Clostridium perfringens type C and Clostridium difcile co-infection in foals

F.A. Uzal
a,
*, S.S. Diab
a
, P. Blanchard
b
, J. Moore
a
, L. Anthenill
a
,
F. Shahriar
a
, J.P. Garcia
a
, J.G. Songer
c
a
California Animal Health and Food Safety Laboratory, San Bernardino Branch, UC Davis, CA, USA
b
California Animal Health and Food Safety Laboratory, Tulare Branch, UC Davis, CA, USA
c
University of Iowa, USA
1. Introduction
Clostridium difcile and Clostridium perfringens type C
are amongst the most important agents of enteric disease
in horses (Magdesian et al., 2002; Songer, 1996; Hurley and
Nguyen, 2002; Kelly and LaMont, 1998). Virulent strains of
C. difcile produce either one or two major toxins, i.e. an
enterotoxin (Tcd A) and a cytotoxin (Tcd B), which is also
an enterotoxin. Foals and adult horses are equally
susceptible to C. difcile disease (Jones et al., 2006;
Madewell et al., 1995; Weese et al., 1999). In foals, the
lesions of C. difcile infection are mostly restricted to the
small intestine, while in adult horses, the lesions are
usually restricted to the cecum and colon (Perrin et al.,
1993). C. perfringens is classied into ve types (AE) on
the basis of production of four major toxins, alpha (CPA),
Veterinary Microbiology 156 (2012) 395402
A R T I C L E I N F O
Article history:
Received 1 September 2011
Received in revised form 21 November 2011
Accepted 24 November 2011
Keywords:
Clostridium perfringens type C
Clostridium difcile
Colitis
Enteritis
Foals
A B S T R A C T
Clostridium perfringens type C is one of the most important agents of enteric disease in
newborn foals. Clostridiumdifcile is nowrecognized as an important cause of enterocolitis
in horses of all ages. While infections by C. perfringens type C or C. difcile are frequently
seen, we are not aware of any report describing combined infection by these two
microorganisms in foals. We present here ve cases of foal enterocolitis associated with C.
difcile and C. perfringens type C infection. Five foals between one and seven days of age
were submitted for necropsy examination to the California Animal Health and Food Safety
Laboratory. The ve animals had a clinical history of acute hemorrhagic diarrhea followed
by death and none had received antimicrobials or been hospitalized. Postmortem
examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically,
the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage
and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal
and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed
that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was
detected by ELISA in intestinal content of all animals and C. difcile toxin A/B was detected
in intestinal content of three animals. C. perfringens (identied as type C by PCR) was
isolated from the intestinal content of three foals. C. difcile (typed as A
+
/B
+
by PCR) was
isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible
synergism of C. perfringens type C and C. difcile in foal enterocolitis. Because none of the
foals had received antibiotic therapy, the predisposing factor, if any, for the C. difcile
infection remains undetermined; it is possible that the C. perfringens infection acted as a
predisposing factor for C. difcile and/or vice versa. This report also stresses the need to
perform a complete diagnostic workup in all cases of foal digestive disease.
2011 Elsevier B.V. All rights reserved.
* Corresponding author at: California Animal Health and Food Safety
Laboratory-San Bernardino Branch, University of California-Davis, 105
West Central Avenue, San Bernardino, CA 92408, USA.
Tel.: +1 909 383 4287; fax: +1 909 884 5980.
E-mail address: fuzal@cahfs.ucdavis.edu (F.A. Uzal).
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology
j our nal homepage: www. el sevi er . com/ l ocat e/ vet mi c
0378-1135/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2011.11.023
beta (CPB), epsilon (ETX) and iota (ITX) (McDonel, 1980;
Rood and Cole, 1991). C. perfringens type C produces, at the
minimum, CPA and CPB, and it is considered the most
common clostridial enteric pathogen in foals in North
America (Bueschel et al., 2003; Drolet et al., 1990; Holland
et al., 1996). CPB is responsible for the intestinal necrosis
and probably also for the systemic alterations seen in type
C infections of several animal species, including horses
(Diab et al., 2011). CPB is a necrotizing toxin which is
