Combined Molecular and Biochemical Identification of Alpha Toxin in Local Isolated Clostridium novyi from the Sheep Liver.

Abstract

Clostridium novyi (C. novyi) causes deadly Black disease in sheep and rarely in other animals. Alpha toxin (α-toxin), the most apparent pathogen of this disease, is produced by C. novyi type B. Economic damages of C. novyi include sheep mortality costs, depreciation of affected farms, and health problems with infected carcasses. The identification of C. novyi and isolation of its pathogens by conventional methods is a time-consuming process, necessitating a simple and rapid method for isolating and detecting pathogenic C. novyi. Therefore, this study aimed to molecularly identify α-toxin in local C. novyi isolates from the sheep livers. In this study, 75 livers suspected of Black disease were sampled. The samples of the liver were cultured under anaerobic conditions. Some of the cultured colonies were used in biochemical tests. For molecular confirmation, the DNA of isolates was extracted, and the isolates were confirmed by the polymerase chain reaction (PCR) on the liver tissue and cultured samples using specific α-toxin primers. The PCR on α-toxin produced a band in the range of 609 bp, indicating that the samples belonged to C. novyi. According to the results, of 75 isolates, 18 isolates were confirmed as C. novyi. C. novyitype B was isolated from the liver and confirmed by biochemical and molecular characterization. The PCR assay ensured a sensitive and specific tool for the detection of C. novyi in the samples.