Establishment And Application Of The Specific Diagnostic Method Based On SEF14Fimbriae For Salmonella Enteritidis

Salmonellae, a member of the family of Enterobacteriaceae, are a major cause of foodborne illness throughout the world. The bacteria are generally transmitted to humans through consumption of contaminated food of animal origin, mainly meat, poultry, eggs and milk, and the prevalence of S.enteritidis has dramaticated increased worldwide. Expression of SEF14fimbriae is limited to Salmonella group D, such as S.enteritidis and S.dublin which is a host-adapted serotype causing infection in clinic. In this study, we analyzed the optimized expression condition and the preparation of monoclonal antibody of SEF14fimbriae protein for establishing a specific diagnotic method of’S. enteritidis infection.Part one:We designed4different cultural methods to express SEF14fimbriae of S. enteritidis and then screened out the optimized expression condition by SDS-PAGE and TEM assay. The results showed that comparing with other methods, S. enteritidis expressed more sufficient of SEF14fimbriae by method4. Briefly, the standard strain of S. enteritidis50336was prepared by direct inoculation of TSB medium from stock cultures, followed by incubation at25℃for24h and transfer to fresh TSB medium with a dilution of1:100and cultured for a further24h at20℃, finally, the culture was grown in static colonization factor antigen broth(CFA, pH6.0) with1:50dilution at37℃for50to60h. We performed immuno-electron microscopy by anti-SEF14monoclonal antibody and goat anti-mouse IgG labeled with colloidal gold to detect SEF14fimbriae. The result showed that there were colloidal gold particles adsorbed on the surface of SEF14fimbriae. And under the optimized expression condition, we obtained SEF14fimbriae protein with size of14.3KD by SDS-PAGE using procedure of homogenate extraction. Result from Western-blotting further showed that the protein can be recognized by anti-SEF14monoclonal antibody, which confirmed SEF14fimbriae.Part two:Three hybridomas cells secreting monocolonal antibodies(MAbs) against SEF14fimbriae were established by fusing SP2/0with spleen cells from BALB/c mice immunized with formaldehyde-inactivated S. enteritidis standard strain50336which expressed SEF14fimbriae, naming3E4,3E7and3H5. All of them belong with ELISA titers of1:128000,1:64000and1:64000for ascites respectively. The result of Western-blotting revealed that the three MAbs could react with SEF14fimbriae protein, S. enleritidis50336and S. dublin C79-84specifically, and have no cross reaction with other four Sallmonella and three E.coli, which revealed high degree of specificity.Part three:A double-antibody sandwich enzyme-linked immunosorbent assay(ELISA) was developed to detect antibodies from the SEF14fimbrial antigen(SEF14-DAS ELISA). using the anti-SEF14monoclonal antibody as capture antibody and anti-SEF14serum samples from chickens as secondary antibody. In a check-board analysis, the working concentration of the capture antibody and SEF14protein were8μg/mL and2μg/mL separately. Furthermore, we detected100sera samples through SEF14-DAS ELISA, there was93%agreement in comparison with PCR assay based on the sdf1gene specific in S.enteritidis. These results demonstrated that SEF14-DAS ELISA was a method for both highly specific and sensitive detection of S. enteritidis infection.