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Pathology of Clostridium perfringens Type C Enterotoxemia in Horses

S. S. Diab, H. Kinde, J. Moore, M. F. Shahriar, J. Odani, L. Anthenill, G. Songer and F. A. Uzal
Vet Pathol 2012 49: 255 originally published online 18 April 2011
DOI: 10.1177/0300985811404710

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 Veterinary Pathology
49(2) 255-263
Pathology of Clostridium perfringens ª The American College of
Veterinary Pathologists 2012
Reprints and permission:
Type C Enterotoxemia in Horses sagepub.com/journalsPermissions.nav
DOI: 10.1177/0300985811404710
http://vet.sagepub.com

S. S. Diab1, H. Kinde1, J. Moore1, M. F. Shahriar1, J. Odani1,

L. Anthenill1, G. Songer2, and F. A. Uzal1

Abstract
Clostridium perfringens type C is an important cause of enteritis and enterocolitis in foals and occasionally in adult horses.
The disease is a classic enterotoxemia, and the enteric lesions and systemic effects are caused primarily by beta toxin, 1 of 2 major
toxins produced by C. perfringens type C. Until now, only sporadic cases of C. perfringens type C equine enterotoxemia have been
reported. We present a comprehensive description of the lesions in 8 confirmed cases of type C enterotoxemia in foals and adult
horses. Grossly, multifocal to segmental hemorrhage and thickening of the intestinal wall were most common in the small
intestine, although the colon and cecum were also frequently affected. All horses had variable amounts of fluid, often hemorrhagic
intestinal contents. The most characteristic microscopic lesion was necrotizing or necrohemorrhagic enteritis, with mucosal and/
or submucosal thrombosis. Numerous gram-positive rods were occasionally seen in affected mucosa. A definitive diagnosis of
C. perfringens type C enterotoxemia in all 8 cases was based on the clinical history, gross and histologic lesions, and detection
of the beta toxin in intestinal contents.

Keywords
beta toxin, Clostridium perfringens, colitis, diarrhea, enteritis, enterotoxemia, horses

Enteritis, enterocolitis, and colitis, manifested clinically by
diarrhea and colic, are important causes of morbidity and
mortality in foals and adult horses. These conditions have been
associated with various causes, including Clostridium spp,
Salmonella spp, Ehrlichia risticii, Aeromonas spp, Lawsonia
intracellularis, cantharidin toxicosis, and larval cyathostomia-
sis.31 Together with Salmonella spp, clostridia, including
Clostridium perfringens and Clostridium difficile, are the most
important agents of equine enterocolitis.
The first reports associating clostridia with enteric disease in
foals were published in the 1930s,13,15 but in the past few
decades, C. perfringens and C. difficile have been increasingly
reported as relevant pathogens involved in equine enteritis and
enterocolitis.2,6,7,18,21,26,31,32
C. perfringens is classified into 5 types (A, B, C, D, and E)
based on the production of 1 or more of 4 so-called major tox-
ins: alpha (CPA), beta (CPB), epsilon (ETX), and iota (ITX).
Two other major toxins, enterotoxin (CPE) and beta 2 (CPB2),
can be produced by all types of C. perfringens but are not used
in its classification. Although C. perfringens types A, B, and C
have been associated with enterocolitis in foals, type C is the
most commonly reported clostridial enteric pathogen in foals
in North America.2,7,10 C. perfringens type C produces major
toxins CPA and CPB. The former is a lecithinase (phospholi-
pase C), which is considered the main virulence factor in
C. perfringens type A–associated myonecrosis in humans and
animals. However, the contribution of CPA to the virulence
of type C isolates is negligible. This was demonstrated by
inoculation of rabbits and mice with C. perfringens type C
CPA-null mutants (type C isolates genetically modified to pro-
duce all toxins except CPA), with no changes observed in the
virulence of the organism.20 CPB, however, is a necrotizing
toxin that forms membrane pores in susceptible cells. It is con-
sidered responsible for the intestinal necrosis and systemic
alterations in type C infections of several animal species,
including horses. The importance of CPB toxin was corrobo-
rated by lack of virulence in animals experimentally inoculated
with CPB-null mutants of type C isolates.20
Disease caused by C. perfringens type C in many mamma-
lian species, including humans, originates when type C strains
proliferate and produce toxins in the intestine.29 C. perfringens
type C causes severe intestinal damage, but death is thought to
1
California Animal Health and Food Safety Laboratory System, San Bernardino
Branch, University of California, Davis, California
2
College of Veterinary Medicine, Iowa State University, Ames, Iowa
Corresponding Author:
F. A. Uzal, California Animal Health and Food Safety Laboratory System,
University of California, Davis, 105 West Central Avenue, San Bernardino,
CA 92408
Email: fuzal@cahfs.ucdavis.edu

