| Mastitis is considered to be one of the most frequently endemic diseases that occured in dairy industry and caused serious economic loss.The disease not only affects the performance of dairy cattle,but also endangers human health through dairy products.Staphylococcus aureus is one of main causative agent of bovine mastitis and could induce antimicrobial resistance and reoccured easily,which brings difficulties to the prevention and treatment of this disease.Adhesion is important virulence factor of S.aureus.It could recognize and bind to host cells by which S.aureus reslized its infection of the host.Agglutination factor A(ClfA),fibronectin-binding protein(FnBPA)are important adhesion factors and antigen factors of S.aureus,which are of great value for its prevention.In this study,the constructed eukaryotic recombinant plasmids pVAX1-CIfA and pVAX1-FnBPA-A that based on ClfA and FnBPA-A of the S.aureus isolates in Xinjiang were used to immuized mice and dairy cows.The immune dose and antigen combination were optimized and the immune effect of the two different animals were tested and evaluated.The mice were immunized with different doses and combinations of these two recombinant plasmids.The antibody levels,the survival rates and pathological observation were performed of liver,spleen and kidney.Antibody detection results showed that the antibody titer of ClfA+FnBPA-A high-dose immunization group could reach to 1:12800.The anti-serum of each immunization group could recognize and bind to the S.aureus(P<0.01).The binding ability of ClfA+FnBPA-A high-dose immunization group was better than other groups(P<0.01).The challenge suirvival rates of mice showed that the ClfA low-dose immunization group,FnPA-A high-dose immunization group and ClfA+FnBPA-A low-dose immunization group could reach to 87.5%,the challenge survival rates of FnBPA-A low-dose immunization group could reach to 75%and ClfA high-dose immunization group and ClfA+FnBPA-A high-dose immunization group could reach to 100%.The pathological observation of liver,spleen and kidney from mice indicated that there was no significant difference between ClfA+FnBPA-A high-dose immunization group and normal control group.But the single immunization group of ClfA and FnBPA-A showed different level of pathological changes.The bacterial burden showed that ClfA+FnBPA-A high-dose immunization group was significantly lower than control group,which indicated that ClfA+FnBPA-A high-dose immunization group had better anti-infection effect compared with other immunization groups.The recombinant plasmids of ClfA,FnBPA-A were used to immunize 44 lactating daily cows.The levels antibody and cellular immunity,the yield and quality of milk,somatic cell count,the incidence of recessive mastitis,bacterial burden and the maintenance time of antibody were monitored for 6 months.The results showed that ClfA+FnBPA-A immunization group had higher level of antibody level(P<0.01).The serum antibody titer of ClfA+FnBPA-A high-dose immunization group can reach to 1:3200.The Cytokine IL-2,IL-4,IL-10,IFN-y detection results showed that cytokine IL-2,IFN-y of combined immunization group were higher than single immunization group and control group(P<0.01),the ClfA+FnBPA-A immunization group mainly induced the Thl cellular immune response.Compared with other immunized groups and control groups,the somatic cell count and the incidence rate of recessive mastitis decreased significantly.Further more,there was a significant increase of ClfA+FnBPA-A high-dose immunization group in milk fat percentage,milk lactose percentage and milk protein percentage compared with other immune groups and control group(P<0.01).The results of bacterial burden of milk showed that the number of colonies from ClfA+FnBPA-A high-dose immunization group was 6×102 cells/mL compared with that of single immunization group 3.47×103-7.73,103 cells/mL and control group 3.73-104 cells/m L.The number of colonies of ClfA+FnBPA-A high-dose immunization group was significantly lower than other groups(P<0.01).In this study,the recombinant plasmids of ClfA and FnBPA-A were optimized and immunized mice and dairy cows.The immunization effect,the quality of milk and the incidence rate of mastitis were analized and evaluated.The results showed that the ClfA+FnBPA-A high-dose immunization group could induce good humoral and cellular immune response in dairy cows.The immunization could give better protection to the immunized mice,reduce the incidence rate of recessive mastitis and improve the quality of milk.The study laid the experimental basis for the further prevention and control of dairy cow mastitis in Xinjiang. |
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