Screening And Functional Research Of Antigens Inducing Th2 Response In Salmonella Pullorum

Pullorum disease is an acute and systemic infectious disease,which brings great harm to the poultry industry.Salmonella enterica serovar Pullorum(S.Pullorum)is the pathogen of pullorum disease,which has a penchant for chickens.S.Pullorum can cause acute septicemia in young chickens,while infected adult chickens often have weight loss and decreased fertility.In addition,S.Pullorum can be vertically transmitted to offspring through eggs.As the facultative intracellular bacteria,the immune response induced by S.Pullorum in chickens is biased towards Th2,which may contribute to the survival of S.Pullorum in macrophages and thus provide favorable conditions for persistent infection.Identifying antigens that responsible for Th2 response induced by S.Pullorum may help to provide a new way to study the persistent infection of S.Pullorum.The M1/M2 phenotype of macrophages is correlated with the T-cell-mediated Th1/Th2 response.IL-12 and chemokines CXCL9 and CXCL10 produced by M1 macrophages can promote the differentiation of Th1 cell.As a production of Th1 response,IFN-? can stimulate macrophages to form M1 polarization.Chemokines CCL17,CCL22 and CCL24 produced by M2 macrophages promote the differentiation of Th2 cells,and IL-4 from Th2 response induces M2 polarization of macrophages.Therefore,the typing of macrophage polarization can indirectly reflect the type of T cell response.The metabolic pathways of arginine in M1 and M2 macrophages are different,iNOS is an important marker of M1 macrophages and metabolizes arginine to NO and ornithine.In this study,mutants which induce the polarization of M1 macrophage were screened from transposable mutation library of S.Pullorum,based on the production of NO from infected HD11.These selected mutants are responsible for promoting Th1 response and thus the products of mutated genes are antigens that inhibits Th1 or induces Th2 response.In vitro and in vivo experiments were conducted to evaluate the Th1/Th2 immune response induced by gene-deleted strains and their scavenging effect on pathogens in chickens.Finally,their potential as vaccine candidates was preliminarily evaluated.This study will provide theoretical basis and new targets for further research on persistent infection of S.Pullorum and help to find better prevention and control methods.1.Screening of genes in S.Pullorum by detecting NO from infected HD11The transposable mutation library of S.Pullorum 449/87 was constructed by the plasmid pSC189.The rate of transposon insertion was 100%by PCR identification,which was consistent with the resistance phenotype.The chicken macrophage HD11 was infected with a single transposable mutant and the production of NO in the culture was detected to screen different strains.2807 transposable mutants were screened and 61 among them showed difference in inducing NO production.Compared with the wild strain 449/87,49 strains up-regulated and 12 strains down-regulated NO secreted by HD11.Gene localization was carried out on the selected mutants,and the genes that might be affected by transposon insertion were determined by PCR,sequencing and alignment analysis.The homologous recombination mediated by suicide plasmid pDM4 was used to construct the gene-deleted strains,and the corresponding complementary strains was constructed using the plasmid pMMB207.Seven gene-deleted strains were determined to up-regulate NO production in infected HD11,and their mutated genes were arcB,cyaA,cysB,hilA,glnD,invE and prgI.2.Effects of NO-related genes in S.Pullorum on macrophage M1/M2 typingPrimary macrophages in chicken PBMCs were obtained when they are stick to the bottom of petri dish.Flow cytometry showed that the final purity of macrophages reached 93%when the inoculation amount of PBMCs in a six-well plate was 7.5×106 cells/well.Primary macrophages were infected with 449/87 and 7 gene-deleted strains,and results showed that deletion of arcB,cyaA,cysB,hilA,glhD,invE or prgI,could significantly up-regulate NO production in mutant-infected primary macrophages.The result is consistent with the conclusion from HD 11 model.The activity of iNOS,mRNA levels of IFN-y,IL-12p40,IL-4 and IL-10,and protein level of IL-12p40 was detected in infected HD11 and primary macrophages.Compared with 449/87,the activity of iNOS induced by 7 gene-deleted strains was significantly increased in both HD 11 and primary macrophages,and so was the level of IL-12p40 protein.In HD11,when compared with 449/87,the infection of seven gene-deleted strains induced almost the same level of Th2 related cytokines IL-4 and IL-10 mRNA