highly sensitive to trypsin. The main predisposing factor
for C. perfringens type C infection in horses seems to be age,
with neonatal foals being at higher risk. The small intestine
is always affected, with 50% of the horses also showing
colitis (Diab et al., 2011).
While infections by C. difcile and C. perfringens type C
are frequently seen in foals, we are not aware of any report
describing the combined infection by these two micro-
organisms in foals. We present here ve cases of foal
enteric disease with C. difcile and C. perfringens type C co-
infection.
2. Materials and methods
Five foals from ve different ranches in Southern
California were submitted for necropsy examination to the
San Bernardino branch of the California Animal Health and
Food Safety laboratory system. Detailed information about
age, gender, breed, and clinical history of each animal is
provided in Table 1. Briey, all the animals were between a
fewhours and seven days of age, and ve of the foals had a
clinical history of acute hemorrhagic diarrhea followed by
death, while the other foal had a history of general malaise
followed by death. All the animals were suckling at the
time of disease onset and neither the foals nor the dams
had received antibiotics. According to clinical history no
signicant stressful conditions (overcrowding or lack of
hygiene) existed in any of the ranches. However, one of the
ranches had a history of C. perfringens type C infection in
foals in previous years.
Postmortem examination was performed and samples
of heart, lung, liver, spleen, kidney, skeletal muscle, small
intestine and large intestine, mesenteric lymph nodes,
thymus, thyroidgland, andthe whole brain were collected
into 10% neutral buffered formalin. After 24 h of xation,
the brain was sub-sampled at the level of parietal cortex,
basal ganglia, thalamus, midbrain, cerebellum, cerebellar
peduncles, pons, and medulla at the obex level. All
samples were processed routinely for histology, sectioned
at 4 mm, andstainedwithhematoxylinandeosin. Selected
intestinal sections were also stained with Gram and
phosphotungstic acid-hematoxylin (PTAH).
Four-micrometer thick parafn-embedded sections of
ileum and colon were processed by an indirect immuno-
peroxidase technique for C. perfringens, using the EnVision
kit (Dako, Carpenteria, CA) according to the manufacturers
instructions. Primary antibody was a rabbit polyclonal
anti-C. perfringens (Genway Bio, San Diego, CA). Small
intestine from a goat inoculated experimentally with C.
perfringens type C was used as positive control. Small
intestine from a goat that was culturally negative for C.
perfringens was used as a negative control. Additional
negative controls consisted of tissue sections from the 4
foals incubated with normal rabbit serum instead of the
specic antibodies.
Small and large intestinal contents from all foals were
inoculated onto pre-reduced, anaerobically sterilized
(PRAS) Brucella Blood agar (Anaerobe Systems, Morgan
Hill, CA), PRAS phenylethyl alcohol sheep blood agar
(Anaerobe Systems), and egg yolk agar (Anaerobe Sys-
tems), and incubated anaerobically at 37 8C for 48 h. Small
and large intestinal contents from all horses were
inoculated onto cycloserine-cefoxitin-fructose agar (CCFA;
Veterinary Media Services, UC Davis, Davis, CA) and
incubated anaerobically at 37 8C for 48 h. Samples from
the same specimens were also inoculated into cycloserine-
cefoxitin-fructose broth (CCFB; Veterinary Media Services)
and incubated anaerobically at 37 8C for 4872 h. After
incubation, these enrichment cultures were subcultured
onto CCFA, which was then incubated as above. Large, rod-
shaped anaerobic bacteria producing spores and a double
zone of hemolysis on sheep blood agar were suspected to
be C. perfringens. Suspect colonies were identied by
standard biochemical techniques. Isolates were typed by a
multiplex PCR technique (mPCR) to amplify segments
specic for the genes encoding toxins CPA, CPB, ETX, ITX,
CPE, and CPB2, as previously described (Bueschel et al.,
2003). Briey, bacteria were grown on brain heart infusion
agar plates, incubated anaerobically overnight at 37 8C and
then processed for mPCR analysis using colony lysates as
templates. The mPCR products were then separated in 2%
agarose gels, stained with ethidium bromide, and exam-
ined by ultraviolet transillumination. C. difcile produced
large, rough colonies on CCFA; these colonies were
