Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes

Abstract

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans.