title: Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei.
abstract: 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 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: J
authors: J
authors: Usharani
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India. Electronic address: vkn111@gmail.com.
authors: Viswas Konasagara
authors: VK
authors: Nagaleekar
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: Prasad
authors: P
authors: Thomas
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: Santosh K
authors: SK
authors: Gupta
authors: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: Sanjeev K
authors: SK
authors: Bhure
authors: Eastern Regional Station, Indian Veterinary Research Institute, Kolkata 700037, India.
authors: Premanshu
authors: P
authors: Dandapat
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: Rajesh K
authors: RK
authors: Agarwal
authors: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.
authors: Vijendra P
authors: VP
authors: Singh
mesh_terms: D000595:Amino Acid Sequence; D000820:Animal Diseases / Q000175:diagnosis* / Q000382:microbiology*; D000818:Animals; D003015:Clostridium Infections / Q000662:veterinary*; D046975:Clostridium chauvoei / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005408:Flagellin / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D005408:Flagellin
keywords: Clostridium chauvoei; Detection; ELISA; Flagellin