A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea

Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.     

Adsorptive effects of di-tri-octahedral smectite on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies

OBJECTIVE: To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies. SAMPLE POPULATION: 3 C perfringens exotoxins and 9 colostral samples. PROCEDURES: Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined via single radial immunodiffusion assay. RESULTS: Di-tri-octahedral smectite decreased the amount of each C perfringens exotoxin in co-incubated samples in a dose-dependent manner and was more effective than bismuth subsalicylate at reducing exotoxins in vitro. Decreases in the concentration of IgG were detected in samples of colostrum that were combined with DTOS at 1:4 through 1:16 dilutions, whereas no significant decrease was evident with DTOS at the 1:32 dilution. CONCLUSIONS AND CLINICAL RELEVANCE: Di-tri-octahedral smectite effectively adsorbed C perfringens exotoxins in vitro and had a dose-dependent effect on the availability of equine colostral antibodies. Results suggested that DTOS may be an appropriate adjunctive treatment in the management of neonatal clostridiosis in horses. In vivo studies are necessary to fully assess the clinical efficacy of DTOS treatment.     

Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic.

OBJECTIVE: To determine prevalence of clostridial enterotoxins in feces of horses with diarrhea and colic, and to determine whether an association exists between detection of clostridial enterotoxins in feces and development of diarrhea as a complication of colic. DESIGN: Prospective case series and case-control study. ANIMALS: 174 horses with diarrhea, colic, or problems not related to the gastrointestinal tract. PROCEDURE: Horses were assigned to 1 of 4 groups: colic with diarrhea (group 1; n = 30); colic without diarrhea (group 2; 30); diarrhea without colic (group 3; 57); and control (group 4; 57). Feces were evaluated by use of ELISA to detect Clostridium perfringens enterotoxin (CPE) and C difficile toxin A (TOXA). Frequency of detection of CPE or TOXA in groups 1 and 3 was compared with that in groups 2 and 4, respectively. RESULTS: Prevalence of enteric clostridiosis in horses in group 3 was 25%. Clostridium perfringens enterotoxin was detected in 9 of 57 (16%), TOXA in 8 of 57 (14%), and both toxins in 3 of 57 (5%) fecal samples collected from these horses. Neither toxin was detected in feces of the age-matched horses in group 4. Clostridial enterotoxins were detected in feces of 7 of 60 (12%) horses with colic (groups 1 and 2), however, a significant association was not found between detection of enterotoxins in feces and development of diarrhea as a complication of colic. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridia are important etiologic agents of diarrhea in horses. Additionally, changes in intestinal flora of horses with colic may allow for proliferation of clostridia and elaboration of enterotoxins regardless of whether diarrhea develops.     

Acute diarrhea in horses of the Potomac River area: examination for clostridial toxins

Fecal specimens from horses in Montgomery County, Md, and in Fairfax and Loudoun counties, Va, were examined for Clostridium perfringens type A enterotoxin and for C difficile cytotoxin (92 and 108 specimens, respectively). The toxins were found in feces from horses that had experienced an acute diarrhea syndrome and from clinically normal horses. The toxins did not appear to be primary determinants of the diarrhea syndrome, although they may have contributed to the spectrum of clinical entities observed.     

Equine grass sickness: serologic evidence of association with Clostridium perfringens type A enterotoxin.

Clostridium perfringens type A enterotoxin seroneutralization was carried out on sera from 50 horses recovered from grass sickness and from 100 other horses with no record of having had the disease. Of the affected horses, 70% had seroneutralizating titers higher than 1:64, half of these being equal or higher than 1:128. More than 88% of the horses with no record of grass sickness had titers lower than 1:64. These data support the theory of association between C perfringens type A toxins and grass sickness.     

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.     

Clostridium perfringens enterotoxicosis in two Amur leopards (Panthera pardus orientalis).

Two 6-yr-old male sibling Amur leopards (Panthera pardus orientalis) housed together at the Pittsburgh Zoo presented for acute onset of diarrhea with no changes in appetite or behavior. Heat-fixed modified Wright-stained and Gram-stained fecal smears revealed a mixed bacterial population with a large number of gram-positive Clostridium perfringens-like spores (>20 per high-power oil immersion field). In addition, C. perfringens enterotoxin was isolated from one leopard at 1:256, confirming the presence of C. perfringens enterotoxicosis. Treatment with oral metronidazole, tylosin tartrate, and psyllium fiber was prescribed, with return of more normal stool by the third day of treatment. Fecal consistency steadily improved and was considered normal by the time all prescribed treatments were complete. Diarrhea has not recurred. Partially thawed meat in the leopards' diet may have precipitated the production of an endogenous clostridial enterotoxicosis by disrupting digestive tract flora with resultant clostridial overgrowth and sporulation.     