uorescent when illuminated by long-wave ultraviolet
light. Identity was conrmed by PCR assays for the genes
for toxins A (tcdA) and B (tcdB) as previously described
(Elliott et al., 2011). C. difcile isolates were evaluated for
their ability to produce TcdAand TcdB in vitro. For this, one
Table 1
Age, gender, breed, clinical history and distribution of gross lesions in the intestinal tract of 5 foals with C. perfringens type C and C. difcile co-infection.
Case # Age Gender Breed Clinical history Distribution of gross lesions
in the intestinal tract
SI Colon Cecum
1 2 days Male Quarter horse Hemorrhagic diarrhea and death Yes Yes Yes
2 6 days Male Paint horse General malaise and death Yes No No
3 7 days Female Quarter horse Depression, hemorrhagic diarrhea and death Yes Yes No
4 <1 day Female Crossbred Hemorrhagic diarrhea and death Yes Yes No
5 1 day Female Quarter horse Hemorrhagic diarrhea and death Yes No No
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 396
colony from CCFA agar was inoculated into CCFB and
incubated anaerobically for 72 h. The culture supernatant
uid was tested for TcdAand TcdB using a capture ELISAkit
(TechLab, Blacksburg, VA). Three C. difcile isolates were
ribotyped according to the method of Stubbs et al. (1999).
Samples of small and/or large intestinal contents fromall
animals, andindividual or pooled samples of two or more of
liver, spleen, kidney, joint uid, and blood, were inoculated
onto Columbia 5% blood sheep agar (Hardy Diagnostics,
Santa Maria, CA) and MacConkey agar plates (Hardy
Diagnostics) and incubated aerobically at 37 8C for 48 h.
Salmonella suspect colonies were identied by standard
biochemical techniques. A real-time PCR to detect a
fragment of the Salmonella-specic invA gene was per-
formed on small and large intestinal content samples from
all foals as previously described (Cheng et al., 2008).
Samples of small and large intestinal contents from all
horses were tested for CPA, CPB, and ETX using a capture
ELISA kit (BIO-X, Brussels, Belgium) and for toxins A and B
of C. difcile, using another capture ELISA kit (TechLab).
Feces from all foals were also examined by a otation
technique for parasite eggs and protozoa, following the
SOP of CAHFS San Bernardino.
3. Results
Postmortemexamination revealed that all foals were in
good nutritional condition. Gross and histological ndings
were similar in the ve animals although the distribution
of the lesions in the gastrointestinal tract varied between
animals (Table 1). Intestinal gross lesions were seen in the
small intestine (jejunum and ileum) of all foals (Figs. 13).
Colonic lesions were present in four foals (Fig. 4), and gross
lesions of the cecum were found in one foal (Table 1).
Strands of brin were observed on the small and large
intestinal serosa of all foals. Gross lesions in the small
intestine were segmental with one or more well-demar-
cated, relatively short (up to approximately 3 m long)
segments of the jejunum and/or proximal ileum affected
(Figs. 1 and 2). The most common gross abnormalities
observed in the small intestine were intense mucosal and
serosal hyperemia and hemorrhage, moderate transmural
edema, dull, dark red, ulcerated mucosa with or without
multifocal, tan-to-yellowish pseudomembranes (Fig. 3),
and brown-to-red, usually foul-smelling, abundant, uid
intestinal contents. Lesions in the colon and cecum were
similar to those described in the small intestine, and the
distribution was multifocal. Colon contents were abun-
dant, clear-to-green and uid. Mesenteric lymph nodes
were moderately enlarged and edematous, hyperemic,
and/or hemorrhagic. Gross lesions outside the gastro-
intestinal tract were similar in all foals and included serous
or serosanguineous pericardial effusion, pulmonary edema
and congestion, and multifocal petechiation and ecchy-
moses of endocardium and multiple serosal surfaces,
including the peritoneum, pleura, and epicardium (Table
2).
Microscopically necrotizing or necrohemorrhagic
lesions were observed in the gastrointestinal tract of all
the foals, and microscopic lesion distribution was gen-
erally consistent with the distribution of the gross lesions
(Figs. 510). Histologic sections of the small and/or large
intestine had at least two, but more commonly a
combination of three or more microscopic lesions, includ-
ing (from most to least commonly present) mucosal
necrosis with thrombosis of mucosal and/or submucosal

Fig. 1. Exposed abdominal cavity: various segments of the small intestine

have transmural, multifocal, necrohemorrhagic enteritis readily observed
from the serosal surface.

Fig. 2. Digestive tract: note the more diffuse distribution of the necrohemorrhagic enteritis affecting jejunum and ileum.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 397
blood vessels (Figs. 6 and 9), mucosal and/or submucosal
hemorrhage (Fig. 5), supercial brinonecrotic pseudo-
membrane (Fig. 7), mucosal and/or submucosal inamma-
tory cell inltration, abundance of Gram-positive rods on
the supercial mucosa (Fig. 8), and brinoid necrosis of
submucosal blood vessels. Severity of the lesions was
similar in small intestine and large intestine when both
portions of the gastrointestinal tract were affected (Table 3).
The mucosal necrosis was always coagulative, often
full-thickness, and characterized by hypereosinophilia,
loss of the mucosal epithelial lining, villus blunting or
collapse of the villi and crypts, moderate to severe
hemorrhage and congestion, and presence of brin, edema,
sometimes neutrophils, and cellular and necrotic debris
within the lamina propria or on the mucosal surface
(pseudomembrane). PTHA-positive stained brin thrombi
were present in small-caliber veins, arterioles, and
capillaries of the lamina propria, and often also in the
small to mid-size arteries and veins of the submucosa
(Fig. 9). Vascular thrombosis was multifocal and, in some
cases, a thorough search of multiple sections of the bowel
was necessary to detect them. Four cases had a diphtheritic
pseudomembrane in the lumen which was usually
attached to the necrotic mucosa in multiple foci, and
was composed of abundant brin mixed with cellular and
necrotic debris, occasionally feed material, and mixed
bacteria. Inltration of neutrophils mixed with lympho-

Fig. 3. Ileum: the mucosal surface is diffusely necrotic.

Fig. 4. Large colon: the mucosa is diffusely hemorrhagic.

Fig. 5. Jejunum: transmural necrohemorrhagic lesion affecting the

mucosa, submucosa and muscularis. The mucosa is diffusely necrotic.
H&E, bar: 100 mm.

Fig. 6. Jejunum: severe necrosis of the jejunal mucosa with thrombosis of

lamina propria vessels. H&E, bar: 20 mm.
Table 2
Most common histopathologic changes observed in 5 foals with C. perfringens type C and C. difcile co-infection.
Horse Necrosis Thrombosis Edema Hemorrhage Hyperemia Inammation Pseudomembrane
observed
Dilated
lymphatics
a
Gram+
rods
a
C. perfringens
IHC
1 +++ + + + + + No ++ M Positive
2 +++ +++ + +++ +++ + Yes +++ M Positive
3 +++ ++ +++ ++ ++ ++ Yes + L Positive
4 +++ ++ ++ ++ ++ ++ No +++ L Positive
5 +++ + ++ + + ++ Yes +++ S Positive
+ = mild; ++ = moderate; +++ = marked/severe.
a
S = small numbers or not observed; M= moderate numbers; L = large numbers.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 398
cytes and plasma cells into the lamina propria or
submucosa was observed in every case although this
lesion was never severe. Large numbers of Gram positive
rods were observed multifocally in the lumen and/or in
close contact with the surface of the small and large
intestinal mucosa in all cases (Fig. 8). These rod-shaped
bacteria were strongly positive for C. perfringens by IHC in
all animals (Fig. 10). Dilated lymphatics were observed in
all foals, commonly in the submucosa of the small intestine
and rarely in the large intestine. Microscopic lesions in
organs other than the large intestine and small intestine
were inconsistently present and included moderate to
marked pulmonary congestion, hemorrhage, and edema,
pulmonary thrombosis, moderate-to-marked subepicar-
dial and subendocardial hemorrhage, marked lymphoid
depletion, mild periportal lymphohistiocytic and neutro-
philic hepatitis, congestion and hemorrhage of multiple
visceral organs and serosal surfaces, mesenteric lymph
node edema, congestion, hemorrhage, and moderate, acute
renal tubular degeneration and necrosis.
C. perfringens was culturedfromtheintestinal contents of
four foals (Table 3). Of these, threeisolates wereidentiedas
type C and one as type A. In addition, a variety of other
aerobic and anaerobic microorganisms were isolated from
all cases. C. difcile was isolated from the small or large
intestine of the ve foals. Two isolates tested for production
of TcdA and TcdB in vitro were positive. The three C. difcile
isolates examined by PCR for tcdA and tcdB were positive for
both, and two of the three isolates were ribotype 056, while
the third isolate was ribotype 027 (Table 3).
A summary of the C. perfringens and C. difcile toxin
ELISA results fromthe intestinal contents is shown in Table
3. All foals tested positive for CPB and three of the foals
tested positive for TcdA and TcdB of C. difcile.
No parasites or parasite eggs were detected in feces of
any foal.

