Construction Of Salmonella Pullorum Attenuated DIVA Vaccine Candidate Strain By Scarless Genome Genetic Modifification And Its Safety And Immunoprotective Efficacy

Salmonella Pullorum is one of the important pathogenic microbes,which seriously endangers the development of poultry industry in China because of extremely high morbidity and mortality in infected chicks.It is difficult to be cleared from chicken farms due to the horizontal and vertical transmission.Thus it is necessary to find simple,efficient and low-cost measures for prevention and control of Salmonella Pullorum.Live attenuated Salmonella vaccine(LASV)is thought as one of effective measures which can solve this urgent problem.After vaccination with LASV,chickens can be protected against Salmonella infection.The spiC gene of Salmonella is an important virulence gene,and its deletion can decrease the virulence significantly and keep the immunogenicity of Salmonella.As a LASV candidate strain,spiC-deleted Salmonella Pullorum has a good balance between the virulence and the immunogenicity.Lipopolysaccharide is an important component of Salmonella outer membrane and one of the important virulence factors of Salmonella.It is often chosen as the target gene of the DIVA vaccines.LASV candidates were constructed mainly by homologous recombination methods to knock out one or more genes from bacterial genomic DNA.The modern genetic engineering technologies have many advantages including clear genetic background,easy genetic operation and rapid production.However,gene-deleted strains often contain antibiotic genes or some remaining foreign fragments by the traditional homologous recombination method,which provides the possibility of the restoration of the missing genes or the insertion of other foreign genes,and this has safety risks.Therefore,in order to meet the requirements of the safety assessment of genetically modified microorganisms,LASV candidates must be constructed without any residues of foreign DNA flagments,even base pairs.In this study,the S06004?spiC?rfaH and S06004?spiC?rfaL LASV candidates were constructed with improved homologous recombination method by deleting the spiC and rfaL or rfaH genes without any scar.The safety and immunoprotective efficacy of S06004?spiC?rfaH and S06004?spiC?rfaL LASV candidates were evaluated.1.Construction of double-deletion strains and study on their biological characteristicsThe S06004?spiC?rfaH and S06004?spiC?rfaL strains of Salmonella Pullorum were constructed by the suicide plasmid pGMB152 mediated homologous recombination method.Their biological characteristics,safety and protective efficacy were further evaluated.PCR and sequencing results showed that the scarless LASV candidates were successfully constructed.The biochemical test showed that they had the same biological characteristics,and their growth rates were not changed significantly.The LD50 test showed that the virulence of LASV candidates was reduced 100-fold compared with the wild strain.In this study,avian macrophage HD-11 cells were used to establish an infection model,and vaccine candidate strains and the wild strain were used for infection respectively.It was found that the vaccine candidate strains had no significant difference in their ability of adhesion,invasion,and proliferation compared to the wild strain.2.Evaluation of the safety and immune protection efficacy of attenuated vaccine candidate strainsTo evaluation of the safety and protective efficacy of the attenuated vaccine candidate strains S06004?spiC?rfaH and S06004?spiC?rfaL,the 3-day-old Hyland White chicks were immunized by intramuscular injection.After immunization,the chicks survived and performed well,and the body weight of the immunized chicks was not changed significantly compared with that of the control group.Compared with the blank control group,no obvious pathological changes were found in the pathological sections of the vaccine group,indicating that LASV candidates had no significant side effects on the chicks and its safety was guaranteed.In vivo dynamic analysis showed that LASV candidates were cleared completely from the livers and spleens at 21 days,while the wild bacteria could still be detected in livers and spleens at 28 days.The results showed that LASV candidates can be cleared faster than the wild strain in vivo.IgG antibody in sera was detected by indirect ELISA and it was found that the Salmonella-specific antibody of immunized chicks could be detected after 2 weeks.The detection of cytokines by quantitative PCR showed that IL-2,IFN-y and IL-1? were expressed largely in the early stage,while the expression of IL-4 was significantly up-regulated at 14 days.After 2 weeks of immunization,chicks were challenged with the wild strain.We found that the survival rate of the immunized chicks can reach about 90%,while only 27%in the control group.The results showed that LASV candidates had good safety and immune protection efficacy.Similarly,the survival rate of oral immunized chicks which were challenged with wild strain after 2 weeks of immunization could reach about 100%,while the control group had only 20%.The PAT results showed that the sera from chickens immunized with one of the LASV candidates could not agglutinate with commercial antigens,which displayed their DIVA ability to differentiate the infected from the vaccinated animals.The S06004?spiC?rfaH and S06004?spiC?rfaL LASV candidates constructed by scarless genome genetic modifification have good safety and immune protection efficacy,and have the DIVA ability to provide help for the prevention and control of Salmonella Pullorum in the poultry industry in our country.