Survival of Clostridium difficile and its toxins in equine feces: implications for diagnostic test selection and interpretation.

Although Clostridium difficile is recognized as a cause of enterocolitis in horses and humans, there has been little work published regarding the lability of C. difficile and its toxins in feces. A significant decrease in recovery of C. difficile from inoculated equine fecal samples occurred during storage. Recovery after storage in air at 4 degrees C decreased from 76% (37/49) after 24 hours to 67% (33/49) at 48 hours and 29% (14/ 49) after 72 hours. In contrast to aerobic storage, 25 of 26 samples stored anaerobically at 4 degrees C yielded growth of C. difficile for 30 days, whereas the organism was only detected for 2.5 +/- 2.52 days (x +/- SD) in paired samples stored aerobically. The use of an anaerobic transport medium was effective in maintaining viability of C. difficile. These findings indicate that poor aerotolerance is the reason for the rapid decrease in culture yield. In contrast to C. difficile organisms stored aerobically at 4 degrees C, C. difficile toxins were considerably more stable and could be detected by enzyme-linked immunosorbent assay in both broth and inoculated fecal samples for at least 30 days. The poor survival of C. difficile but the stability of its toxins when feces are stored aerobically must be considered when submitting samples for diagnosis of C. difficile-associated enterocolitis in horses and when interpreting laboratory results.     

The association of erythromycin ethylsuccinate with acute colitis in horses in Sweden

In Sweden there are several reports of mares developing acute colitis while their foals were being treated orally for Rhodococcus equi pneumonia with the combination of erythromycin and rifampicin. In this study 6 adult horses were given low oral dosages of these antibiotics, singly or in combination. Within 3 days post administration of erythromycin, in one case in combination with rifampicin, 2 horses developed severe colitis (one fatal). Clostridium difficile was isolated from one of the horses, whereas no specific pathogens were isolated from the other. Both horses had typical changes in blood parameters seen in acute colitis. Clostridium difficile was also isolated from the faeces of a third horse given an even lower dosage of erythromycin in combination with rifampicin. This horse developed very mild clinical symptoms and recovered spontaneously. In the fourth horse given erythromycin only, very high numbers of Clostridium perfringens were isolated. The horses given rifampicin only did not develop any clinical symptoms and there were no major changes in their faecal flora. In conclusion, it has been demonstrated that low dosages of erythromycin ethylsuccinate can induce severe colitis in horses associated with major changes of the intestinal microflora. Clostridium difficile has been demonstrated as a potential aetiological agent in antibiotic-induced acute colitis.     

Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs

Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution.     

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.     

Clostridial enteric infections in pigs.

Clostridium perfringens types A and C and Clostridium difficile are the principal enteric clostridial pathogens of swine. History, clinical signs of disease, and gross and microscopic findings form the basis for a presumptive diagnosis of C. perfringens type-C enteritis. Confirmation is based on isolation of large numbers of type-C C. perfringens and/or detection of beta toxin in intestinal contents. Diagnosis of C. perfringens type-A infection, however, remains controversial, mostly because the condition has not been well defined and because type-A organisms and their most important major (alpha) toxin can be found in intestinal contents of healthy and diseased pigs. Isolation of large numbers of C. perfringens type A from intestinal contents, in the absence of other enteric pathogens, is the most reliable criterion on which to base a diagnosis. Recently, beta2 (CPB2) toxin-producing C. perfringens type A has been linked to disease in piglets and other animals. However, implication of CPB2 in pathogenesis of porcine infections is based principally on isolation of C. perfringens carrying cpb2, the gene encoding CPB2, and the specific role of CPB2 in enteric disease of pigs remains to be fully defined. Clostridium difficile can also be a normal inhabitant of the intestine of healthy pigs, and diagnosis of enteric infection with this microorganism is based on detection of its toxins in feces or intestinal contents.     

