Purification of recombinant IpaJ to develop an indirect ELISA-based method for detecting Salmonella enterica serovar Pullorum infections in chickens

Abstract

BackgroundSalmonella enterica serovar Pullorum is a host-restricted serotype causing infection in poultry. The pathogen can not only cause acute infection in young chicks with high mortality and morbidity, but also persist in adult chickens without evident clinical symptoms and lead to vertical transmission. To eradicate S. Pullorum in poultry farms, it is necessary to establish an efficient method to monitor the prevalence of the pathogen in adult chickens. The protein IpaJ is a specific immunogen in S. Pullorum and is not detected in closely related serotypes, such as S. Gallinarum and S. Enteritidis.ResultsIn the present study, IpaJ was expressed as a recombinant fusion protein MBP-IpaJ in E. coli. The purified MBP-IpaJ was used as a coating antigen to develop an indirect ELISA assay, which was applied to the detection of S. Pullorum infection in chickens. The indirect ELISA assay demonstrated that antibodies produced against IpaJ were detectable in antisera of chickens infected with S. Pullorum in the second week, stably increased until the tenth week, and persisted at a high level in the following two weeks. Furthermore, the ELISA method detected four positive samples out of 200 clinical antiserum samples collected from a poultry farm, and the positive samples were confirmed to be reacted with S. Pullorum using the standard plate agglutination test.ConclusionsThe established indirect ELISA using the IpaJ protein is a novel method for specific detection of S. Pullorum infection, and contribute to eradication of pullorum disease in the poultry industry.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1753-0) contains supplementary material, which is available to authorized users.

Application of Monoclonal Antibodies Developed Against the IpaJ Protein for Detection of Chickens Infected With Salmonella enterica Serovar Pullorum Using Competitive ELISA

“…Our previous study showed that chickens could produce antibodies against IpaJ protein 1 week after infection with S . Pullorum and kept at a stable level for up to 12 weeks (9). In order to detect S .…”

“…The bacteria with recombinant plasmids were grown in Luria Bertani (LB) broth with Ampicillin (100 μg/ml). The purified MBP-IpaJ was collected and preserved in our laboratory (9).…”

“…The positive hybridoma clones expressing antibodies against His-IpaJ were screened using the previously established indirect ELISA method, with MBP-IpaJ recombinant protein as the coating antigen (9). Positive clones were sub-cloned three times by the limiting dilution method.…”

“…The samples were preserved at −70°C and subsequently monitored using the established competitive ELISA assay. An assay for each sample was performed in triplicates, and the detected positive samples were confirmed by PAT and the indirect ELISA assay (9).…”

“…Additionally, the antibodies against IpaJ protein could be detected from the first to the twelfth week after S . Pullorum infection in chicken sera (9).…”