PrejitNIMISHA SOMAN2020-07-172020-07-172018http://krishikosh.egranth.ac.in/handle/1/5810149300Salmonellosis is one of the most frequently reported poultry disease worldwide. Screening and identification of salmonellosis among poultry flock have assumed principal role in preventing transmission of this pathogen to humans from chickens. The objective of this study was to develop a highly promising and reliable diagnostic tool for detection of Salmonella antibodies using rOmpF protein. The ompF gene of S. Typhimurium was amplified, cloned in pRham expression vector and expressed in E. coli as a fusion protein. The rOmpF protein was expressed in E. coli at an optimal temperature of 25°C with 0.2 per cent rhamnose for 16 h auto induction. The expression was analysed by SDS-PAGE and confirmed by western blot using anti-His-tag conjugate. This confirmed that rOmpF is a fusion protein with a size of ~57kDa. The expressed rOmpF was in insoluble form and was purified under denaturation condition by Ni-NTA purification and further concentrated by dialysis for obtaining single protein band. The rOmpF was assessed for its immunoreactivity as a diagnostic antigen by immunoblotting. The immunogenic reactivity of expressed rOmpF was optimized in indirect ELISA using known true positive and negative sera of poultry with respect to OmpF antibodies. The relative diagnostic sensitivity and specificity of the assay (95.24 per cent and 100 per cent respectively) was observed at a cut off value of 0.897. The diagnostic efficiency was comparable with the reference test (rOmpC based I-ELISA). Standardized rOmpF based IELISA was used for analysing 210 poultry serum samples from Wayanad distirct, of that 42 sera were tested positive for Salmonella antibodies. In this study, the overall occurrence of Salmonella in poultry stock of Wayanad was found to be 20 per cent. This study revealed that indirect rOmpF based I-ELISA is a promising diagnostic tool for diagnosis of salmonellosis in poultry and also a safe antigen to be used for sero-diagnosis.ennullThesis