| At present, E. coli and avian cholera are common diseases of poultry. Due to the irrational use of antibiotics, antimicrobial resistance of bacteria was serious, drug treatment effect is not ideal, therefore effective vaccine to prevent bacterial infection is more and more attention and was widely adopted. Protoplast fusion strains DB4 was prepared using duck E. coli ZYD2 (O78) and Pasteurella multocida YB2 (5:A) by Li mu-zi, with parents of the genetic material and can be stably inherited. In this study, by using DB4 producted inactivated vaccine and vaccination experiments duck, detecting the levels of duck immunity from two aspects of cellular immunity and humoral immunity, with a view to the adoption of once immunity can acquire immunity of two kinds of diseases, providing a more convenient way for the vaccine preparation of duck E. coli disease and duck cholera, simplify immunization programs. The results were as follows:1. In this study,2 fusion strains DB1 and DB4 is selected which containing part of the two parental strains outer membrane protein A(OmpA) and H(OmpH) genes, inoculated in containing neomycin 20 ?g/mL MacConkey medium for culture and purification, examinated the cell morphology by Gram staining and microscope, micro biochemical reaction, DB1 and DB4 were isolated, the purified and preserved from the dead mice by grouping and attacking, DB4 strains with stable and highly pathogenic characteristics were selected for follow-up tests.2. ZYD2 LD50 was 1×1010.63 CFU and YB2 LD50 was 1×102.309 CFU by bacterial virulence test. As the vaccine strain, DB4 was made into 109CFU/mL and 1010 CFU/mL concentrations of propolis adjuvant inactivated vaccine; Observed 14 days after inoculation with high dose, test duck without abnormal,suggests that vaccine is safe and reliable; Ducks were immunized again with the two concentrations of vaccine by groups; 14 days later, ducks were injected with 10 LD50 of ZYD2 and 10 LD50 of YB2.The protection rates of two concentrations for ZYD2 were 40%,80%, and YB2 were 50%,80%, indicating that the vaccine concentration in 1×1010 CFU/ml or more could have play a good effect for protection.3. In this experiment,5.15 ?g/mL protein concentration ZYD2 cell lysates and 8.02 ?g/mL protein concentration YB2 cell lysates as coating antigen, serum 2-fold serial dilutions to 1:212, HRP-IgG anti-duck working concentration 0.25 ?g/mL, using an indirect ELISA method for detecting parent antibody titer. The results of cross reaction between the other 6 kinds of different antigen positive sera were negative;the result of blocking test was negative.4. In this study, ducks were immunized with the inactivated vaccine of DB4, compared difference between control group, immunohistochemistry and different immune times in the level of serum antibody, immune cell and cytokine, comprehensive evaluation DB4 propolis off live vaccine immunogenicity:(1) The antibody titers in serum of immunized ducks were detected by Indirect ELISA method. ZYD2 antibody titer change trend:after immunization 3 days antibody titers were reduced; after that, the secondary immunization group rose to 1:27.5 gradually reduced to the level of the control group, the control group continued to rise, and finally continued above 1:29. YB2 antibody titer change trend:the primary immunization group increased first, then decreased to the level of the blank control group, the serum antibody titer of the secondary immunization group continued to rise to 1:28, and finally maintained at 1:27. In the control group, the changes were not obvious before and after the immunization.(2) The proliferation activity of ducks'lymphocyte in vitro was detected by CCK-8 test kit. Peripheral blood lymphocytes were isolated and cultured in vitro. Con A and parental antigen could stimulate lymphocytes to enhance their proliferative activity, the proliferation ability of the immune group was significantly higher than that of the control group (P<0.05), the difference between the secondary immunization group and the control group was extremely significant (P<0.01). The lymphocyte proliferation could be stimulated by DB4 vaccine immunization, the two stimulation of the immune function is more obvious.(3) We detected the consistence of IL-4 and IFN-y in the peripheral blood serum of ducks at 0,3,7,10,15,20 and 25 days after immunization by ELISA kits. The content of IL-4 and IFN-y in immune group was gradually increased after immunization, compared with the control group, the difference was significant (P<0.05); The rise of primary immunization group is gentle, the secondary immunization rapid rise in 25 days after immunization, and the difference was very significant compared with the control group and primary immunization group (P<0.01). The control group was not changed significantly before and after immunization.(4) At 0,7,15 and 25 days after immunization, the phagocytic rate (PP) and phagocytic index(PI) of the immune group and control group all increased with the increase of day age, and the rate of rise of the immune group was much higher than that of the control group, in particular, the difference between secondary immunization group and control group were extremely significant (P<0.01); After immunization with PP continued to increase, but not immune group did not change significantly; After immunization, PI increased more significantly, and enhanced PI increased more significantly after immunization (P<0.01). |
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