Fig. 7. Colon: necrotic mucosa showing a supercial pseudomembrane.

H&E, bar: 100 mm.

Fig. 8. Jejunum: large numbers of Gram positive rods are observed on the
necrotic mucosal surface of the jejunum. Gram, bar: 20 mm.

Fig. 9. Jejunum: PTAH stained brin thrombi in the lamina propia of the
jejunum. Bar: 20 mm.

Fig. 10. Jejunum: C. perfringens positive indirect immunoperoxidase

staining in a segment of necrotic small intestine which had abundant
Gram positive rods on the supercial mucosa. Bar: 20 mm.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 399
4. Discussion
The objective of this study was to comprehensively
document the pathologic lesions of conrmed cases of C.
perfringens type C and C. difcile co-infection in foals. In all
cases, a presumptive diagnosis of C. perfringens type C or C.
difcile was established based on gross and microscopic
lesions of necrotizing enteritis and/or enterocolitis, and it
was conrmed by detection of CPB in all cases and
detection of C. difcile TcdA and TcdB and/or isolation of
toxigenic strains of this microorganism.
C. difcile-associated disease should be suspected in any
case of necrotizing enteritis, enterocolitis, or colitis in
horses. No disease denition has been established for C.
difcile infection in horses, but the diagnosis of the disease
in this species is usually contingent on isolation of
toxigenic strains of this pathogen from fecal samples,
detection of C. difcile toxins, or both (Keel and Songer,
2006). A history of antibiotic use and/or hospitalization
may increase the suspicion of infection by this micro-
organism. In this case, there was no history of antibiotic
treatment in any of the foals and none of the animals had
been hospitalized. No history of previous cases of C. difcile
on the farms of origin of these foals was available, but it is
possible that environmental contamination from previous
equine cases at those farms was the source of the
infections.
The carrier rate of C. difcile is nil or low in
asymptomatic foals and adult horses. The rate of isolation
from clinically normal adults is between 0 and 4.3%,
whereas normal foals are generally reported to be culture
negative (Baverud et al., 1997; Jones et al., 1987; Madewell
et al., 1995; Weese et al., 2001). Therefore, isolation of this
microorganism from the intestinal tract of horses with
gastrointestinal disease is considered by some authors as
highly suggestive of C. difcile-associated disease. How-
ever, a small percentage of healthy carriers can occur and
also some healthy or sick animals may harbor strains of C.
difcile that do not encode and/or produce either of the two
major toxins of this microorganism (A and B). In order to
overcome this diagnostic problem, many human diagnos-
tic laboratories regularly characterize C. difcile isolates for
their ability to produce these toxins (Blake et al., 2004;
Mathis et al., 1999). This practice is rarely performed in
veterinary diagnostic laboratories. In this study, conrma-
tion of the role of C. difcile in the pathogenesis of the
lesions described was achieved by (a) detection of
preformed TcdA and TcdB in small and/or large intestinal
content, (b) isolation of C. difcile followed by toxin testing
of the isolates, and (c) demonstration of the capacity of
these isolates to produce TcdA and TcdB in vitro.
C. difcile and its toxins have been detected in clinically
healthy human babies. However, human babies are known
to be resistant to the action of C. difcile toxins. On the
contrary, newborn foals are highly susceptible to C. difcile