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256 Veterinary Pathology 49(2)

Table 1. Signalment, History, and Lesion Distribution in the Intestinal Tract in 8 Horses With Clostridium perfringens Type C Enterotoxemia

Lesion Distribution

No. Age at Death Breed Sex History Death Small Intestine Colon Cecum

1 5 months Paint Male Sudden death, no clinical signs Spontaneous Yes No Yes
observed
2 3 years Friesian Male Acute colic, fever, cardiovascular Spontaneous Yes Yes Yes
collapse with tachycardia,
tachypnea, purple mucous
membranes, poor peripheral
pulse
3 7 days Crossbred Female Acute foul diarrhea and colic Spontaneous Yes Yes Yes
4 6 years Thoroughbred Female Acute colic during surgery Euthanasia Yes No No
recovery, profuse sweating,
tachypnea, tachycardia;
antibiotic treatment
5 2 days Unknown Female Acute diarrhea, colic, profound Euthanasia Yes No No
weakness and dehydration
6 2 days Thoroughbred Male Acute diarrhea, colic, acidosis, Spontaneous Yes No No
hyponatremia and azotemia
7 16 days Paint Male Acute illness Euthanasia No Yes No
8 5 months Miniature Male Endotoxemia, depression Euthanasia Yes No No

result mainly from absorption of toxins from the intestine into
the circulation.23,24,28 Therefore, type C infections are consid-
ered true enterotoxemias (ie, diseases produced by toxins gen-
erated in the intestine but absorbed into the systemic circulation
and acting on organs distant from the gastrointestinal tract).
Because CPB is highly susceptible to the action of tryp-
sin,12,14 neonatal animals are particularly susceptible to type
C infections, due to the low level of intestinal trypsin in the first
days of life and the presence of trypsin inhibitors in colostrum.
Type C disease also occurs in animals and humans that ingest
food with trypsin inhibitors and in patients with pancreatic
disease.12
In horses, a presumptive diagnosis of C. perfringens type
C enterotoxemia can be based on clinical history (acute onset
of diarrhea, colic, or sudden death) and gross and microscopic
lesions (necrotizing enteritis or enterocolitis). This presumptive
diagnosis can be reinforced by isolation of numerous C. per-
fringens type C from the small or large intestine. However,
because C. perfringens type C can be isolated from some
healthy horses, definitive diagnosis should be based on detec-
tion of CPB in intestinal contents.24 CPB can be detected in
enteric content by assays in mice or guinea pigs (less common
nowadays) or by in vitro methods based on enzyme immuno-
assays such as ELISA.24
Published descriptions of equine enterotoxemia caused by
C. perfringens type C, confirmed by CPB detection, have been
limited to case reports.6,9,16,18,22 A few reports7,10,27,32 have
described a larger number of animals, but the diagnosis was
based on pathology and isolation of C. perfringens type C with-
out toxin detection. In this retrospective study, we describe the
pathologic and other laboratory findings in 8 horses with C.
perfringens type C enterotoxemia that was confirmed by CPB
detection in intestinal contents.
Materials and Methods
Case Selection and Clinical History
Eight cases were selected from the archives of the San
Bernardino Branch of the California Animal Health and Food
Safety Laboratory System. All cases fulfilled the following
2 conditions: (1) equine carcasses received for necropsy
between 2003 and 2010 with a history of gastrointestinal
disease and death within 48 hours or sudden death without
clinical gastrointestinal disease and (2) positive ELISA test for
C. perfringens CPB in intestinal contents.
Table 1 presents age, breed, sex, clinical history, and distri-
bution of lesions in the gastrointestinal tract. Briefly, the 5 male
and 3 female horses included 6 foals (n ¼ 4,  16 days old) and
2 adults distributed among crossbred and 4 different breeds.
Most horses had acute illness, followed by death or euthanasia
within 2 days of the onset of clinical signs. One foal was found
dead without any clinical signs observed. Only 1 horse (No. 4)
had received antibiotics (drug not reported) 1 day before onset
of clinical signs. The 4 youngest foals (2 to 16 days old) had
been nursed by their dams from birth until onset of disease
and had not received any additional feed or supplement.
The remaining 4 horses (5 months to 6 years of age) had been
on a diet of alfalfa hay and a mixture of grains for at least
1 month before onset of disease. No major dietary changes or
stress factors were reported in any of the 8 cases.
Gross and Microscopic Pathology
A necropsy was performed on all horses within 6 to 24 hours of
death, and samples of lung, liver, kidney, spleen, stomach, and
small and large intestine in all cases, as well as brain, adrenal
gland, and skeletal muscle in most cases, were collected and

