Construction And Immunoprotective Effect Evaluation Of Two Vaccine Candidates Based On Salmonella Enteritidis CZ14-1

Salmonella enterica serovar Enteritidis(S.Enteritidis)is an important foodborne pathogen with a wide spectrum of hosts and invasiveness,which has important public health significance.The use of antibiotic intervention can be used to treat the disease caused by S.Enteritidis,but the treatment can lead to the emergence of multi-drug resistant strains and drug residues in meat products.Therefore,the development of safe and effective vaccines is an effective measure to prevent and control Salmonella Enteritidis infection.With the development of genetic engineering and molecular biotechnology,construction of attenuated vaccine candidate based on knockout of the key virulence genes from pathogenic bacteria have been widely used worldwide.The live attenuated vaccine has many advantages,including the convenient use,good immunogenicity,low virulence,and production of perfect immunoprotective effect even in the "immune blank period".In addition,serologically negative markers can be obtained by gene knockout,and DIVA(Differentiating Infected and Vaccinated Animals)detection methods can be established to distinguish vaccinated animals from those infected with wild-type pathogen in animals during the breeding and/or production processes.1.Construction and verification of recombinant S.Enteritidis strains CZ14-1?spiC?nmpC and CZ14-1?spiC?rfaLIn this study,the virulence-related gene spiC of S.Enteritidis CZ14-1,and the outer membrane protein-related gene nmpC,or the lipopolysaccharide-related gene rfaL were knocked out by the combination of ?-Red homologous recombination system and the suicide plasmid-mediated homologous recombination method.Based on the construction of CZ14-1?spiC single gene deleted strain,the nmpC or rfaL was knocked out to produce two double gene deleted strains.The CZ14-1?ipiC deletion strain was successfully constructed by knocking out the spiC gene of S.Enteritidis CZ14-1 by using the suicide plasmid pGMB152-?spiC/Cm mediated homologous recombination system.Next,PCR was used to amplify the DNA fragments of both the upper and lower homologous arms of the S.Enteritidis gene nmpC with the chloramphenicol resistance gene in both ends,or the two ends of the S.Enteritidis gene rfaL.In the middle,the amplified DNA fragments were then electroporated into CZ14-1?spiC containing plasmid pKD46 to obtain a positive clone replacing nmpC or rfaL(CZ14-1?spiC?nmpC::Cat or CZ14-1?spiC?rfaL::Cat),and then transferred the temperature sensitive plasmid pCP20 into CZ14-1?spiC?nmpC::Cat and CZ14-1?spiC?rfaL::Cat to eliminate the chloramphenicol resistance gene.By PCR identification and sequencing analysis,the S.Enteritidis double gene deletion strain CZ14-1?spiC?nmpC and CZ14-1?spiC?rfaL were successfully constructed for the subsequent analysis.The physiological and biochemical characteristics of two double gene mutants were determined to reveal that knockout of spiC and nmpC showed no evident changes to the wild type strain.However,deletion of rfaL caused the decreased growth ability and the change in metabolism of mannitol compared to wild type strain.At the same time,the immunoprotective efficacy of double mutant strains was investigated,providing materials and evidence for the study of live attenuated Salmonella vaccine2.Evaluation of the immunoprotective efficacy of attenuated S.Enteritidis strains CZ14-1?spiC?nmpC and CZ14-?spiC?rfaLCZ14-1?spiC?nmpC and CZ14-1?spiC?rfaL deletion strains were immunized by intramuscular injection at the dose of 1.0 × 105 CFU per 7-day-old Hyland white chicken,which were boosted at 14 days.Ten days later,immunized chickens were was artificially infected by intramuscular injection at the dose of 2.0×109 CFU per chicken.The results showed that CZ14-1?spiC?nmpC and CZ14-1?spiC?rfaL were able to produce 80%and 75%protection against large-dose challenge of wild-type strains,and only 5%was detected in the unvaccinated group.The net increase in body weight of vaccinated groups was also significantly higher than those of the unimmunized group.The in vivo study was performed to determine the distribution and colonization of Salmonella in tissues or organs of chickens.The wild-type CZ14-1 and two double-deletion strains were inoculated intramuscularly in 3-day-old Hyland white chickens,21 days later,the bacteria was not detected in the liver and ileum of two groups infected with two deletion strains,respectively.The indirect ELISA results of IgG against S.Enteritidis showed that the IgG were detected in the serum of groups infected by either of two deletion strains one week after immunization,and the antibody titres were gradually increased in the next few weeks to reach a peak at the fifth week post-immunization.Quantitative PCR was used to detect the expression of cytokines in spleen cells of groups infected with CZ14-1?spiC?nmpC or CZ14-1?spiC?rfaL.The results showed that the expression levels of IFN-? and IL-6 were significantly up-regulated compared with the control group.An indirect ELISA assay using SpiC protein as a coating antigen can clearly distinguish wild-type strain-infected chickens and vaccinated chickens.Chickens infected with wild-type strain and chickens immunized with CZ14-1?spiC?rfaL deletion strains were clearly distinguished by the slide plate agglutination test(SPA).The results showed that the two double deletion strains have DIVA ability,and the above mentioned methods can be used to distinguish vaccinated animals from wild-type infected animals.The above results showed that the two double-deletion strains not only have good safety,but also have good protection ability against high-dose challenge of wild-type strain,laying a foundation for the development of live attenuated Salmonella Enteritidis vaccines.