparation of antisera capable of sorting spe-
cific antigenic types, the identification of this
gram-negative anaerobe should be possible in
laboratories concerned with anaerobic infec-
tions.
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