ABSTRACT Clostridium perfringens and C. difficile have been associated with acute and chronic large and small bowel diarrhoea, and acute haemorrhagic diarrhoeal syndrome in dogs. The objective of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. perfringens and 10 C. difficile isolates from 95 canine faeces (n = 36, diarrhoeic and n = 59, non-diarrhoeic). Concerning C. perfringens, 13 strains (92.9%) were type A, of which 3 (23.1%) also possessed the beta 2 toxin (CPB2)-encoding gene. One isolate (7.1%) was type D and possessed CPB2 gene. On the whole, 4 of the 14 strains (28.6%) tested cpb2-positive. Six C. difficile isolates (60.0%) demonstrated tcdA+/tcdB+ and cdtA+/vcdtB+ genotype and tested positive for, in vitro, toxin production by EIA. Eight distinct ribotypes were observed. In conclusion, the PCR assays may provide useful and reliable tools for C. perfringens and C. difficile molecular typing in routine veterinary diagnostics.
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M. Cristina Ossiprandi, M. Buttrini, E. Bottarelli and L. Zerbini, "A Preliminary Molecular Typing by PCR Assays of Clostridium perfringens and Clostridium difficile Isolates from Dogs," Advances in Microbiology, Vol. 2 No. 2, 2012, pp. 87-92. doi: 10.4236/aim.2012.22011.
 S. L. Marks and E. J. Kather, “Antimicrobial Susceptibilities of Canine Clostridium difficile and Clostridium perfringens Isolates to Commonly Utilized Antimicrobial Drugs,” Veterinary Microbiology, Vol. 94, No. 1, 2003, pp. 39-45. doi:10.1016/S0378-1135(03)00061-0
 S. L. Marks and E. J. Kather, “Bacterial-Associated Diarrhea in the Dog: A Critical Appraisal,” Veterinary Clinics of North America: Small Animal Practice, Vol. 33, No. 5, 2003, pp. 1029-1060.
 C. G. Baums, U. Shotte, G. Amtsberg and R. Goethe, “Diagnostic Multiplex PCR for Toxin Genotyping of Clostridium perfringens Isolates,” Veterinary Microbiology, Vol. 100, No. 1-2, 2004, pp. 11-16.
 J. G. Smendley III, D. J. Fisher, S. Sayeed, G. Chakrabarti and B. A. McClane, “The Enteric Toxins of Clostridium perfringens,” Reviews of Physiology, Biochemistry and Pharmacology, Vol. 152, 2004, pp. 183-204.
 J. G. Songer and R. R. Meer, “Genotyping of Clostridium perfringens by Polymerase Chain Reaction Is a Useful Adjunct to Diagnosis of Clostridial Enteric Disease in Animals,” Anaerobe, Vol. 2, No. 4, 1996, pp. 197-203.
 S. L. Marks, “Bacterial Gastroenteritis in Dogs and Cats,” Proceedings of 28th World Congress of the World Small Animal Veterinary Association, Bangkok, 24-27 October 2003.
 S. B. Melville, R. E. Collie and B. A. McClane, “Regulation of Enterotoxin Production in Clostridium perfringens,” In: J. I. Rood, B. A. McClane, J. G. Songer and R. W. Titball, Eds., The Clostridia: Molecular Biology and Pathogenesis, San Diego, Academic Press, 1997, pp. 471-485.
 D. M. Bueschel, B. H. Jost, S. J. Billington, H. T. Trinh and J. G. Songer, “Prevalence of cpb2, Encoding Beta2 toxin, in Clostridium perfringens Field Isolates: Correlation of Genotype with Phenotype,” Veterinary Microbiology, Vol. 94, No. 2, 2003, pp. 121-129.
 N. Chouicha and S. L. Marks, “Evaluation of Five Enzyme Immunoassays Compared with the Cytotoxicity Assay for Diagnosis of Clostridium difficile-Associated Diarrhoea in Dogs,” Journal of Veterinary Diagnostic Investigation, Vol. 18, No. 2, 2006, pp. 182-188.
 P. Fach and M. R. Popoff, “Detection of Enterotoxigenic Clostridium perfringens in Food and Faecal Samples with a Duplex PCR and the Slide Latex Agglutination Test,” Applied and Environmental Microbiology, Vol. 63, No. 11, 1997, pp. 4232-4236.
 P. Spigaglia and P. Mastrantonio, “Molecular Analysis of the Pathogenicity Locus and Polymorphism in the Putative Negative Regulator of Toxin Production (TcdC) among Clostridium difficile Clinical Isolates,” Journal of Clinical Microbiology, Vol. 40, No. 9, 2002, pp. 3470-3475. doi:10.1128/JCM.40.9.3470-3475.2002
 S. Stubbs, M. Rupnik, M. Gilbert, J. Brazier, B. Duerden and M. Popoff, “Production of Actin-Specific ADP-Ribosyltransferase (Binary Toxin) by Strains of Clostridium difficile,” FEMS Microbiology Letters, Vol. 186, No. 2, 2000, pp. 307-312.
 P. Bidet, F. Barbut, V. Lalande, B. Burghoffer and J. C. Petit, “Development of a New PCR-Ribotyping Method for Clostridium difficile Based on Ribosomal RNA Gene Sequencing,” FEMS Microbiology Letters, Vol. 175, No. 2, 1999, pp. 261-266.
 P. Bidet, V. Lalande, B. Salauze, B. Burghoffer, V. Avesani, M. Delmée, A. Rossier, F. Barbut and J. C. Petit, “Comparison of PCR-Ribotyping, Arbitrarily Primed PCR, and Pulsed-Field Gel Electrophoresis for Typing Clostridium difficile,” Journal of Clinical Microbiology, Vol. 38, No. 7, 2000, pp. 2484-2487.
 L. Zerbini and M. C. Ossiprandi, “Prevalence of Clostridium spp. in Diarrhoeic and Healthy Dogs,” In: Annali della Facoltà di Medicina Veterinaria dell’Università di Parma, Parma, Vol. 27, 2007, pp. 143-156.
 S. Thiede, R. Goethe and G. Amtsberg, “Prevalence of β2 Toxin Gene of Clostridium perfringens Type A from Diarrhoeic Dogs,” Veterinary Record, Vol. 149, No. 9, 2001, pp. 273-274. doi:10.1136/vr.149.9.273
 U. Schotte, U. Truyen and H. Neubauer, “Significance of ?2-Toxigenic Clostridium perfringens Infections in Animals and Their Predisposing Factors—A Review,” Journal of Veterinary Medicine, Vol. 51, No. 10, 2004, pp. 423-426. doi:10.1111/j.1439-0450.2004.00802.x
 R. M. Batt and C. Rutgers, “Bacteria and Intestinal Disease in Dogs,” GDBA Technical Review No 11, UK, 1997.
 K. Keel, J. S. Brazier, K. W. Post, S. Weese and J. G. Songer, “Prevalence of PCR Ribotypes among Clostridium difficile Isolates from Pigs, Calves, and Other Species,” Journal of Veterinary Medicine, Vol. 45, No. 6, pp. 2007, pp. 1963-1964.
 F. A. Uzal and J. G. Songer, “Diagnosis of Clostridium perfringens Intestinal Infections in Sheep and Goats,” Journal of Veterinary Diagnostic Investigation, Vol. 20, No. 3, 2008, pp. 253-265.