Clostridium difficile: prevalence in horses and environment,and antimicrobial susceptibility

REASONS FOR PERFORMING STUDY: Clostridium difficile has been associated with acute colitis in mature horses. OBJECTIVES: To survey C. difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C. difficile in the environment and antimicrobial susceptibility of isolated strains. METHODS: A total of 777 horses of different breeds, age and sex were studied. Further, 598 soil samples and 434 indoor surface samples were examined. Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics. RESULTS: In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C. difficile culture and 12 of these (28%) were positive in the cytotoxin B test. Furthermore, C. difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 [6%]) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic. An interesting new finding was that, in normal healthy foals age < 14 days, C. difficile was isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C. difficile. On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C. difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C. difficile (P = < 0.001). By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C. difficile. The experimental study conducted here found that C. difficile survived in nature and indoors for at least 4 years in inoculated equine faeces. The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l). CONCLUSIONS: C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furthermore, C. difficile was isolated from 1 in 3 normal healthy foals age < 14 days. POTENTIAL RELEVANCE: Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C. difficile.     

Prevalence of Diarrhea and Enteropathogens in Racing Sled Dogs

Background: Diarrhea is highly prevalent in racing sled dogs, although the underlying causes are poorly understood.
Hypothesis: Clostridium perfringens enterotoxin (CPE) and Clostridium difficile Toxin A and B are associated with diarrhea in racing sled dogs.
Animals: One hundred and thirty-five sled dogs.
Methods: Freshly voided feces were obtained from 55 dogs before racing and from 80 dogs after 400 miles of racing. Samples were visually scored for diarrhea, mucus, blood, and melena. CPE and C. difficile Toxin A and B were detected by ELISA. Samples were cultured for C. perfringens, C. difficile, Campylobacter, Salmonella , and Escherichia coli 0157; Giardia and Cryptosporidium spp. were detected via immunofluorescence.
Results: Diarrhea occurred in 36% of dogs during racing, and hematochezia, fecal mucus or melena, or all 3 occurred in 57.5% of dogs. Salmonella was isolated from 78.2% of dogs before racing, and from 71.3% of dogs during racing. C. perfringens and C. difficile were isolated from 100 and 58.2% of dogs before racing, and from 95 and 36.3% of dogs during racing. Dogs were more likely to test positive for CPE during than before racing (18.8 versus 5.5%, P = .021); however, no enteropathogens or their respective toxins were significantly associated with hematochezia or diarrhea.
Conclusions and Clinical Importance: Sled dogs participating in long distance racing have a high prevalence of diarrhea and hematochezia that is not associated with common enteropathogens. It is possible that diarrhea and hematochezia represent the effect of prolonged exercise on the gastrointestinal tract.     

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.     

The emergence of Clostridium difficile as a pathogen of food animals

Clostridium difficile causes pseudomembranous colitis in humans, usually after disruption of the bowel flora by antibiotic therapy. Factors mediating the frank disease include the dose and toxigenicity of the colonizing strain, its ability to adhere to colonic epithelium, the concurrent presence of organisms that affect multiplication and toxin production or activity, and the susceptibility of the host. Toxins A (an enterotoxin) and B (a cytotoxin) play the major role in pathogenesis and the detection of toxins in gut contents is the gold standard for diagnosis. Disease in horses takes the form of often-fatal foal hemorrhagic enteritis. Nosocomial, antibiotic-associated, disease is increasingly common in adult horses. Enteric clinical signs are reported in ostriches, companion animals and recently calves. Clostridium difficile colitis is now a common diagnosis in neonatal pigs in the USA and elsewhere. Clinical features include onset at 1-5 days of age, sometimes with dyspnea, mild abdominal distension and scrotal edema, and commonly with yellow, pasty diarrhea. There is mesocolonic edema grossly, with microscopic diffuse colitis, mucosal edema, crypt distension, epithelial necrosis and superficial mucosal erosion. Neutrophil infiltration of the lamina propria is common, and fibrin and numerous rod-shaped bacteria are observed on the surface. About two-thirds of litters and one-third of piglets will be affected (based upon positive toxin tests), although this appears to vary with the season. The case fatality rate is probably low if considering only direct effects of C. difficile infection. The significance of toxin-positive non-diarrheic pigs and the nature of the interaction of toxins A and B with enterocytes are unknown. Given the widespread occurrence of the disease, there is substantial effort to develop immunoprophylactic products.     