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Diab et al
fixed by immersion in 10% buffered formalin, pH 7.2, for 24 to
72 hours. All tissues were processed by standard histologic
techniques for the production of 4-mm-thick sections stained
with hematoxylin and eosin. Selected sections from the small
intestine, cecum, and colon were also stained with Hucker and
Conn’s Gram and phosphotungstic acid–hematoxylin.
Bacteriology
Small and large intestinal contents or swabs collected from
grossly affected areas of the intestine of each horse were
inoculated onto prereduced anaerobically sterilized Brucella
Blood Agar (Anaerobic Systems, Morgan Hill, California), pre-
reduced anaerobically sterilized phenylethyl alcohol sheep
blood agar (Anaerobic Systems), and egg yolk agar (Anaerobic
Systems) and incubated anaerobically at 37 C for 48 hours.
Small and large intestinal content from each horse was inocu-
lated onto cycloserine-cefoxitin-fructose agar (Veterinary
Media Services, University of California, Davis, Davis,
California) and incubated anaerobically at 37 C for 48 hours.
Samples from the same specimens were also inoculated into
cycloserine-cefoxitin-fructose tube (Veterinary Media
Services) and incubated aerobically and anaerobically at 37 C
for 48 to 72 hours. All isolates were identified by conventional
biochemical techniques. Samples of small and/or large intestinal
contents from each animal, as well as individual or pooled sam-
ples of liver, spleen, kidney, joint fluid or blood, were inoculated
onto Columbia 5% sheep blood agar (Hardy Diagnostics, Santa
Maria, California) and MacConkey agar plates (Hardy Diagnos-
tics) and incubated aerobically at 37 C for 48 hours. A real-time
polymerase chain reaction (PCR) to detect a fragment of the
Salmonella-specific invA gene was performed on small and
large intestinal content samples from 3 horses (Nos. 6–8) as
described.4 Briefly, enriched overnight cultures were centri-
fuged and processed for real-time PCR using the sediment as
template. C. perfringens isolates were typed by a multiplex PCR
technique to amplify segments specific for the genes encoding
CPA, CPB, ETX, ITX, CPE, and CPB2, as described.1 Briefly,
bacteria were grown on brain-heart infusion agar plates (Hardy
Diagnostics), incubated anaerobically overnight at 37 C and
then processed for multiplex PCR analysis using colony lysates
as templates. The multiplex PCR products were then separated
in 2% agarose gels, stained with ethidium bromide, and examined
by ultraviolet transillumination.
C. perfringens Toxins ELISA
Samples of small and/or large intestinal contents from all
horses were tested for CPA, CPB, and ETX via a commercial
capture ELISA kit (BIO-X, Brussels, Belgium), following the
manufacturer’s instructions. Briefly, the test used 96-well
plates sensitized by specific monoclonal antibodies for CPA,
CPB, or ETX. Samples were added to wells, and plates were
incubated for 60 minutes at room temperature, followed by
washing and incubation for 60 minutes with peroxidase-labeled
anti-CPA, anti-CPB, or anti-ETX polyclonal antibodies. Plates
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257
were washed again, and a mixture of chromogen-substrate
(hydrogen peroxide and tetramethyl benzidine) was added.
The enzymatic reaction was stopped by acidification with
phosphoric acid. Optical densities were read using an ELISA
reader with a 450-nm filter. Purified CPA, CPB, or ETX was
used in positive control wells; toxins were replaced by buffer
in negative control wells. Results were calculated according
to the manufacturer’s instructions.
C. difficile Toxins ELISA
Samples of small and/or large intestinal contents from all horses
were tested for toxins A and B of C. difficile using a commercial
ELISA kit (Techlab, Blacksburg, Virginia) according to manu-
facturer’s instructions. This kit has antibodies against toxin
A and B in the same well. A mixture of purified toxin A and B
was used in the positive control wells; toxins were replaced by
buffer in negative control wells.
C. perfringens Immunohistochemistry
Paraffin sections, 4 mm in thickness, of ileum and/or colon from
2 of the horses with moderate or large numbers of gram-
positive rods on the mucosal surface were processed by an indi-
rect immunoperoxidase technique for C. perfringens, using the
Dako EnVision Kit (Dako, Carpenteria, California) according
to the manufacturer’s instructions. The primary antibody was
rabbit polyclonal anti–C. perfringens (GenWay Bio, San Diego,
California). Small intestine from a goat inoculated experimen-
tally with C. perfringens type C was used as positive control
tissue. Small intestine from a goat that was culturally negative
for C. perfringens was used as a negative control tissue. Addi-
tional negative controls consisted of serial tissue sections of the
test tissue, incubated with normal rabbit serum instead of the
specific antibodies.
Results
Gross Pathology
Gross lesions (Table 1) were seen in the small intestine of 7 of
8 horses (all but No. 7), in the colon of 3 (Nos. 2, 3, and 7), and
in the cecum of 3 (Nos. 1, 2, and 3). Small intestinal lesions
were segmental (Fig. 1) in 4 horses (Nos. 1, 3, 6, and 8), with
one or more lesions of the jejunum and/or ileum well demar-
cated and up to 3 m long. Lesions were diffuse (Fig. 2) in
2 horses (Nos. 1 and 5), in which the affected area was more
extensive, involving most of the mid and aboral jejunum and/
or ileum and usually sparing the duodenum and oral jejunum.
The most common small intestinal lesions were intense muco-
sal and serosal hyperemia and hemorrhage; moderate trans-
mural thickening; dull and dark red ulcerated mucosa (Fig. 3)
with or without multifocal, tan to yellowish pseudomembranes
(Fig. 4); and abundant brown to red, foul-smelling, fluid intestinal
contents. A gelatinous, clear, transmural edema was associated
multifocally with areas of severe ulceration and necrosis in
2 horses (Nos. 1 and 2). In horse No. 4, the only gross abnormality
258 Veterinary Pathology 49(2)

