Preparation Of Bigeminal Polyvalent Propolis Inactived Vaccine Of Duck Pasteurella Multocida And Escherichia. Coli And Studies On Its Dynamic Immune Response

This research use duck pathogenic Escherichia.coli serotyoes O1,O2,O18,O78and 3 strains of Pasteurella multocida D22,D23,D157which were isolated from dead or diseased ducks.These strains had strong pathogenicity and good immunogenicity.We used them as vaccine-making bacterium,to prepare antigens by solid surface culture on 5%blood agar medium and Martin agar medium;and then add propolis adjuvant,emulsified with high speed so as to obtaine Escherichia.coli-Pasteurella multocida bigeminal Polyvalent Propolis inactived vaccin.The research studied the security and immunogenicity of the vaccine by the immunity efficiency test of immuned ducks which were challeged by homologous of P.M and E.coli,the research used Indirect enzyme-linked immunosorbent assay(ELISA) to test the changes of P.M's antibody and E.coli' s antibody responses in ducks inoculated with vaccine,MTT method was used to study the transformation ability of T lymphocyte in peripheral blood after vaccination.The result of security test and best immunizing dose test of E.coli-P.M multivalence combined vaccine in ducks showed that the vaccine is safe and reliable,the best immunizing dose is 4.2×10~9 CFU/mL,0.5mL per duck(with a content of E.coli:8.4×10~9 CFU,P.M:6.3×10~9 CFU).The vaccine can be stored in 4℃over 12 months.The result of the immunity efficiency test of immuned ducks which were challeged by homologous of P.M and E.coli demonstrated that 14 days after vaccination the immuno-protective rates were 90%and 100%,14 days after revaccination,ducks had strong immunity,the immuno-protective rates were both 100%.The research use P.M and E.coli whole thallus split antigen to coat microdilurion plates,developed an ELISA to detect the specific antibody in ducks inoculated with vaccine.Established the antigen covering concentration:D22:11μg /mL,D23.171μg/mL,D157:16bμg/mL,P.M mixing antigen:14.7μg/mL,horseradish peroxidase-labelled goat-anti-duck IgG:0.25μg/mL;O1:18.86μg/mL,O2:16.76μg/mL,O18:17.43μg/mL,O78:16.32μg/mL,E.coli mixing antigen:20μg/mL, horseradish peroxidase-labelled goat-anti-duck IgG:0.5μg/mL.The changes of antibody responses in ducks inoculated with vaccine showed that 3 days after vaccination,vaccination groups '4 serotyoes E.coli antibody were very significantly higher(P＜0.01) than control group,twice vaccination group were significantly higher(P＜0.05) or very significantly higher(P＜0.01) than once vaccination group,specific antibody peaked 20 days post vaccination in titer,then decline gradually,till 54th days post vaccination,the titer is still significantly higher than the same group's ninth day's level,and similar with 13th day's level;vaccination groups' 3 strains P.M antibody were very significantly higher(P＜0.01) than control group,twice vaccination group were significantly higher(P＜0.05)or very significantly higher(P＜0.01) than once vaccination group,specific antibody peaked 20 days post vaccination in titer,then decline gradually,till 54th days post vaccination,the titer is similar or significantly higher than the same group's 13th day's level.The result of transformation ability of T lymphocyte in peripheral blood after vaccination showed that the notably enhanced cell mediated immunity in the experiment group was different(P＜0.05)or very different(P＜0.01)from the control group,twice vaccination group were significantly higher than once vaccination group and was about 3 weeks in duration.