| Mycoplasma gallisepticum disease and mycoplasma synovial disease caused by Mycoplasma gallisepticum and Mycoplasma synoviae has brought enormous economic cost of the poultry industry. At present the prevention and control of the two diseases is by inactivating vaccine, the use of single union vaccine not only has large stress on chickens and affects the production effeciency,but also waste time and energy.Therefore, the preparation of Mycoplasma synoviae and Mycoplasma gallisepticum bivalent inactivated vaccine has important practical significance.MS WVU1853 strains and MGRlow stains bought from China Institute of Veterinary Drugs Control were used to prepare the bivalent inactivated vaccine.By optimizing the conditions, the growth curve of MS was drew in different culture mediums to screen the best culture medium for MS.MS inactivated vaccine was prepaerd by concentration and inactivation.After immunization the amount of IFN-γ in chickens’ serum were dected each week from the age of 7 days to the age of 28 days were 79 pg/m L, 265 pg/m L, 290 pg/m L, 435 pg/m L that were significantly higher than that of the control group(P<0.05).The amount of CD4+ T cells and CD8+ T cells of spleen from tne immunized chickens were tested by flow cytometry and the radio of CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes showed an increasing trend by the increase of immune antigen. Chicken dendritic cells and T lymphocyte were stimulated by immunizing antigen in vitro,the amount of MHC II in dendritic cells were the highest when stimulated with 5 MOI MS,it could reach 74.4%,the proliferation rate of CD4+ T cells was 51.2% and the proliferation rate of CD8+ T cells was 36.2%.The result showed that MS inactivated immunogen can promote significant proliferation of CD4+ T lymphocytes,MS was cultured with the most suitable culture which screened before, MS inactivated antigen was prepaerd by concentration and inactivation. According to the laboratory research about the culture cuve of MG,the culture condition of MG were optimized. MG inactivated antigen was prepaerd by cultureing and concentration and inactivation. According to the amount of those two Mycoplasma,inactivated antigen were mixed by 1:1 to prepare the bivalent inactivated vaccine. Then add oil adjuvant emulsion to prepare MS and MG bivalent inactivated vaccine.Then physical properties tests of the vaccine and safety tests were carried out,the results showed that the vaccine is safe and useful.In order to study the immune protective effect of the bivalent inactivated vaccine, the SPF chickens were immuned with different dose bivalent inactivated vaccine, serum titer of MS and MG were detected by ELISA to evaluate the protective effect of different dose vaccine.The result showed that the titer of two species of Mycoplasma were higher than those of the control group(P <0.05) and 4 weeks after the immunization, chickens were attacked with the international standard strain MG strain Rlow(2×108 CFU) each chicken in the air bag and with MS WVU1853(2×108 CFU) each chicken in joint,two weeks later joint fluid and airbag were separated. The amount of load mycoplasma in air bag and joint were quantified by fluorescence quantitative PCR,the log of copies detected of MG was 1.06, and the control group was 3.33, the differences between them were significant(P<0.05); the log of copies detected of MS was 0.76 and the control group was 2.6 and the differences were significant(P<0.05), the results showed that the amount of Mycoplasma of the immune groups were higher than that of the control group suggested that the vaccine has shown good protective quality against Mycoplasma. After the prime immune, 5 weeks later the chickens were immuned twice, the dynamic change of the chickens serm titer in 20 weeks was studied.The result showed that the immune protection can persisted 6 mouths. High and sustained antibody response of chickens can be induced by the bivalent inactivated vaccine。... |
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