| The research were selected Staphylococcus aureus DS12 and DS13 which were isolated from sick ducks of duck farms in Sichuan and Pasteurella multocide D22,D23 and D157,development of the inactivated propolis bivalent vaccine.The physical properties of vaccine meet the requirements of vaccine production.Optimization immunizing dose of the vaccine is 5×10~9CFU/ mL,0.5 mL/dose.The guarantee period of the vaccine is twelve-month when stored at 4℃.The research developed an indirect ELISA method to detect the antibody of Pasteurella multocide and Staphylococcus aureus and studied the ducks immunity response to the bivalent vaccine.Pasteurella multocide and Staphylococcus aureus were treated with ultrasonic to obtain the whole thallus antigen,and the ELISA method to detect the duck's specific antibody to P.multocide -S.aureus propoli inactivated bivalent vaccines was established successfully.The optiomal antigen condition was determined,the DS12 antigen covering concentration was 5.5μg/mL,the DS13 was 6.25μg/mL,the D22 was 11μg/mL, the D23 was 17μg/mL,the D157 was 16μg/mL.The optimum dilution of goat anti-duck IgG labeled horseradish peroxidase was 0.625μg/mL.The indirect ELISA were confirmed to be reproducible and specific by cross-assay,The coefficient of variation within plate were 1.71%to 4.06%(S.aureus) and 1.93%to 3.55%(P.multocide),and the coefficient of variation between plates were 1.82%to 4.18%(S.aureus) and 1.96%to 4.75%(P.multocide). The sensitivity of indirect ELISA were 50 to 100 times(S.aureus) than micro-agglutination test and 50 to 100 times(P.multocide) than micro-agglutination test.The research were conduced to study the humoral immune response by bivalent vaccine in ELISA.The result were as follows.Preliminary immunity was conducted after inoculated 3 days,The level of antibody increased gradually,the antibody titers reach highest point by days 26,then decline gradually and slowly.The antibody titers of days 75 reach almost the same level of two weeks.Immune once group was very significantly higher than saline control group at 3-75 days(P＜0.01),Immune twice group was significantly higher than immune once group at 9 day(P＜0.05),and very significantly higher than immune once group at 12-75 days(P＜0.01).Challenges were performed by homologous challege of P.multocide and S.aureus to measure their protective abilities,the results were as follows.After once immune 14 days, immunity protective rates against homologous challege of P.multocide and S.aureus reached 90%and 70%respectively.After twice immune 14 days,immunity protective rates against homologous challege of P.multocide and S.aureus reached 100%and 80% respectively.MTT method was used to study the transformation ability of T lymphocyte in peripheral blood after vaccination,obtaining the following experiment result.3 to 19 days after vaccination,immune once group was very significantly higher than saline control group(P＜0.01).Immune twice group was very significantly higher than immune once group(P＜0.01) at 9 to 12 days,immune twice group was significantly higher than immune once group(P＜0.05) at 19 days.At 26 to 33 days,there was no significant difference during immune once group,immune twice group and saline control group.In conclusion,the inactivated propolis bivalent vaccine of Pasteurella multocide and Staphylococcus aureus was safety,utility and no side effect,which could elicit humoral immunity and cellular immunity.
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