and up-regulated Thl related cytokines IL-12 p40 mRNA;only five gene-deleted strains(prgI,invE,hilA,cyaA and arcB)can significantly up-regulate IFN-y mRNA level.In chicken primary macrophages,449/87?cyaA,449/87?hilA and 449/87?invE induced up-regulated IFN-y mRNA,down-regulated IL-4 mRNA when compared with the wild strain.The results showed that 449/87?cyaA and 449/87?hilA promoted M1 polarization of primary macrophages by up-regulating IFN-y and inhibited M2 polarization of primary macrophages by down-regulating IL-4.3.The immune responses induced by S.Pullorum 449/87?cyaA and 449/87?hilA in chickensHylan white chickens were infected with 449/87,449/87?hilA and 449/87?cyaA by intramuscular injection,respectively,and at different time,the liver and spleen were collected for checking histopathological changes.The humoral and cellular immune responses induced by 449/87?cyaA and 449/87?hilA were assessed by detecting serum antibodies,the proportion of CD4/CD8+T cells,the levels of lymphocyte proliferation and cytokine expression in spleen.The detection of antibodies in serum showed that chickens infected with 449/87 and 449/87?hilA produced similar levels of antibodies,while 449/87?cyaA induced lower levels of antibodies.This suggests that the deficiency of cyaA inhibit S.Pullorum induced humoral immune response in chickens.Through testing the proportion of CD4+and CD8+T cells in spleen and the proliferation ability of splenocytes,we found that,compared with the 449/87 group,CD4+T cells decreased and CD8+T cells increased in spleen of chickens infected with 449/87?cyaA(at 21 dpi),and the proportion of CD4+T cells increased in spleen of chickens infected with 449/87?hilA(at 28 dpi).The proliferation of splenocytes in 449/87?cyaA and 449/87?hilA infected chickens was significantly enhanced at 21 and 28 dpi,respectively,suggesting that the two gene-deleted strains could enhance the ability to induce the T lymphocyte response.The results of splenic cytokines show that 449/87?hilA infection induced significantly higher levels of IFN-y mRNA(at 3 and 7 dpi);449/87?cyaA infection inhibited the expression of IL-4 mRNA(at 3 dpi)and induced higher levels of IFN-y mRNA(at 7 dpi).In view of results above,both 449/87?cyaA and 449/87?hilA induced Thl-biased responses in Hylan white chicken.The bacterial loads in the liver and spleen of 449/87?cyaA infected group was less,and the 449/87?cyaA in the liver and spleen were cleared at 7 and 21 dpi,respectively.The amount of Salmonella colonization in liver and spleen of 449/87?hilA group was lower than that of wild strain group,and on the 21 dpi,449/87?cyaA were cleared from livers.All of this suggest that Thl-bias response induced by 449/87?cyaA and 449/87?cyaA may accelerate bacterial clearance.4.Preliminary assessment of 449/87?cyaA as a vaccine candidateThe virulence of 449/87?cyaA in Hylan white chickens was determined and the results showed that the LD50 of 449/87?cyaA was 69 times higher than that of its parent strain 449/87.1×107 CFU 449/8 7?cyaA were intramuscularly injected into Hylan white chickens for vaccine evaluation.The immunized chickens have no obvious clinical symptoms and histopathologic changes in liver and spleen,and their body weight have no difference with those of PBS-treated chickens.The humoural and cellular immune responses induced by 449/87?cyaA were evaluated.Serum antibody levels showed that the anti-S.Pullorum antibodies produced at 14 and 21 dpi.The results of splenic lymphocyte proliferation assay showed that compared with the PBS-treated group,the splenocytes of 449/87?cyaA immunized group had an increase in proliferation ability when stimulated by ConA and SPAgP.The cytotoxic lymphocyte(CTL)test showed that the killing ability of CD8+T cells in 449/87?cyaA immunized chickens was stronger than that in the PBS-treated chickens.Immunized chickens were challenged at 14 dpi to evaluate the protection role of 449/87?cyaA.The results showed that after challenge,the chickens immunized with 449/87?cyaA had no obvious symptoms,and the mortality and morbidity were very low;while the chickens in PBS-treated group showed high morbidity and mortality,with obvious clinical symptoms.The bacteria load in the liver of 449/87?cyaA immunized Hylan white chickens was significantly lower than that in PBS-treated chickens,which suggest that Thl-bias response induced by 449/87?cyaA may play a scavenging effect on challenged bacteria.The results above show that the attenuated 449/87?cyaA could induce both humoral and cellular immune responses and provide good protective effect for chickens,and thus has the potential as a vaccine candidate for pullorum disease.