infection (Keel and Songer, 2006).
A presumptive diagnosis of C. perfringens type C
infection in horses is based upon clinical signs and gross
and histological lesions. Isolation of C. perfringens type C
from the intestine provides stronger evidence of infection
by this microorganism, as this type of C. perfringens is
rarely isolated fromhealthy horses (Diab et al., 2011). Final
conrmation of C. perfringens type C disease, however, is
based on detection of CPB in intestinal contents. In this
study, CPB was detected in intestinal content of all horses
and C. perfringens was isolated from the small and/or large
intestine of the four cases on which anaerobic culture was
performed. Three of these ve isolates were identied as
type C. Failure to isolate C. perfringens type C in one case
(horse #4) does not preclude a diagnosis of type C
infection, because CPB was present in all cases. C.
perfringens type C can grow in the intestine in a segmental
fashion (Diab et al., 2011), with some segments devoid of
this microorganism. This was observed before in a study of
eight cases of C. perfringens infection in horses (Diab et al.,
2011). It is also possible that a mixture of type A and C was
present on the plates and a type A colony was picked for
PCR typing. C. perfringens type A, but not type C, was
isolated from the intestine of 1 horse 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 nding. However, several authors
Table 3
Main microbiological and toxicological ndings in 5 foals with C. perfringens type C and C. difcile co-infection.
Case # Anaerobic culture C. perfringens toxins ELISA C. difcile
toxins
ELISA
Small intestine Colon SI colon SI colon
C. perfringens isolated C. difcile isolated C. perfringens
isolated
C. difcile
isolated
Alpha Beta Alpha Beta A/B A/B
1 Yes, type C, CPE+, CPB2+ Yes, A
+
/B
+
, 027 A/B toxin
production in vitro
ND ND + + ND ND ND
2 Yes, type C, CPE, CPB2 Yes, A
+
/B
+
, 056 A/B toxin
production in vitro +
ND ND + + ND ND ND
3 Yes, type C, CPE, CPB2 No Yes, not typed Yes, not
typed
+ + + + + +
4 Yes, type A, CPE, CPB2 Yes, A
+
/B
+
, 056, A/B toxin
production in vitro+
ND ND + + + + + +
5 ND Yes, not typed ND ND + + + + + +
ND= not done; + = positive; = negative.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 400
(Bueschel et al., 1998; East et al., 1998; Kanoe et al., 1990)
have suggested a pathogenic role for C. perfringens type Ain
equine enterocolitis, so a possible synergism between C.
perfringens type A and C. perfringens type C cannot be ruled
out. CPAwas detected in the intestine of all ve foals in this
study. All types of C. perfringens carry the gene that encodes
this toxin, but not all C. perfringens strains produce
detectable concentrations of CPA. However, CPA is
frequently found in the intestine of healthy horses
(authors unpublished observation) and it has been
demonstrated recently that this toxin plays a negligible
part in the intestinal virulence of C. perfringens type C in
mammals (Sayeed et al., 2008).
In this study, other potential agents of enteritis in foals
were ruled out. Known agents of enteritis in foals, which
were ruled out in this study include Salmonella spp.,
Clostridium cadaveris, and Escherichia coli. Salmonella spp.
was ruled out by negative culture and PCR test results. E.
coli was isolated fromthe gastrointestinal tract of only two
of our foals (results not shown) and the gross and
histological lesions in this study are very different from
those described in E. coli-associated enterocolitis (Holland
et al., 1996), which rules out this organism as etiological
agent in our cases. C. cadaveris was associated with
experimental colitis in horses treated with lincomycin
(Staempi et al., 1992). That microorganism was ruled out
in our study by anaerobic culture. In addition, none of our
foals had received antibiotic treatment, a predisposing
factor that was considered responsible for colitis in the
cited study (Staempi et al., 1992).
All the foals were very young and, therefore, not
expected to have had mature normal microbiota; it is
almost certain that they had not acquired the organisms
that are most important in preventing establishment of C.
difcile in the gut. It is also possible that the C. perfringens
infection acted as a predisposing factor for C. difcile and/or
vice versa. It has recently been shown that virulent strains
of C. perfringens type A in poultry clear out the normal ora
as they multiply and move towards disease production
(Barbara et al., 2008). In the same way, C. perfringens type C
could have had a similar effect clearing out the way for C.
difcile establishment. Alternatively, an yet undetermined
factor may have predisposed to both C. perfringens and C.
difcile overgrowth. In summary, this report suggests a
possible synergismof C. perfringens type C and C. difcile in
foal enterocolitis and it also stresses the need to perform a
complete diagnostic work up in all cases of foal digestive
disease.
Conict of interest statement
The authors express that there are no conicts of
interest that could bias the work presented in this
manuscript.
Acknowledgements
We thank Ms S. Fitisemanu for excellent secretarial
support, and E.J. Hurley, J. Saputo and A. Curtis for excellent
technical help.
References
Barbara, A.J., Trinh, H.T., Glock, R.D., Songer, J.G., 2008. Necrotic enter-
itis-producing strains of Clostridium perfringens displace non-necro-
tic enteritis strains from the gut of chicks. Vet. Microbiol. 126, 377
382.
Baverud, V., Gustafsson, A., Franklin, A., Lindholm, A., Gunnarsson, A.,
1997. Clostridium difcile associated with acute colitis in mature
horses treated with antibiotics. Equine Vet. J. 29, 279284.
Blake, J.E., Mitsikosta, F., Metcalfe, M.A., 2004. Immunological detec-
tion and cytotoxic properties of toxins from toxin A-positive, toxin
B-positive Clostridium difcile variants. J. Med. Microbiol. 53, 197
205.
Bueschel, D., Jost, B., Billington, S., Trinh, H., Songer, J.G., 2003. Prevalence
of cpb2, encoding beta2 toxin, in Clostridium perfringens eld isolates:
correlation of genotype with phenotype. Vet. Microbiol. 94, 121129.
Bueschel, D., Walker, R., Woods, L., Kokai-Kun, J., McClane, B., Songer, J.G.,
1998. Enterotoxigenic Clostridiumperfringens type A necrotic enteritis
in a foal. J. Am. Vet. Med. Assoc. 213, 13051307.
Cheng, C.M., Lin, W., Van, K.T., Phan, L., Tran, N.N., Farmer, D., 2008. Rapid
detection of Salmonella in foods using Real-Time PCR. J. Food. Protect.
71, 24362441.
Diab, S.S., Kinde, H., Moore, J., Shahriar, M.F., Odani, J., Anthenill, L., Songer,
J.G., Uzal, F.A., 2011. Pathology of Clostridium perfringens type C
enterotoxemia in horses. Vet. Pathol. Epub ahead of print.
Drolet, R., Higgins, R., Ce cyre, A., 1990. Necrohemorrhagic enterocolitis
caused by Clostridium perfringens type C in a foal. Can. Vet. J. 31, 449
450.
East, L., Savage, C., Traub-Dargatz, J., Dickinson, C.E., Ellis, R.P., 1998.
Enterocolitis associated with Clostridium perfringens infection in neo-
natal foals: 54 cases (19881997). J. Am. Vet. Med. Assoc. 212, 1751
1756.
Elliott, B., Squire, M.M., Thean, S., Chang, B.J., Brazier, J.S., Rupnik, M., Riley,
