OBJECTIVE This study reports on comparisons between polymerase chain reaction (PCR) and conventional diagnostic methods for typing Clostridium perfringens toxins collected from Central Saudi Arabia. METHODS Fecal samples from 150 animals showing signs of enterotoxaemia were collected from 24 April 2009 to 25 September 2009, from different farms located in Riyadh, Kingdom of Saudi Arabia. Twenty-seven toxigenic strains of Clostridium perfringens were recovered from 150 fecal and intestinal content samples were identified and typed by conventional methods. All the strains were analyzed by PCR using specific primers for alpha, beta, epsilon and iota toxin genes. The experimental work was carried out at the Center of Excellence in Biotechnology, King Saud University, Riyadh, Kingdom of Saudi Arabia. RESULTS The results revealed alpha toxin gene Clostridium perfringens type A in 22 (81.5%) strains out of 27 toxigenic strains, however, only 20 (74.1%) of them were identified previously as type A by classical method. One strain (3.7%) was identified as type C and 3 strains (11.1%) were identified as D by PCR typing. Moreover, PCR results confirmed the traditional methods in typing one strain as type B (3.7%). Also, PCR method can detect 2 other strains of type A directly from the feces and intestinal contents of the examined chicken, which provide negative results in bacteriological examination. CONCLUSION Polymerase chain reaction technique can be used as an alternative diagnostic method for detection and typing of Clostridium perfringens.

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.

Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways:(i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-gamma) and interleukin 2 (IL-2), suggesting a Th1 type immune response.

Cattle enterotoxaemia is one of numerous pathologies caused by Clostridium perfringens. These anaerobic Gram-positive bacteria are naturally present in the intestinal flora of mammals, but their uncontrolled multiplication under certain conditions results in the overproduction of toxins in the intestinal tract. Major clinical signs are induced by the systemic spread of these toxins in the blood and tissues. Enterotoxaemia may be acute or peracute, and sudden death is often reported in rapidly growing, apparently healthy cattle. Enterotoxaemia can be prevented only with better understanding of its risk factors and pathogenesis. This paper provides an up-to-date overview of knowledge concerning the aetiology of the syndrome, its epidemiological context, pathogenesis, clinical signs and lesions, the diagnostic procedures and prophylactic tools, with specific attention to field aspects that are directly relevant to practitioners and clinical researchers.

This study was carried out in order to quantify enteric bacteria and identify the presence of Salmonella spp., Escherichiacoli, Clostridiumperfringens and Erysipelothixrhusiopathiae in the liquid fraction of excreta generated from a small scale swine farm by means of a primary treatment system, consisting of the separation of solids and the sedimentation of liquids. Samples were collected at the following stages of the treatment: collection basin (CB), liquid obtained from a solids separator (SLF) and liquid from the sedimentation basin (SB). In each sample, enteric bacteria (cfu/g wet weight) and E. coli were quantified, Salmonella spp. was isolated and typified, and C. perfringens, and E. rhusiopathiae were isolated. No significant differences (p>0.05) were found in the enteric bacteria and E. coli population levels at any treatment stage. S.choleraesuis was found in 20% of CB samples analyzed, 40% of SLF samples and 30% of SB samples. C. perfringens was isolated from SLF and SB. E. rhusiopathiae was not isolated (below the minimum detection limit). Results suggest that primary treatment does not reduce the amount of enteric bacteria, or eliminate Salmonella spp., E. coli and C. perfringens. Therefore, it is necessary to apply additional treatments to allow safe use of the liquid obtained in the farm.

The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to approximately 90-kb plasmids in most type B isolates, although a few isolates carried a approximately 65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located approximately 3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.

The main aim of this study was to investigate the occurrence and ribotypes of Clostridium perfringens in broiler flocks reared in 2 European countries that apply European Union Regulation 1831/2003. A total of 1,532 cecum contents were collected between June 2005 and November 2006 from birds belonging to 51 intensively reared flocks produced in the Czech Republic and 41 intensive production, organic, and free-range flocks reared in Italy. Clostridium perfringens was detected in 64.7 and 82.9% of the Czech Republic and Italian flocks, respectively, at mean loads ranging between 3.65 and 4.77 log10 cfu per gram of cecum content. More than 1 ribotype was identified among isolates belonging to the same flock in 57.1 and 76.5% of the Czech Republic and Italian flocks, respectively. Moreover, common ribotypes were identified between strains belonging to 2 up to 8 different flocks. In particular, 4 ribotypes were shared between strains isolated in the 2 European countries. The results of this study report on C. perfringens occurrence and mean populations in broilers reared on diets devoid of antibiotic growth promoters. Moreover, these findings show for the first time the presence of common ribotyping profiles among isolates collected from birds reared more than 1,000 km apart.

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.

AIMS To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing. METHODS AND RESULTS For this purpose, alpha (cpa), beta (cpb), beta 2 (cpb2), epsilon (etx), iota (iA) and enterotoxin (cpe) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa, gene but in none of the isolates cpb, etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative. CONCLUSIONS Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.

Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

Clostridium perfringens is a well-characterized bacterial species which can be both commensal and pathogenic in humans and many animals. Genetic typing of the bacterium is often used for molecular epidemiological purposes, and can be useful for observing population structures as well. Analysis of the variable number of tandem repeats (VNTRs) within the genome, called multiple-locus VNTR analysis (MLVA) provides genetic information useful for molecular typing. A MLVA typing method has been developed recently by Sawires and Songer [Sawires, Y.S., Songer, J.G., 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe 11, 262-272] for C. perfringens. A novel MLVA protocol is described here, with the aim of investigating the discriminatory potential of the method, and to obtain preliminary data on the population structure of C. perfringens from a wide variety of C. perfringens sources. This protocol uses new loci in noncoding regions of the chromosome, and also makes use of capillary electrophoresis for more precise results and for high-throughput typing. DNA sequencing of amplicons was performed to ensure inclusion of conserved tandem repeats within each locus. Fifty-four epidemiologically unrelated isolates from a local collection obtained from 11 different animal species were typed at 6 loci. Thirty-five unique MLVA types were obtained, resulting in a Simpson's index of diversity of 0.975. Epidemiologically related isolates (n=27) previously typed by pulsed-field gel electrophoresis (PFGE) were also examined with MLVA and the congruency of the two methods was found to be very high. All 81 isolates were successfully typed with MLVA, and polymerase chain reactions (PCR) were automated using robotics and 96-well plates, with PCR product sizes determined using capillary electrophoresis. Reproducibility was also shown to be very high.

A gastroenteritis outbreak occurred in a military camp where a laboratory and epidemiological investigation was carried out. The early onset of symptoms indicated probable food contamination with Clostridium perfringens. Stool samples collected from affected patients were tested within 4 h via real-time polymerase chain reaction (PCR) for the presence of the C. perfringens plc gene. Ten out of the 12 stool samples were positive. Confirmation of the molecular test results was carried out by enumeration of C. perfringens in stool by culture and shown to be in excess of 106 spores/g stool. The isolates obtained from culture were further analysed by PCR for the presence of the chromosomal enterotoxin (cpe) gene. Based on the clinical symptoms, epidemiological and laboratory investigations, C. perfringens was implicated as the aetiological agent. The ability to conduct real-time PCR analysis greatly shortens the time to diagnosis and allows for preventive and control measures to be effected quickly.

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium associated with a wide variety of diseases in domestic animals and humans. We have developed dual-labeled fluorescence hybridization probe (TaqMan((R)))-based real-time multiplex PCR assay for detection of toxin genes alpha (cpa), beta (cpb), iota (ia), epsilon (etx), beta2 (cpb2) and enterotoxin (cpe) of C. perfringens directly from cattle feces. The assay was standardized using ATCC reference strains of C. perfringens producing alpha, beta, iota, epsilon and enterotoxin, respectively. The assay for detection of beta2 toxin gene was standardized using a field strain of C. perfringens producing beta2 toxin. The minimum detection limit for the real time PCR assay ranged from 5 to 70 pg of DNA for the six toxin genes. A total of 307 fecal samples collected from seven dairy herds in Pennsylvania were analyzed using the multiplex assay. The real-time PCR assay revealed that cpa, cpb, ia, etx, cpb2 and cpe were detected in 68 (28.2%), 6 (2.5%), 6 (2.5%), 4 (1.6%), 164 (68%) and 11 (4.5%) of 241 PCR positive samples, respectively. The findings of the study revealed that C. perfringens beta2 toxin producing strains were widely prevalent in lactating cows in Pennsylvania and they may play an important role in C. perfringens associated diarrheal diseases.

Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.

We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance observed in natural cases of necrotic enteritis (NE)[Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257-266]. Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C. perfringens. Birds developed typical NE lesions in response to administration (2x per day for 4 days) of a combined inoculum comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable. Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited each other and normal flora strains (n=17), while normal flora strains inhibited neither NE strains nor each other. Thus, it appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests that other factors may be partially or completely responsible for the dominance.

Clostridium perfringens type D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding epsilon-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, including C. perfringens enterotoxin and beta2 toxin, encoded by the cpe and cbp2 genes, respectively. In the current study we demonstrated that the etx, cpe, and cpb2 genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that the etx gene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. The etx plasmids also typically carried IS1151 and tcp open reading frames (ORFs) known to mediate conjugative transfer of C. perfringens plasmid pCW3. PCR studies revealed that other than their tcp ORFs, etx plasmids of type D isolates do not carry substantial portions of the conserved or variable regions in the cpe plasmids of type A isolates. Southern blotting also demonstrated that in type D isolates the cpe and cpb2 genes are sometimes present on the etx plasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids,(ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) many etx plasmids should be capable of conjugative transfer.