Development of PCR protocols for specific identification of Clostridium spiroforme and detection of sas and sbs genes

Abstract

Rabbit diarrhoea caused by toxigenic Clostridium spiroforme is responsible for significant losses in commercial rabbitries but the accurate identification of this micro-organism is difficult due to the absence of both a commercial biochemical panel and biomolecular methods. The aim of this study was therefore to develop PCR protocols for specific detection of C. spiroforme and its binary toxin encoding genes. The C. spiroforme specie-specific primers were designed based on its 16S rDNA published sequences and the specificity of these primers was tested with DNA extracted from closely related Clostridium species. The sa/bs_F and sa/bs _R C. spiroforme binary toxin specific primers were designed to be complementary, respectively, to a sequence of 21 bases on the 3′ and of sas gene and on the 5′ of the sbs gene. The detection limits of in house developed PCR protocols were 25 CFU/ml of bacterial suspension and 1.38 × 10 4 CFU/g of caecal content for specie-specific primers and 80 CFU/ml of bacterial suspension and 2.8 × 10 4 CFU/g of caecal content in case of sa/bs primers. These results indicated that the described PCR assays enable specific identification of C. spiroforme and its binary toxin genes and can therefore be considered a rapid, reliable tool for the diagnosis of C. spiroforme-related enterotoxaemia.