DEVELOPMENT OF RECOMBINANT OmpF PROTEIN BASED INDIRECT ELISA FOR THE DETECTION OF SALMONELLA ANTIBODIES IN POULTRY
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD
Abstract
Salmonellosis 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.
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