Construction And Immune Efficacy Evaluation Of Salmonella Enterica Serovar Pullorum Deletion Mutations With SipB,crp And RfaG Genes

Salmonella enterica serovar pullorum(S.Pullorum)is an important pathogen that causes serious damage to poultry industry in our country and results in serious economic losses every year.The National Medium-and-Long-Term Plan for Animal Disease Control(2012-2020)clearly suggested the eradication of pullorum disease in breeding chickens.The breeding enterprises usually quarantine the pullorum disease by serological detection and eliminate the positive chickens to achieve the purpose of clean-up.Vaccine can be used as a supplementary method for clean-up.Compared with inactivated vaccines and subunit vaccines,live attenuated vaccines have the characteristics of long-term stimulation to humoral,cellular and mucosal immune responses.The DIVA vaccines to distinguish between natural infections and vaccine-immunized animals can also be designed by gene deletion strategy.This kind of vaccine is of great significance for the clean-up of pullorum disease and shows broad application prospects.Previously,S.pullorum aroA gene-deleted mutant S004?aroA was constructed.In this study,we successfully constructed the sipB,crp and rfaG multiple genes-deleted mutants based on S004?aroA through ?-Red homologous recombination.We also evaluated their biological characteristics,pathogenicity and immune efficacy to screen out the ideal attenuated vaccine candidate.This study lays the foundation for the further development of attenuated S.pullorum vaccine.1.Construction and biological characteristics of S.pullorum sipB,crp and rfaG gene deletion mutantsThree genes sipB,crp and rfaG were selected and the related primers were designed respectively.The gene sipB is related to the invasiveness and high-permeability resistance,the gene crp is related to the expression of pili,while the gene rfaG is related to lipopolysaccharide synthesis.Based on the parent strain S004?aroA,four gene-deleted mutants were constructed through ?-Red homologous recombination and named separately as S004AaroA?sipB,S004?aroA?crp,S004?aroA?and S004 AaroA?crp?rfaG.Growth curve,cell adhesion and invasion were determined for each gene deletion strain.The results showed that the growth rate of S004?aroA?sipB and S004?aroA?sipB?rfaG were not significantly different from that of wild-type.The growth rate of S004?aroA?and S004?aroA?crp?rfaG were slower in the LB culture medium and could be restored when adding 0.05%glucose in the medium.The genetic stability tests revealed that all the gene deletion mutants were genetically stable without recovery or natural recombination after passage for 50 generations.Adhesion and invasion ability assay indicated that deletion of sipB gene could significantly weaken the adhesion and invasion ability of bacteria.These dates laid the foundation for further screening of the attenuated vaccine for pullorum disease.2.Pathogenicity and immune efficacy evaluation of S.pullorum gene deletion mutantsIn order to determine the pathogenicity of each mutant,two-day-old SPF chickens were challenged with each mutant by intraperitoneal injection and the half-lethal dose(LD50)were measured respectively.The results showed that the LD50 of wild strain S004 was 6.0×107 CFU,the LD50 of the parent strain S004AaroA is>1.3×109 CFU,the LD50 of double gene deletion mutants S004?aroA?and S004AaroA?crp were>1.4×109 CFU and>1.7×109 CFU,respectively,and the LD50 of the three gene deletion mutants S004AaroAAsipBArfaG and S004?aroA?crpArfaG were>2.1×109 CFU and 1.4×109 CFU,respectively.The virulence of S004AaroAAsipB and S004?aroAsipB?rfaG were further attenuated when compared with the parental strain S004?aroA.To further evaluate the immune efficacy of gene deletion mutants,the two-day-old SPF chickens were orally immunized with a dose of 108 CFU for each deletion mutant and were challenged intraperitoneally with a dose of 109 CFU for the wild type strain S004 at 14 days post-immunization.The body weights of the chickens before and after challenge were monitored.The results showed that the weight gain of S004?aroA,S004?aroAAsipB and S004AaroA?sipB?rfaG immunized groups were not significantly different from that of the healthy control group during the immunization period(P>0.05).The results of immune protection test indicated that the S004?aroA?sipB and S004?aroA?sipBArfaG groups had the best immune efficacy,an 83.3%protection.The S004?aroA,S004?aroA?crp and S004?aroA?crp?rfaG provided less immue efficacy,66.7%,16.7%,33.3%,respectively,while the S.enteritidis vaccine control strain Sm24 could provide 50%protection.In summary,S004?aroA?sipB and S004?aroA?sipB?rfaG both provided superior immune efficacy.Furthermore,S004?aroA?sipB?rfaG contained the rfaG gene deletion,which has been proved that this kind of gene deletion mutant has a characteristic of DIVA.Therefore,the strain S004?aroA?sipB?rfaG can be used as a candidate strain for S.pullorum DIVA attenuated vaccine.