Development Of The Multivalent Propolis-adjuvant Inactivated Vaccine Of Riemerella Anatipesttifer And Pathogenic Escherichia Coli And Study On Its Dynamic Immune Response

Riemerella anatipestifer(RA) infection and colibacillosis are probably the most economically important infectious diseases in the duck industry through high mortality ,poor feed conversion,and high treatment costs. In this study,three strains (B₁₈,B₁₁₂,B₁₁₅,, isolated from ducks with infectious serositis of Sichuan ) RA and four strains (O₁,O₂,O₁₈,O₇₈) Escherichia coli (E-Coli) were chosen,we developed the bivalent propolis adjuvant-inactivated vaccine of Riemerella anatipesttifer and pathogenic Escherichia coli. Optimization immunizing dose of the vaccine is 1×10⁹CFU/mL in erery bacterium strain, 0.5mL/dose, The guarantee period of the vaccine is ten monthes when stored at 4℃. Ducks immunized one time (group 1) and twice (group 2) were challenged by homologous bacteria to measure their protective abilities, the results showed that immunity protective rates against homologous challege of RA and E-Coli reached 60% and 50% after once immune 7 days,respectively ,and immunity protective rates against homologous challege of RA and E-Coli reached 90% and 70% after twice immune 7 days,respectively.We have used the lymphocyte transformation test (LTT) to study the expression of cell-mediated immunity,and the indirect enzyme-linked immunosorbent assay (ELISA) to monitor antibody production following administration of the vaccines. Lymphocytes (2×10⁶ in 100μl of RPMI-1640 + 10% bovine serum, in 96 well trays) were stimulated with ConA (5μg/mL) , Cells were cultured for 66h at 39℃, We showed that the lymphocyte transformation result of groups 1 and 2 surpass (P<0.05 or P<0.01) control group at 3d, 6d, 9d and 13d, and arrived top level after 6d of postvaccination . So,we can see that the vaccine stimulated strong but transient (about 3 weeks) LTT response. Serum antibodies after vaccine inoculation 3d, 6d, 9d, 13d, 20d, 27d, 34d,41d, 48d, 55d and 69d later were examined by indirect ELISA.Serum antibodies of ducks about 7 strain bacterium arrived top level after 20d of postvaccination and kept up the antibody titers till 48d, respectively ,and then, were generally rather low. Serum antibodies of ducks in group 1 and group 2 maintained high titers in 69d contrast with control group (P<0.05),while the antibodies of group 2 was higher than that of group 1 beginning 13d(P<0.05). The sensitivity of indirect ELISA were 50 to 200 times (RA) and 25 to 100 times (E-Coli) than IHT, The Ab response to vaccine was longer-lived than the LTT response.In conclusion, the bivalent propolis adjuvant-inactivated vaccine of Riemerella anatipesttifer and pathogenic Escherichia coli was safety,utility and no side effect , whick could elicit humoral immunity and cellular immunity,but humoral immunity was important.The observations are important in terms of our understanding of immunopathogenesis, immunoprophylaxis, and immunodiagnosis in RA and E-Coli.