Figure 1. Abdominal cavity, horse No. 6. Segments of small intestine have transmural, multifocal, necrohemorrhagic enteritis visible from the
serosal surface. Figure 2. Abdominal cavity, horse No. 3. Note the more diffuse distribution of the necrohemorrhagic enteritis in a segment of
the jejunum and ileum. The apex of the cecum (thin arrow) and the colon (thick arrow) have multifocal bluish discoloration indicating
focal necrosis and hemorrhage of the mucosa and submucosa. Figure 3. Ileum, horse No. 3. The mucosal surface is diffusely hemorrhagic and
necrotic, with coalescing ulcers. Figure 4. Jejunum, horse No. 6. A yellow-brown pseudomembrane covers the mucosal surface. Figure 5.
Gastrointestinal tract, horse No. 7. Diffuse reddening extends from the mid jejunum through the large colon. Note the normal color of the
duodenum and orad jejunum. Figure 6. Ileum and duodenum, horse No. 7. The affected ileum (I) is diffusely reddened and contains abundant
hemorrhagic fluid; the unaffected duodenum (D) contains abundant yellow-green fluid.
was diffuse reddening of the jejunal/ileal mucosa and serosa with distribution was multifocal in 3 horses (Nos. 1, 3, and 7), locally
abundant watery, red-brown contents (Figs. 5, 6). Lesions in the extensive to diffuse in 1 horse (No. 2), and not described in the
colon and cecum resembled those in the small intestine; the other 4 horses (Nos. 4, 5, 6, and 8). Colonic contents were