T.V., 2011. New types of toxin A-negative, toxin B-positive strains
among clinical isolates of Clostridium difcile in Australia. J. Med.
Microbiol. 60, 11081111.
Holland, R.E., Grimes, S.D., Walker, R.D., Wilson, R.A., 1996. Experimental
inoculation of foals and pigs with an enterotoxigenic E. coli isolated
from a foal. Vet. Microbiol. 52, 249257.
Hurley, B.W., Nguyen, C.C., 2002. The spectrum of pseudomembranous
enterocolitis and antibiotic-associated diarrhea. Arch. Intern. Med.
28, 21772184.
Jones, R.L., Adney, W.S., Alexander, A.F., Shideler, R.K., Traub-Dargatz,
J.L., 2006. Hemorrhagic necrotizing en-Keel and Songer. Vet. Pathol.
43, 3.
Jones, R.L., Adney, W.S., Shideler, R.K., 1987. Isolation of Clostridiumdifcile
and detection of cytotoxin in the feces of diarrheic foals in the absence
of antimicrobial treatment. J. Clin. Microbiol. 25, 12251227.
Kanoe, M., Inoue, S., Abe, T., Abe, T., Anzai, T., Kamada, M., Imagawa, H.,
Kanemaru, T., 1990. Isolation of Clostridium perfringens from foals.
Microbios 64, 153158.
Keel, M.K., Songer, J.G., 2006. The comparative pathology of Clostridium
difcile-associated disease. Vet. Pathol. 43, 225240.
Kelly, C.P., LaMont, J.T., 1998. Clostridium difcile infection. Annu. Rev.
Med. 49, 375390.
Madewell, B.R., Tang, Y.J., Jang, S., Madigan, J.E., Hirsh, D.C., Gumerlock,
P.H., Silva Jr., J., 1995. Apparent outbreaks of Clostridium difcile-
associated diarrhea in horses in a veterinary medical teaching hospi-
tal. J. Vet. Diag. Invest. 7, 343346.
Magdesian, K.G., Hirsh, D.C., Jang, S.S., Hansen, L.M., Madigan, J.E.,
2002. Characterization of Clostridium difcile isolates from foals
with diarrhea: 28 cases (19931997). J. Am. Vet. Med. Assoc. 220,
6773.
Mathis, J.N., Pilkinton, L., Mc Millin, D.E., 1999. Detection and transcrip-
tion of toxin DNA in a nontoxigenic strain of Clostridium difcile. Curr.
Microbiol. 38, 324328.
McDonel, J.L., 1980. Clostridium perfringens toxins (type A, B, C, D E).
Pharmacol. Ther. 10, 617655.
Perrin, J., Cosmetatos, I., Gallusser, A., Lobsiger, L., Straub, R., Nicolet, J.,
1993. Clostridium difcile associated with typhlocolitis in an adult
horse. J. Vet. Diag. Invest. 5, 99101.
Rood, J.I., Cole, S.T., 1991. Molecular genetics and pathogenesis of Clos-
tridium perfringens. Microbiol. Rev. 55, 621648.
Sayeed, S., Uzal, F.A., Fisher, D.J., Saputo, J., Vidal, J.E., Chen, Y., Gupta, P.,
Rood, J.I., McClane, B.A., 2008. Beta toxin is essential for the virulence
of Clostridium perfringens type C isolate CN3685 in a rabbit ileal loop
model. Mol. Microbiol. 67, 1530.
Songer, J.G., 1996. Clostridial enteric diseases of domestic animals. Clin.
Microbiol. Rev. 9, 216234.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 401
Staempi, H.R., Prescott, J.F., Brash, M., 1992. Lincomycin-induced severe
colitis in ponies: association with Clostridium cadaveris. Can. J. Vet.
Res. 56, 168169.
Stubbs, S.L., Brazier, J.S., ONeill, G.L., Duerden, B.I., 1999. PCR targeted to
the 16S23S rRNAgene intergenic spacer region of Clostridiumdifcile
and construction of a library consisting of 116 different PCR ribotypes.
J. Clin. Microbiol. 37, 461463.
Weese, J.S., Parsons, D.A., Staempi, H.R., 1999. Association of Clostridium
difcile with enterocolitis and lactose intolerance in a foal. J. Am. Vet.
Med. Assoc. 214, 229232 205.
Weese, J.S., Staempi, H.R., Prescott, J.F., 2001. A prospective study of the
roles of Clostridium difcile and enterotoxigenic Clostridium perfrin-
gens in equine diarrhoea. Equine Vet. J. 33, 403409.
F.A. Uzal et al. / Veterinary Microbiology 156 (2012) 395402 402