Establishment and application of an indirect enzyme-linked immunosorbent assay based on tandem expression PrgH-PagN protein to detect Salmonella infection in ducks.

Abstract

Salmonella, an important foodborne zoonotic pathogen, can be transmitted both vertically and horizontally. It is difficult to control and effectively decontaminate Salmonella, and it poses a serious threat to food safety. Therefore, to prevent the spread of salmonellosis, there is an urgent need for a rapid, accurate and sensitive assay to detect the prevalence of Salmonella in duck flocks. In this study, we utilized biological software to predictively screen the highly conserved Salmonella-specific proteins SpiC, PrgH and PagN. The recombinant proteins PrgH, SpiC, PagN were screened for sensitivity based on individual proteins and pairwise combinations (SpiC + PrgH, SpiC + PagN and PrgH + PagN). A specific and sensitive dual-protein combination, PrgH + PagN, was used as an antigen. Subsequently, PrgH-PagN was produced by tandem expression and employed as the coating antigen in an indirect ELISA (iELISA) for detecting Salmonella antibodies in duck serum. The optimal antigen coating concentration was determined to be 1 μg/ml, with a critical value of OD₄₅₀ = 0.154. The cross-reactivity test results showed no evidence of cross-reactivity with known positive serums from ducks infected with S. Enteritidis, S. Typhimurium, S. Kottbus, E. coli, Streptococcus or Staphylococcus. Screening of 611 duck serums was performed to determine an overall positive rate of 22.09%. The final compliance rate of 93.1% was determined by comparison with that of the commercial kit. In conclusion, the PrgH-PagN-iELISA established in the present study was an accurate and reliable method, with high sensitivity and specificity for detecting antibody responses to systemic Salmonella infections in ducks.