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

<i>Salmonella,</i> an important foodborne zoonotic pathogen, can be transmitted both vertically and horizontally. It is difficult to control and effectively decontaminate <i>Salmonella</i>, 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 <i>Salmonella</i> in duck flocks. In this study, we utilized biological software to predictively screen the highly conserved <i>Salmonella</i>-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 <i>Salmonella</i> 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 <i>S</i>. Enteritidis, <i>S.</i> Typhimurium, <i>S</i>. Kottbus, <i>E. coli</i>, <i>Streptococcus</i> or <i>Staphylococcus</i>. 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 <i>Salmonella</i> infections in ducks.