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Diab et al 259

Figure 7. Small intestine, horse No. 3. Transmural necrohemorrhagic enteritis. The mucosa is diffusely necrotic, and there is loss of mucosal/
submucosal boundary, submucosal emphysema, and transmural hemorrhage. HE. See Figure 11 for box. Figure 8. Small intestine, horse
No. 2. Necrotic mucosa covered by a pseudomembrane (PM) with thrombosis in lamina proprial vessels (arrows). HE. Figure 9. Colon, horse
No. 2. The submucosa is expanded by edema without leukocytic infiltration. The mucosa has multifocal hemorrhage. HE. Figure 10. Colon,
horse No. 2. Higher magnification of Figure 9 depicting loss of superficial epithelium, lamina proprial hemorrhage, and thrombosis in subepithelial
capillaries (arrows). HE. Inset: Higher magnification of superficial mucosa showing thrombotic subepithelial capillaries. HE. Figure 11.
Small intestine, horse No. 6. Higher magnification of the area within the box in Figure 7. Numerous gram-positive rods in the necrotic mucosa
are morphologically compatible with Clostridium perfringens. Figure 12. Small intestine, horse No. 3. C. perfringens indirect immunoperoxidase staining
in a segment of necrotic small intestine with numerous positive rods in the superficial mucosa.

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260 Veterinary Pathology 49(2)

Table 2. Histologic Lesions in the Small or Large Intestine of 8 Horses With Clostridium perfringens Type C Enterotoxemia

Dilated Gramþ C.
No. Necrosis Thrombosis Edema Hemorrhage Congestion Inflammation Pseudomembrane Lymphatics Rods perfringensa

1 þþþ þ þþ þþ þþ þþ No þ S NP
2 þþ þþþ þþ þþþ – þ Yes – M Positive
3 þþ þþ þ þþ þþþ – Yes þþþ L Positive
4 þ þþþ – – – – No – S NP
5 þþþ þþ þ – – þ No þþ S NP
6 þþþ þþ þ þ þþþ þ Yes þþ S NP
7 þþþ þþþ – – – – No – S NP
8 þþþ þþ – þþ þþ – No – S NP
–, not observed; þ, mild; þþ, moderate; þþþ, marked/severe; S, small numbers or not observed; M, moderate numbers; L, large numbers; NP, not performed.
a
Immunohistochemistry.

