Abstract:

【Objective】The aim for this study was to identify the predominant antigen of Salmonella and to develop a sensitive, specific and universal iELISA assay method for the rapid and accurate detection of Salmonella antibodies in equidae.【Method】For the purpose of screening out dominant antigens for Salmonella Abortusequi, the immunoprecipitation (pull down) tests were performed using Salmonella Abortusequi positive/negative sera with whole bacterium antigens of Salmonella Abortusequi. Then, amino acid sequence alignment of the dominant antigen were compared with Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis to verify itsconservative. Three pairs of specific primers were designed and synthesized according to the nucleotide sequence of the full ompA gene published in GenBank. Three ompA genes with different lengths were amplified by PCR, and then cloned into pET28a vector and transformed Escherichia coli Rosetta (DE3) competent cell. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by IPTG. The reactivity of the purified protein was verified using S. Abortusequi, Salmonella Typhimurium, Salmonella Dublin (S. Dublin), and S. Enteritidis serums, and one negative serum by Western blot. An indirect ELISA method for the diagnosis of equine abortion salmonellosis was developed by optimizing the amount of coating antigen, serum and secondary antibody concentrations using the purified ompA3 protein as the coating antigen, and evaluating the specificity and sensitivity of the iELISA, and finally applying the iELISA to detection of clinical samples.【Result】In this study, the ompA dominant antigen of S.Abortusequi was screened. S.Abortusequi ompA was conservative and showed 99.4%-100% identical with Salmonella Typhimurium, Salmonella Dublin and S.Enteritidis strains at the amino acid level. Three target genes were successfully obtained by PCR amplification. Three recombinant plasmids, including pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 were successfully constructed. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by addition of IPTG to a final concentration of 0.6 mmol·L^-1 for 5 h at 24 ℃. The recombinant ompA1 and ompA2 were obtained in an inclusion and soluble recombinant ompA3 proteins. The soluble recombinant ompA3 were identified by Western blot, which had a specific reaction with four Salmonella positive serums, therefore, the ompA3 was considered as a potential target candidate for serological detection of Salmonella. An iELISA method was developed in a maximum P/N ratio using the coating antigen at a concentration of 1 μg·mL^-1, a serum dilution of 1﹕200 and secondary antibody was 1﹕10000. The cutoff value was 0.143, and an OD₄₅₀ value over 0.143 was considered as positive. The specificity test showed that the coated antigen did not cross-react with the positive serum of common equine infectious diseases. The iELISA provided better sensitivity by detecting antibodies in intravenously infected horses, as the iELISA could continue to monitor antibody positivity up to 116 days, 47 days longer than microagglutination test (69 days). The established iELISA method was used to detect antibodies in 180 serum samples from 8 different farms. The average positive rate of iELISA antibody was 63.3%, which was 53.9% higher than that of micro agglutination test.【Conclusion】The soluble ompA3 protein was successfully expressed, and a universal indirect ELISA antibody method was established for the diagnosis of equine abortus salmonellosis. The method enables detection of antibodies to Salmonella Abortusequi in clinical samples.The method has good specificity and sensitivity and could be a promising candidate tools for use in the monitoring of the equine abortus salmonellosis epidemic.

Key words: Equine abortus salmonellosis, ompA, universal, iELISA, diagnostic