Genotyping and Phenotyping of Beta2-Toxigenic Clostridium perfringensFecal Isolates Associated with Gastrointestinal Diseases in Piglets

ABSTRACTAlthough Clostridium perfringensis recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringenstoxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringensisolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringensisolates 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. perfringensisolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2gene, yet no clonal relationships were detected between the cpb2-positive pig GI 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 GI 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. perfringensisolates and diarrhea in piglets.