| Mycoplasma bovis infection is an important causative pathogen of the bovine, mainly caused by the bovine pneumonia and mastitis, can also lead to Bull arthritis, keratoconjunctivitis, otitis,reproductive tract inflammation,abortion and notprogesterone and other various diseases.The United States has been isolated the pathogen from mastiffs milk in 1961 for the first time,and first reported with bovine respiratory disease in 1976.China was isolated from the lungs of calves suffering from pneumonia to M.bovis in 2008 for the first time,and there after began to spread in the country.The pathogen was resulting in huge economic losses in a wide range of domestic and international transmission.On the basis of the consolidated pre-analytical study, this system is carried out Culture characteristics, Concentration of bacterial suspension, Preparation of Mycoplasma bovis inactivated vaccine and Evaluation of Protective Immunity in Calves which obtained much antigen and provided technical support in the development of M.bovis inactivated vaccine.1 Study on the Application Effect of TTC for colony enumeration and Determination of Amplification Culture of Mycoplasma bovisIn the present study, an attempt was made to analyse the possibility of using TTC for colony enumeration of M.bovis and verified the growth characteristics of Self-made medium. CFU and CCU methods were used. OD value and p H value of the culture was recorded every 6 hours and the growth curve was drawn. The results showed when the ultimate concentration of TTC was 0.3~0.6g/L, M.bovis could grow normally, and the effect of color development was visible. When the concentration of TTC was lower, M.bovis could grow better and there was no obvious difference between test group and control group. The growth of M.bovis colony was inhibited when the concentration of TTC was higher. The characteristics of M. bovis colony was very significant when the concentration of TTC was 0.4g/L. The concentration of the M.bovis culture was 1.8~2.42×108 CFU/m L, and the growth titer was 5.0×108 CCU/m L. The OD value ranged from 0.024~0.213 at different quantum and the p H value reduced urgently in 48 h. These findings mean that TTC can be used for colony enumeration of M.bovis and indicated that these two methods have preferably coherence. M.bovis strain could grow rapidly and with a good adaptability to the self-made medium which should have important implications for the determination of effect on amplification culture of M.bovis and lay a foundation of further study on the vaccine development of Mycoplasma bovis.2 Growth characteristics comparison of different mediums and Determination of the growth Curve of Mycoplasma bovisIn the present study, an attempt was made to verify the growth characteristics of self-made medium. The titer was detected and growth curve was drawn with CCU method. The growth curves of M.bovis strain in the improved Thiaucourt’ s medium,the cattle-heart fusion medium, the serum glucose broth medium and self-made medium were determined by viable count method,respectively. The results showed M.bovis strain could grow rapidly and with a good adaptability to the self-made medium. It indicated that in the self-made medium the logarithmic growth phase of the M.bovis strain was 0-24 h. On the hour 56 it reached plateau phase and then slowed down after 96 hours. The average highest growth titers of the M.bovis strain were 4.0×109 ccu/m L, 7.0×108 ccu/m L, 7.0×107 ccu/m L and 7.0×107 ccu/m L, respectively.It provided a useful data for the development of inactivated vaccine against M.bovis.3 Preparation and detection of Mycoplasma bovis inactivated vaccineThe aim was to establish a set of antigen concentration technology for producing inactivated vaccine of M.bovis. Comparative study was taken with dfferent methods such as Dialysis, high speed centrifugation and ultrafitration concentration. The protein contents with different trentments were 0.625 mg/m L, 0.889 mg/m L and 1.624 mg/m L, respectively. The concentration of concentrated solution were 3.65×108 CFU/m L and 4.0×108 CCU/m L and indicated that these two methods have preferably coherence. M.bovis pneumonia inactivated vaccine was prepared with the M.bovis strain isolated from Xinjiang Province. The vaccine was inoculated when it was proved safe to use after aseptic test, animal test and emulsifying effect test. These results showed that the vaccine has good physical properties and could be kept 180 days at 4℃ and 3 days at 37℃. The animals were no any adverse reactions after vaccination and the injection site were normal. And there were no festering and swelling phenomenon. These findings indicated that ultrafitration concentration could may be the best method in the concentration of M.bovis and laid a foundation of further study on the vaccine development of Mycoplasma bovis. It indicated the vaccine was safe and reliable for large-scale production and provided technical supports in the development of M.bovis vaccine.4 Determination of the Minimum dose and Antibody dynamics of an Inactivated Mycoplasma bovis Vaccine in MiceIn the present study, we determined the minimum dose of Mycoplasma bovis inactivated vaccine and antibody dynamics in immunized mice.The mice were injected intramuscularly with 0.1, 0.2, 0.3 or 0.4m L of Mycoplasma bovis inactivated vaccine. The results demonstrated that the minimum dose of this vaccine was 0.2 m L per mouse. Vaccination after 3 weeks, the antibody titers reached a peak and then showed a downward trend. All of these data provided a very good basis for further develop ment of an inactivated M.bovis vaccine.5 Experimental Study on M.bovis Inactivated Vaccine and Evaluation of Protective Immunity in CalvesThis study was designed to detect the protective immunity of Mycoplasma bovis pneumonia and arthritis vaccine in calves. Through amplification culture in self-made medium, membrane separation concentration and inactivated with formalin, M.bovis pneumonia inactivated vaccine was prepared with the M.bovis strain isolated from Xinjiang Province. The vaccine was inoculated through intramuscular injection route into 10–15 days-old calves when it was proved safe to use after aseptic test, animal test and emulsifying effect test. Serum antibodies of Ig G against M.bovis were detected by ELISA at the time of pre-immunization and after the second immunization. The calves were challenged three weeks later with a broth culture of M.bovis by tracheal and nasal mucosal route on two successive days. The titre was 1.8×109(CFU)/m L. Fever and other clinical signs were observed and recorded after challenged. The calves were monitored for clinical signs and serological responses then post mortemed 4 weeks after challenge. The vaccine was shown to be highly immunogenic in calves and did not cause adverse effects. Vaccinated calves showed few clinical signs while all unvaccinated calves developed signs of pneumonia. There was a significant decrease in body weight gain in unvaccinated calves compared to vaccinates and a significant increase in lung lesions and rectal temperatures in unvaccinated calves. In conclusion the M. bovis vaccine produced a significant level of protection against a large virulent challenge. |
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