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Journal of Clinical Microbiology, August 2003, p. 3584-3591, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3584-3591.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331,¹ Department of Biomedical Sciences, Defense Science and Technology Laboratory Chemical and Biological Sciences, Porton Down SP4 0JQ, United Kingdom,² Department of Veterinary Sciences and Microbiology, University of Arizona, Tucson, Arizona 95721,³ Unite des Toxines Microbiennes, Institute Pasteur, 75724 Paris, Cedex 15, France,⁴ Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261⁵

Received 23 December 2002/ Returned for modification 9 March 2003/ Accepted 26 March 2003

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. perfringens 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 GI disease isolates). PCR and restriction fragment length polymorphism 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 GI disease isolates included in this study all carry a plasmid cpb2 gene, 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. perfringens isolates and diarrhea in piglets.

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