A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia?

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the alpha toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described beta2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the beta2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the beta2 probe. Pure and mixed cultures of four C. perfringens (one alpha+beta2+, one alpha+enterotoxin+ and two alpha+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the alpha and beta2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the alpha and beta2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of beta2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.     

Prevalence of cpb2, encoding beta2 toxin,in Clostridium perfringens field isolates: correlation of genotype with phenotype

Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens. By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C. perfringens. Of these, 37.2% were PCR positive for the cpb2 gene. The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C. perfringens isolated from cases of porcine neonatal enteritis (91.8%). In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases. The correlation between enteritis in other animal species and the presence of cpb2 was not so strong. cpb2 was found in 21.4% of C. perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis. The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases. Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2. We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2. Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting. In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%). However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein. The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections. However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species.     

Toxinotypes of Clostridium perfringens isolated from sick and healthy avian species.

Currently, the factors/toxins responsible for Clostridium perfringens-associated avian enteritis are not well understood. To assess whether specific C. perfringens' toxinotypes are associated with avian enteritis, the isolates of C. perfringens from 31 cases of avian necrotic or ulcerative enteritis submitted between 1997 and 2005 were selected for retrospective analysis using multiplex PCR. C. perfringens was isolated from chickens, turkeys, quail, and psittacines. The toxinotypes of isolates from diseased birds were compared against the toxinotype of 19 C. perfringens isolates from avian cases with no evidence of clostridial enteritis. All C. perfringens isolates were classified as type A regardless of species or disease history. Although many isolates (from all avian groups) had the gene encoding the C. perfirngens beta2 toxin, only 54% produced the toxin in vitro when measured using Western blot analysis. Surprisingly, a large number of healthy birds (90%) carried CPB2-producing isolates, whereas over half of the cpb2-positive isolates from diseased birds failed to produce CPB2. These data from this investigation do not suggest a causal relationship between beta2 toxin and necrotic enteritis in birds.     

Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in Italy

A study was carried out in the South of Italy to assess the role of clostridia in neonatal diseases of lambs and kids. Eighty-seven lambs and 15 kids belonging to 25 flocks were examined and Clostridium perfringens was the microorganism most commonly identified. C. perfringens isolates were analysed by polymerase chain reaction (PCR), in order to determine the prevalence of the genes cpa, cpb, cpb2, etx, iap and cpe. The most prevalent toxin-type of C. perfringens was found to be type A found in 84% of the cases with clostridial enterotoxaemia. No C. perfringens type B, C or E were found. C. perfringens type D was isolated in 16% of the cases. About 24% of the isolates were cpb2 positive. The prevalence of cpb2 across the different C. perfringens types varied. The beta(2)-toxin gene cpb2 was detected in 4/21 (19%) type A isolates, in 1/2 type D isolates, and in 1/2 type DE (cpe-carrying type D) isolates. The high rate of positivity to cpb2 among the isolates suggests that a vaccine based on the beta(2)-toxin, should be included in the vaccination schedule of the animals to confer adequate protection and to prevent the disease.     

Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS)

This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature.     

The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed

This study examined the prevalence and expression of the "consensus" and the "atypical"cpb2 genes in Clostridium perfringens isolates from cattle, chickens, dogs, goats, horses, pigs and sheep using polymerase chain reaction (PCR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. Almost all porcine isolates (12/14) carried and expressed the consensus form of cpb2 but, when present in 108 non-porcine isolates, the gene was usually the atypical form (40 atypical versus 9 consensus). Western blotting showed expression in 30 of 40 (75%) atypical cpb2-positive isolates, considerably more frequently than reported previously. CPB2 was expressed by almost all (20/21) the consensus cpb2-positive isolates, regardless of source.     

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.     

