Development Of An Indirect ELISA For Detection Of Antibody Against Salmonella And Construction And Immunogenicity Of Recombinant Swinepox Virus Vector Vaccines Of Salmonella

Salmonella are Gram-negative flagellated bacteria that cause a variety of diseases in humans and animals,ranging from mild gastroenteritis to severe systemic infection.Salmonella contains 2 species(S.enterica and S.bongori),7 subspecies and approximately 2500 serovars and most of them are highly pathogenic.A wide range of serovars make the diagnosis and prevention of Salmonella more difficult.In this study,a novel diagnostie method was developed and two novel vaccines of Salmonella were constructed.1.Establishment of an indirect ELISA based on PagC for detection of antibody against SalmonellaScreening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of Salmonella infection.In this study,outer membrane protein PagC was found to be the perfect detection antigen of Salmonella anti-serum by using bioinformatics methods.Analysis of the amino acid sequence of PagC indicates that this protein is widely distributed in Salmonella spp.and conserved among different Salmonella serovars.The pagC gene without signal peptide was cloned into the prokaryotic expression vector pET-28a and the correct recombinant plasmids pET-28a?pagC was transformed into E.coli BL21.The fusion protein PagC was purified by gel filtration and aflinity chromatography.An indirect enzyme-linked immunosorbent assay(ELISA)was developed based on a purified recombinant PagC.The assay was established using 200 ng of PagC protein as coating antigen per well,with the optimal dilution of 1:50 for testing serum and 1:1 000 for HRP-labeled Goat Anti-Chicken IgG.5%skimmed milk powder blocked for 2 h,and the reaction time of serum and HRP-labeled Goat Anti-Chicken IgG were both 1 h.It was proved that the method was rather specific and repeatable by cross experiment and repeated experiment.The coincidence rate between the PagC-ELISA and Biochek(?)commercial detection kit was determined as 83.0%;the coincidence rate between the PagC-ELISA and pullorum slide agglutination test was 80.6%.The coincidence rate between the PagC-ELISA and standard positive sera and negative sera was 98.3%.The 482 swine serum samples were detected by the established indirect ELISA and the positive rate was 46.7%.The 252 chicken serum samples were also detected by PagC-ELISA and the positive rate was 42.9%.2.Construction and immunogenicity of recombinant swinepox virus expressing outer membrane protein L of Salmonella.To explore development of a potent vaccine against Salmonella infections,the gene encoding outer membrane protein L(ompL)was inserted into the swinepox virus(SPV)genome by homologous recombination.PCR,western blot and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL.Immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model.Forty mice were assigned to four groups,which were immunized with rSPV?OmpL,inactive almonella(positive control),wild-type SPV(wtSPV;negative control),or PBS(challenge control),respectively.The OmpL-specific antibody in the rSPV-OmpL immunized group increased dramatically and continuously over time post-vaccination,and was present at a significantly higher level than in positive control group(P<0.05).The concentrations of IFN-? and IL-4 which represent Thl-type and Th2-type cytokine responses,were significantly higher(P<0.05)in the rSPV-OmpL-vaccinated group than in the other three groups.After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542,eight out of ten mice in the rSPV-OmpL-vaccinated group were protected,whereas all the mice in the negative control and challenge control groups died within 3 days.The protective efficiency in the rSPV-OmpL immunized group was significantly higher(P<0.01)than in the other three groups.The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.3.Construction and immunogenicity of recombinant swinepox virus co-expressing outer membrane protein L and FliC of SalmonellaTo explore development of a better vaccine against Salmonella infections,the gene encoding outer membrane protein L(ompL)and FliC were inserted into the swinepox virus(SPV)genome by homologous recombination.PCR,western blot and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV?OF.Immune responses and protection efficacy of rSFV?OF were assessed in a mouse model.Forty mice were assigned to four groups,which were immunized with rSPV-OF,inactive Salmonella(positive control),wild-type SPV(wtSPV;negative control),or PBS(challenge control),respectively.The OmpL-specific antibody and FliC-specific antibody in the rSPV-OF immunized group increased dramatically and continuously over time post-vaccination,and was present at a significantly higher level than in positive control group(P<0.05).The concentrations of IFN-? and IL-4 which represent Thl-type and Th2-type cytokine responses,were significantly higher(P<0.05)in the rSPV-OF-vaccinated group than in the other three groups.After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542,seven out of ten mice in the rSPV-OF-vaccinated group were protected,whereas all the mice in the negative control and challenge control groups died within 3 days.The recombinant swinepox virus rSPV?OF might serve as a promising vaccine against Salmonella infection and deserve to be further developed.