Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei

• Variable region of the flagellin was targeted to develop an ELISA for detection of C. chauvoei.
• The test developed is specific for C. chauvoei.
• The sensitivity of the test was determined to be 104 CFU/ml.
• The assay developed may be used for detection of C. chauvoei.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 104 CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.