Genotyping and phenotyping of beta2-toxigenic Clostridium perfringens fecal isolates associated with gastrointestinal diseases in piglets

Although Clostridium perfringens is recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringens toxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C perfringens isolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfiingens isolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringens isolates from pigs with GI disease (pig G1 disease isolates). PCR and restriction fragment length polyrnorphism analysis reconfirmed the presence of cpb2 gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig G1 disease isolates included in this study all carry a plasmid cpb2 gene, yet no clonal relationships were detected between the cpb2-positive pig G1 disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig G1 disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringens isolates and diarrhea in piglets.