| Mycoplasma bovis (M.bovis) is a pathogenic microorganism, which causes bovine respiratory disease, and makes huge economic losses to the livestock industry worldwide, but there is not enough intensive study about the M.bovis vaccine. This study is about the infection of M.bovis with the base of isolation and identification, and the inactive vaccine is prepared after optimization of the inactivation method. The effect of different immunizing dose and the immunization routes were analyzed based on the infecion model, in order to get the best adjuvants. The method and results are shown below:1. To establish the infection model, the infection effect was analyzed with the clinical symptom, autopsy, by comparing the rabbits before and after infection. The M.bovis was isolated from the lung and blood of the rabbits infected, and the isolation was tested with PCR method after culture, and the isolation and antibody was analyzed. The results showed that the rabbits could be infected by M.bovis with the concentration of 1.0×108ccu/ml, and the rabbits infected was observed deprementia; there were oedema, status marmoratus of lung, and pleural effusion by autopsy; the bleeding alveolar lumen, increasing gap among alveolar lumens, necrosis and drop of bronchiole epithelial cell were observed; the sequencing and specific PCR test results proved that the M.bovis was isolated from rabbits infected, and the antibodies to M.bovis were detected. All the results proved the infection model of rabbit succeeded.2. M.bovis was inactivated with formaldehyde of the 0.3%,0.4%,0.5% concentration. To evaluate theimmunogenicity of inactive M.bovis by immunization test. The results showed:0.5% formaldehyde could inactivate M.bovis in short time incubation, at the same time the inactive M.bovis was not fit for immunization because of reduced immunogenicity; 0.3% formaldehyde could inactivate part of M.bovis after 60-hour incubation; and 0.4% formaldehyde could inactivate M.bovis completely after 48-hour incubation, and the immunogenicity is similar to that before inactivation, so 0.3% formaldehyde was the best condition for inactivation.3. After inactivation with 0.4% formaldehyde, the M.bovis was washed with PBS then mixed with white oil, aluminum hydroxide, Freund’s Complete Adjuvant at the concentration of 5.0×109 ccu/ml respectively. The diluted inactive M.bovis with concentration of 1.0×1010ccu/ml was tested for the stability and sterility was test, and the inactive vaccine was safe for use.4. Rabbits were immunized with different immunizing doses, immunization routes, and immunization adjuvants. The clinical situation and immunization sites were observed to adjudge the safety of the vaccine, and the immunoglobulin (IgG, IgM), Cytokine IL-4 and IFN-γ was tested for evaluation the optimized immunization dose, route and the adjuvant. The results showed:the effect of 2ml 1.0×1010ccu/ml inactive vaccine can be immunized for protective effect; the best immunization route is intramuscular injection; the white oil mixed as adjuvant with ration 1:1 could generate the better protective effect than aluminum hydroxide, Freund’s Complete Adjuvant.5. The challenge test was taken with the culture of M.bovis from different sources, in order to evaluate the protective effect. The inactive vaccine could stimulate the protective effect to the same isolation of M.bovis, and the different isolation of M.bovis, by Pathologic analysis, after challenging the immunized rabbits. |