described as abundant, clear to clear green, and fluid (Nos. 2, 5, 7,
and 8); brown or red and fluid (Nos. 1 and 3); and pasty yellow or
normal looking (No. 4). Mesenteric lymph nodes were moder-
ately enlarged and edematous and/or hemorrhagic in 1 case
(No. 2). Gross lesions other than those in the gastrointestinal tract
included serous or serosanguineous pericardial effusion (Nos. 1,
2, 3, and 8), pulmonary edema and congestion (No. 2), and multi-
focal petechiae and ecchymoses of endocardium and serosal sur-
faces, including the peritoneum, pleura, and epicardium (Nos. 1,
2, and 6).
Microscopic Pathology
Histologically, necrotizing or necrohemorrhagic lesions were
observed in the gastrointestinal tract of all the horses; lesion
distribution was generally consistent with the distribution of
the gross lesions. Histologic sections of the small and/or large
intestine had at least 2, but usually 3 or more, microscopic
lesions, including (from most to least commonly present)
mucosal necrosis (8 of 8), mucosal and/or submucosal throm-
bosis (8 of 8), mucosal and/or submucosal hemorrhage (5 of
8), submucosal edema (5 of 8), mucosal and/or submucosal
congestion (4 of 8), mucosal and/or submucosal leukocytic
infiltration (4 of 8), fibrinonecrotic pseudomembranes (3 of
8), numerous gram-positive rods on the superficial mucosa
(2 of 8), and fibrinoid necrosis of submucosal blood vessels
(1 of 8). Severity of the lesions was similar in the small and
large intestine when both portions of the gastrointestinal tract
were affected (Table 2).
Mucosal necrosis was always coagulative, often full thick-
ness, and characterized by mucosal hypereosinophilia, loss of
the mucosal epithelial lining, attenuation or collapse of the villi
and crypts, and moderate to severe hemorrhage and congestion
(Figs. 7–10). Fibrin thrombi (Figs. 8, 10), stained with phos-
photungstic acid–hematoxylin, were present in small veins,
arterioles, and capillaries of the lamina propria and often also
in the small to midsize arteries and veins of the submucosa.
Vascular thrombosis was multifocal, and in some cases, a thor-
ough search of multiple sections of the bowel was necessary to
detect it. Three cases (Nos. 2, 3, and 6) had a diphtheritic pseu-
domembrane that was multifocally attached to the necrotic
mucosa and composed of abundant fibrin mixed with cellular
debris, feed material, and mixed bacteria. Infiltration of leuko-
cytes into the lamina propria or submucosa was not a prominent
feature, and in only 4 cases (Nos. 1, 2, 5, and 6), a mild to mod-
erate number of neutrophils mixed with lymphocytes and
plasma cells, as well as cellular debris, diffusely infiltrated the
mucosa and submucosa. Numerous gram-positive rods (Fig. 11)
were observed multifocally in the lumen and/or along the
surface of the small and large intestinal mucosa in 2 horses
(Nos. 2 and 3). These rod-shaped bacteria were strongly positive
for C. perfringens by immunohistochemistry (Fig. 12). Dilated
lymphatic vessels were observed in 4 horses (Nos. 1, 3, 5, and
6), commonly in the submucosa of the small intestine and rarely
in the large intestine. One case (No. 6) had severe fibrinoid
necrosis of the submucosal blood vessels. Microscopic lesions
in organs other than the intestine were inconsistently present and
included pulmonary congestion, hemorrhage, and edema
(Nos. 1, 2, 5, 6, and 7); pulmonary thrombosis (Nos. 1 and 7);
embolic pneumonia (No. 5); subepicardial and subendocardial
hemorrhage (1, 2, and 6); lymphoid depletion (Nos. 5 and 7);
periportal lymphohistiocytic and neutrophilic hepatitis (No. 1);
congestion and hemorrhage of multiple visceral organs and sero-
sal surfaces (Nos. 1, 2, and 6); mesenteric lymph node edema,
congestion, and hemorrhage (Nos. 1 and 2); and acute renal
tubular degeneration and necrosis (No. 1).
Bacteriology
C. perfringens was cultured from the intestinal contents from 7
of 8 horses (Table 3). Of these, 4 isolates were identified as
type C and 4 as type A. Mixed aerobic and anaerobic flora,
without growth of C. perfringens, was obtained from the
remaining horse. In addition, a variety of other aerobic and/or
anaerobic microorganisms were isolated from all cases. No
C. difficile was isolated from any horse. No Salmonella spp was
isolated or detected by PCR in any horse.
C. perfringens and C. difficile Toxins ELISAs
The C. perfringens toxin ELISA results from the intestinal
contents are summarized in Table 3. All 8 horses tested positive

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Diab et al 261

Table 3. Laboratory Test Results From 8 Horses With Clostridium perfringens Type C Enterotoxemia