Clostridium difficile associated with acute colitis in mature horses treated with antibiotics

Clostridium (C.) difficile, or its cytoxin, was demonstrated in faecal samples from 10 of 25 (40%) mature horses investigated with acute colitis treated primarily with antibiotics for disorders other than diarrhoea. C. difficile was not found in faecal samples from 140 horses without signs of enteric disorders, from 21 nondiarrhoeic horses treated with antibiotics, nor from 22 horses with colitis untreated with antibiotics. Except for C. difficile neither Salmonella nor any other investigated intestinal pathogen was isolated in any of the diarrhoeic horses. The findings strongly support some earlier reports that C. difficile is associated with acute colitis in mature horses treated with antibiotics. Of the 10 horses, 4 proved positive for C. difficile both in culture and in the cytotoxin test, 4 in culture only and 2 only in the cytotoxin test. Eight of 10 horses positive for C. difficile were or had recently been hospitalised, indicating that C. difficile may be a nosocomial infection in horses. All horses positive for C. difficile were treated with beta-lactam antibiotics.     

Evaluation of a routine diagnostic fecal panel for dogs with diarrhea

OBJECTIVES: To assess the diagnostic yield of a routine fecal panel and determine whether Clostridium perfringens or C difficile toxin production is associated with acute hemorrhagic diarrheal syndrome (AHDS) in dogs. DESIGN: Case-control study. ANIMALS: 260 dogs with diarrhea and 177 dogs with normal feces. PROCEDURE: Medical records were reviewed for results of culture for C difficile, Campylobacterspp, and Salmonella spp; C perfringens fecal enterotoxin (CPE) assay via ELISA or reverse passive latex agglutination (RPLA) assay; fecal endospore enumeration; C difficile toxin A assay; and parasite evaluation. RESULTS: Prevalence of CPE in dogs with diarrhea was 22/154 (14.3%) via ELISA and 47/104 (45.2%) via RPLA assay, versus 9/74 (12%) via ELISA and 26/103 (25%) via RPLA assay in control dogs. Prevalence of C difficile was 47/260 (18%) in dogs with diarrhea and 41/74 (55%) in control dogs. Prevalence of C difficile toxin A was 26/254 (10.2%) in dogs with diarrhea and 0/74 in control dogs. Diagnosis of AHDS was made in 27 dogs; 8 had positive results for CPE, 7 had positive results for toxin A, and 1 had positive results for both toxins. Campylobacter spp were isolated from 13 of 260 (5%) dogs with diarrhea and 21 of 74 (28.4%) control dogs. Salmonella spp were isolated from 3 (1.2%) dogs with diarrhea. CONCLUSIONS AND CLINICAL RELEVANCE: Diagnostic value of a fecal panel in dogs with diarrhea appears to below.     

Molecular characterization of Clostridium difficile isolates from horses in an intensive care unit and association of disease severity with strain type

OBJECTIVE: To determine molecular characteristics, antimicrobial susceptibility, and toxigenicity of Clostridium difficile isolates from horses in an intensive care unit and evaluate associations among severity of clinical disease with specific strains of C difficile. DESIGN: Prospective study. ANIMALS: 130 horses. PROCEDURES: Feces were collected from horses admitted for acute gastrointestinal tract disease with loose feces and submitted for microbial culture and immunoassay for toxin production. Polymerase chain reaction assays were performed on isolates for toxins A and B genes and strain identification. RESULTS: Isolates were grouped into 3 strains (A, B, and C) on the basis of molecular banding patterns. Toxins A and B gene sequences were detected in 93%, 95%, and 73% of isolates of strains A, B, and C, respectively. Results of fecal immunoassays for toxin A were positive in 40%, 63%, and 16% of horses with strains A, B, and C, respectively. Isolates in strain B were resistant to metronidazole. Horses infected with strain B were 10 times as likely to have been treated with metronidazole prior to the onset of diarrhea as horses infected with other strains. Duration from onset of diarrhea to discharge (among survivors) was longer, systemic inflammatory response syndromes were more pronounced, and mortality rate was higher in horses infected with strain B than those infected with strains A and C combined. CONCLUSIONS AND CLINICAL RELEVANCE: Horses may be infected with a number of heterogeneous isolates of C difficile. Results indicated that toxigenicity and antimicrobial susceptibility of isolates vary and that metronidazole-resistant strains may be associated with severe disease.