Development and evaluation of an indirect enzyme-linked immunosorbent assay based on a recombinant SifA protein to detect Salmonella infection in poultry

Abstract

Salmonella is an important zoonotic pathogen that not only endangers food safety and human health, but also causes considerable economic losses to the poultry industry. Therefore, it is essential to establish a rapid, sensitive, and specific diagnostic method for the early detection of Salmonella infection in poultry. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Salmonella antibodies using a recombinant SifA protein. Amino acid sequence comparison revealed that SifA is a relatively conserved secretory protein across Salmonella serotypes. Therefore, we hypothesized that SifA can serve as a detection antigen for diagnostic testing. The SifA protein was expressed in Escherichia coli and used as a coating antigen to establish an SifA-ELISA. Control sera from specific-pathogen-free (SPF) chickens infected with Salmonella or several other non-Salmonella pathogens were then tested using the SifA-ELISA. Specificity testing demonstrated that the SifA-ELISA could detect antibodies against 3 different serotypes of Salmonella, whereas antibodies against other non-Salmonella pathogens could not be detected. Compared to the SifA-ELISA, the Salmonella plate agglutination test (PAT) failed to detect antibodies in serum samples from chickens infected with Salmonella Typhimurium. This result suggests that our SifA-ELISA may be better than PAT at detecting Salmonella infection. Comparing clinical sera, we observed a similar rate of Salmonella positivity between SifA-ELISA and PAT (92.6%). In addition, anti-SifA antibodies were continuously detected during Salmonella infection of SPF chickens, demonstrating that SifA-ELISA could consistently detect high levels of antibodies for at least 8 wk. Furthermore, the intra-assay and interassay coefficients of variation (CV) of the SifA-ELISA were below 10%, which is considered acceptable. In summary, the SifA-ELISA established here is a promising and reliable method for detection of anti-Salmonella antibodies in poultry and may contribute to the early diagnosis of Salmonella infection.