C. perfringens

C. perfringens Isolation PCR Genotyping Toxin ELISA

No. SI Colon Cecum Type CPE Beta 2 CPA CPB ETX Other Bacteria Isolateda

1 R NP NP A Pos Neg Neg Pos Neg SI: mixed aerobic flora (L) / Streptococcus equi beta haemolyticum (R)
2 R NP NP A Pos Neg Pos Pos Neg SI: mixed anaerobic/aerobic flora (R)
Colon: mixed anaerobic flora (R)
3 L L NP C Neg Neg Pos Pos Neg SI: Clostridium ramosum (L), mixed aerobic flora (L)
Colon: mixed aerobic flora (L)
4 NP NP L A Pos Neg Neg Pos Neg Cecum: No other anaerobic organisms were isolated.
5 L L NP C Pos Neg Neg Pos Neg SI: mixed aerobic flora (L), Escherichia coli (L), Enterococcus (L)
Colon: E. coli (M), mixed aerobic flora (L)
6 L NP NP A Pos Pos Pos Pos Neg SI: mixed aerobic flora (L)
7 Neg Neg NP C NP NP Neg Pos Neg SI: mixed aerobic/anaerobic flora (L)
Colon: mixed anaerobic flora (L), E. coli (L)
8 L L NP C NP NP Pos Pos Neg SI: mixed anaerobic flora (L)
Colon: mixed anaerobic flora (L)
PCR, polymerase chain reaction; SI, small intestine; R, rare; L, large; NP, not performed; Neg, negative; Pos, positive.
a
Salmonella culture and/or PCR was negative in all samples of the small and/or large intestine analyzed.

for CPB; 4 were positive for CPA; and none was positive
for ETX. No toxins (A/B) of C. difficile were detected in the
intestinal contents of any horse.
Discussion
The objective of this study was to document the lesions of con-
firmed type C enterotoxemia in a series of equine cases because
previous reports have been based on individual cases, often
with incomplete laboratory workup.6,9,16,18 In all cases in this
retrospective study, a presumptive diagnosis of C. perfringens
type C enterotoxemia was based on gross and microscopic
lesions of necrotizing enteritis and/or enterocolitis and con-
firmed by detection of CPB in the intestinal content. Detection
of CPB is considered the gold standard for diagnosis of
C. perfringens type C infections in humans and animals.24
The ‘‘molecular Koch’s postulates’’ for C. perfringens type C
infection have been fulfilled in several species, including
rabbits and mice,20 via elimination of virulence by removing
the gene that encodes CPB from virulent type C isolates and via
restoration of virulence by reinserting the gene.20 In this case
series, C. perfringens was isolated from the small and/or large
intestine of 7 of 8 horses, and 4 of these 8 isolates were identified
as type C. Failure to isolate C. perfringens type C in some of our
cases does not preclude a diagnosis of type C enteritis, because
detection of CPB was positive in all cases. C. perfringens type C
can grow in the intestine in pockets,23 with some segments
devoid of this microorganism. It is therefore possible that in the
culturally negative cases, the sample was collected from areas of
the intestine where C. perfringens type C was not present or was
present in numbers below the sensitivity of the culture method.
C. perfringens type C is rarely isolated from the intestine of nor-
mal foals (our unpublished observation), which lends support to
the idea that isolation of this microorganism from foals with
necrotizing enteritis has at least presumptive diagnostic value.
C. perfringens type A (CPE positive), but not type C, was
isolated from the intestine of 4 horses in our study. C. perfringens
type A is considered a normal inhabitant of the equine intestine,
so isolation of this microorganism was probably an incidental
finding. However, several authors2,7,11 have suggested a
pathogenic role for C. perfringens type A in equine enterocoli-
tis, so a possible synergism between C. perfringens type A and
C. perfringens type C cannot be ruled out. The 4 C. perfringens
type A isolates from our horses carried the CPE gene. Entero-
toxigenic C. perfringens type A has been associated with
equine enterocolitis,2 and it is possible that CPE acted syner-
gistically with CPB to produce the lesions observed in this
study. It is also possible that the CPE produced by type C
isolates (1 of our type C isolates carried the CPE gene) was also
implicated in the pathogenesis of enterocolitis in these cases.
However, the presence of CPE in the intestinal content was not
investigated, so this possibility remains only speculative. CPA
was detected in 4 of the 8 horses in this study. Although all
types of C. perfringens carry the gene that encodes this toxin,
not all C. perfringens strains produce detectable concentra-
tions of CPA. Because similar lesions were observed in horses
with and without CPA in the intestinal content, it is unlikely
that this toxin contributed much to lesion development.
Moreover, it was recently demonstrated that this toxin has
negligible action in the intestinal virulence of C. perfringens
type C isolates.20 However, as for CPE, we cannot completely
rule out a synergistic action between CPA and CPB.
The most consistent gross and histologic finding in this
series of cases was necrotizing enteritis with mucosal and/or
submucosal thrombosis. It is noteworthy that the identification
of mucosal necrosis, grossly or histologically, may be hampered
by autolysis, in which case, histologic evidence of mucosal
or submucosal thrombosis was the most important indicator
of intestinal damage. Although neither mucosal necrosis nor
thrombosis is specific for type C enterotoxemia in horses, the