A new PCR followed by MboI digestion for the detection of all variants of the Clostridium perfringens cpb2 gene

Clostridium perfringens which is a causative agent of several diseases in animals and humans is capable of producing a variety of toxins. Isolates are typed into five types on the basis of the presence of one or more of the four major toxins genes, i.e. cpa, cpb, etx, and iap. A decade ago another toxin termed beta2 (beta2) and its gene (cpb2) were identified. Two alleles of cpb2 are known and a possible link between differences in gene expression and allelic variation has been reported. A correlation between the level of expression and the origin of the isolates has also been suggested. The demonstration and typing of the cpb2 gene in the genome of isolates can be seen as a vital part of research on the role of the beta2 toxin in the pathogenesis of disease. This study describes a PCR with a single primer set which in contrast to published primer sets recognizes both alleles. Subsequent restriction enzyme analysis of the PCR product enables typing of the alleles. Applying this protocol on a total of 102 isolates, a sub-variant was found which occurred only in C. perfringens isolates from pigs and appeared to be the predominant variant found in C. perfringens isolates from this species.     

携带非典型cpb2基因牛源A型产气荚膜梭菌重组α毒素的免疫保护性研究

为验证重组α毒素对携带非典型cpb2基因的A型产气荚膜梭菌的免疫保护性,本研究应用PCR技术,从某牛场牛源A型产气荚膜梭菌G1分离株中扩增出1 194 bp的α毒素编码基因(cpa)和795 bp的β2毒素编码基因(cpb2)。经BLAST分析显示,G1分离株携带的cpb2基因与14个菌株的非典型cpb2基因的氨基酸序列同源性为95.1%～98.9%,与典型cpb2基因(L77695)的氨基酸序列同源性为61.7%。这表明,G1的cpb2基因为非典型cpb2基因。同时分别将cpa和cpb2基因扩增产物克隆于原核表达载体中,构建重组表达质粒pET-a和pET-b2,重组菌经IPTG诱导表达重组蛋白,将其纯化后单独及联合免疫小鼠进行免疫保护试验。结果显示,单独免疫重组α毒素蛋白组以及联合免疫重组β2毒素蛋白组的小鼠,均可以抵抗至少6倍最小致死量(MLD)的G1外毒素(包含α毒素和非典型β2毒素)的攻击,也可以完全抵抗至少6 MLD的G2外毒素(不包含β2毒素)的攻击。表明,重组α毒素蛋白对含有及不含有非典型β2毒素的A型产气荚膜梭菌均具有良好的免疫保护作用。     

Significance of beta 2-toxigenic clostridium perfringens infections in animals and their predisposing factors--a review

The novel beta 2-toxin of Clostridium perfringens has recently been described as the cause of enteric diseases in animals. The biological activity of beta 2-toxin is similar to that of the beta1-toxin with a possibly weaker cytotoxic activity. However, the production of beta 2-toxin in vitro is not seen in all beta 2-toxin-gene (cpb2)-positive C. perfringens strains, and to deduce a clinical importance solely from the detection of cpb2 is difficult. Detection of cpb2-positive C. perfringens from various animal species with and without enteric diseases demonstrates the wide distribution of cpb2 in nature, and the presence of cpb2 gene is therefore not considered a risk by itself. Predisposing factors like low trypsin activity in the intestinal tract, antibiotic and/or antiphlogistic treatment or changes in diet can result in the selection of beta 2-toxigenic C. perfringens which may lead to enteritis or enterotoxaemia.     

Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves

The enterotoxaemia syndrome in Belgian Blue calves is characterised by a high case fatality rate, sudden death, lesions of haemorrhagic enteritis of the small intestine and, quite often an absence of other clinical signs but its cause has not been yet identified. As a first step in this identification, the aerobic and anaerobic intestinal flora of a population of 78 calves, originating from farms located in southern Belgium and that died in circumstances defined as "calf enterotoxaemia" (study population) and of 64 calves that died in other circumstances (control population) were studied qualitatively and quantitatively. The colonies were identified after subcultures with appropriate API sugar sets. Anaerobically Clostridium perfringens was isolated in higher numbers (mean values of 10(7)-10(7.5) colony forming units (CFU) versus 10(4)-10(5) CFU per ml of intestinal content) and from more animals (79 versus 19%) in the study population than in the control population, although individual results from both populations could overlap. Other clostridial species, i.e. mainly urease-negative C. sordellii and C. bifermentans, were isolated in high numbers (>10(6) CFU per ml of intestinal content) from a few animals in the study population only. All but one of the 705 C. perfringens isolates from both populations belonged to the A toxin type and none of the urease-negative C. sordellii was toxigenic. Gram-negative anaerobes were not isolated in high numbers from any of the samples. Aerobically beta-haemolytic E. coli were significantly more frequent among the study population, but were isolated from only 25% of the animals. Salmonella Typhimurium was isolated from only two animals in the study population. Less than 1% of the E. coli isolated were verotoxigenic and one-third were necrotoxigenic. At this stage only non-enterotoxigenic type A C. perfringens are thus statistically associated with the enterotoxaemia syndrome in Belgian Blue calves and fulfil the first of the Koch's postulates.     

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.     

Association of genes encoding beta2 toxin and a collagen binding protein in Clostridium perfringens isolates of porcine origin

Clostridium perfringens is a cause of economically significant enteritis in livestock. Beta2 toxin, encoded by one of two cpb2 alleles, is implicated as a virulence factor in this disease. Previous studies determined that the consensus cpb2 allele is preferentially associated with C. perfringens isolated from pigs. In C. perfringens strain 13, the consensus cpb2 allele is found on the plasmid pCP13, which also carries cna, encoding a putative collagen binding protein, CpCna. This protein was shown to be a bona fide collagen adhesin, as recombinant, HIS-tagged CpCna bound collagen type I as determined by Far Western blotting. Genomic DNA from C. perfringens isolated from a variety of host species were subjected to PCR to determine the prevalence of cna in these strains and correlate its carriage with the presence and type of cpb2 allele. The cna gene was found in 55.8% of isolates from all host species (n=208) and 68.1% of porcine isolates (n=119). In cpb2+ isolates, cna was present in 69.9% of isolates from all hosts (n=153), but was found in 98.7% of porcine isolates (n=75). Furthermore in porcine isolates, the consensus cpb2 allele and cna were absolutely correlated with the presence of pcp12, a pCP13-encoded gene, and pcp12 was never found in any isolate that lacks either cpb2 allele. The finding that CpCna binds collagen and that the cna gene is associated with the consensus cpb2 allele implicates CpCna as a potential virulence factor in porcine enteritis caused by C. perfringens.     

Survival of clostridium perfringens during simulated transport and stability of some plasmid-borne toxin genes under aerobic conditions

Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4 degrees C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpbl and etx genes were detected in all isolates from samples stored either at room temperature or at 4 degrees C for 24-44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem.     

An investigation into the association between cpb2-encoding Clostridium perfringens type A and diarrhea in neonatal piglets

To investigate the possible role of cpb2-positive type A Clostridium perfringens in neonatal diarrheal illness in pigs, the jejunum and colon of matched normal and diarrheic piglets from 10 farms with a history of neonatal diarrhea were examined grossly and by histopathology, and tested for C. perfringens, for C. perfringens beta2 (CPB2) toxin, as well as for Clostridium difficile toxins, Salmonella, enterotoxigenic Escherichia coli, rotavirus, transmissible gastroenteritis (TGE) virus, and coccidia. Clostridium perfringens isolates were tested using a multiplex real-time polymerase chain reaction (PCR) to determine the presence of cpa, consensus and atypical cpb2, and other virulence-associated genes. The numbers of C. perfringens in the intestinal contents were lower in diarrheic piglets (log₁₀ 5.4 CFU/g) compared with normal piglets (log₁₀ 6.5 CFU/g) (P < 0.05). The consensus cpb2 was present in 93% of isolates in each group, but atypical cpb2 was less common (56% healthy, 32% diarrheic piglets isolates, respectively, P < 0.05). The presence of CPB2 toxin in the intestinal contents of normal and diarrheic piglets did not differ significantly. Clostridium difficile toxins and rotavirus were each detected in 7 of the 21 (33%) diarrheic piglets. Rotavirus, C. difficile toxins, Salmonella, or enterotoxigenic E. coli were concurrently recovered in different combinations in 4 diarrheic piglets. The cause of diarrhea in 8 of the 21 (38%) piglets on 6 farms remained unknown. The etiological diagnosis of diarrhea could not be determined in any of the piglets on 2 of the farms. This study demonstrated that the number of cpb2-positive type A C. perfringens in the intestinal contents was not a useful approach for making a diagnosis of type A C. perfringens enteritis in piglets. Further work is required to confirm whether cpb2-carrying type A C. perfringens have a pathogenic role in enteric infection in neonatal swine.     

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.     

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.     

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats

Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.