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262
presence of both lesions narrows the differential diagnosis to a
few conditions. Besides C. perfringens type C, agents that can
produce acute onset of diarrhea or sudden death, with intestinal
necrosis and mucosal and/or submucosal thrombosis in horses,
include Salmonella spp and C. difficile. In our study, Salmonella
spp was ruled out by negative culture and PCR test results.
Neither C. difficile nor its toxins were detected in any horse in
this study.
E. coli has been associated with enterocolitis in horses.
However, the gross and histologic lesions in this study are very
different from those described in E. coli–associated enterocoli-
tis.8 This difference in lesions, coupled with the fact that E. coli
was isolated from the gastrointestinal tract in only 2 of our
horses, strongly suggests that this microorganism was not
responsible for the lesions described in this study.
C. cadaveris was associated with experimental colitis in
horses treated with lincomycin.25 That microorganism was
ruled out in our study by negative anaerobic culture results.
In addition, only 1 horse had received antibiotic treatment, a
predisposing factor that was considered responsible for colitis
in the cited study,25 before onset of clinical signs.
Traditionally, type C infections have been reported in
newborn animals of several species, including horses.23,24,29
The reason for this age predisposition is thought to be the very
low level of trypsin activity in the intestine of newborn
animals, which allows CPB to remain active. This toxin is
exquisitely sensitive to the action of trypsin and other proteases
normally present in the intestinal content; therefore, intestinal
trypsin is considered an important innate defense mechanism
against type C infections.14 However, malnutrition, trypsin
deficiency, and diets rich in trypsin inhibitors, such as sweet
potato or soy bean products, predispose people and animals
to type C infections.12 The sensitivity of CPB to trypsin
explains why attempts to reproduce type C enterotoxemia in
animal models by inoculating CPB toxin orally or intraduoden-
ally have consistently failed or produced only low lethality.28
In contrast, experimental mice and rabbits inoculated with CPB
or type C cultures mixed with trypsin inhibitor developed clin-
ical signs and/or lesions characteristic of type C infections.20,30
Four of the 8 cases in this study were older foals or adult
horses. The pathogenesis of type C enterotoxemia in horses
after the first few weeks of life is poorly understood, but low
intestinal trypsin activity, as in the neonates, could have been
an important factor. Low intestinal trypsin activity may be a
consequence of trypsin inhibitors in feed; acute pancreatic
necrosis, pancreatitis, or exocrine pancreatic insufficiency;
changes in the intestinal microflora; and administration of cer-
tain antibiotics.3,5,17,19 Antimicrobials are known predisposing
factors for colitis in horses, especially that produced by
C. difficile. The role of previous antibiotic treatment in the
pathogenesis of C. perfringens type C colitis is not known.
Nevertheless, only 1 horse in our study had received antibiotics
before the onset of intestinal disease, which rules out antimi-
crobial treatment as a predisposing factor in the majority of the
cases. Importantly, type C enterotoxemia in the nonneonatal
horse may not be as uncommon as once believed.
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Veterinary Pathology 49(2)
In summary, C. perfringens type C enterotoxemia occurs
most commonly in foals but also in adult horses and should
be included in the differential diagnosis in cases with a history
of sudden death or acute onset of colic, with or without diar-
rhea. The hallmark lesions were necrotizing enteritis and
mucosal/submucosal thrombosis. Although isolation of numer-
ous C. perfringens type C from the intestinal tract of affected
horses is highly suggestive of the disease, the gold standard for
diagnosis of type C enterotoxemia is the detection of CPB toxin
in the intestinal contents.
Acknowledgements
We thank J. Saputo, A. Curtis, E. Hurley, D. Paulson, P. Yant,
R. Cazares, S. Fitisemanu, and M. Rhea for excellent technical
assistance.
Declaration of Conflicting Interests
The authors declared that they had no conflicts of interest with respect
to their authorship or the publication of this article.
Financial Disclosure/Funding
The authors declared that they received no financial support for their
research and/or authorship of this article.
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