| Mycoplasma bovis (M. bovis) is an important pathogen for respiratory relevant disease and arthritis in feedlot cattle and dairy calves. The diseases associated with M.bovis have caused huge economic losses in beef and dairy cattle farming throughout the world. M. bovis was firstly separated from the lesioned lung of diseased beef cattle in Hu Bei province in 2008, it was then formally proved to be extensive existence in China, due to more frequent animal trade that caused M.bovis epidemic and a big loss in cattle industry.There are no effective ways to prevent and treat this terrible disease since M.bovs is not sensitive to commonly used antibiotics and easily develops drug resistance to drugs. In addition, there are no available commercial vaccines currently in China. As to the vaccine development, inactivated vaccine can significantly reduce the clinical signs and pulmonary pathological changes, but can not significantly reduce M.bovis infection; Meanwhile, this vaccine has a short immune duration. Therefore, many research people hope to build new strategies in the new vaccine research and development, but the main reason for the slow progress is that the pathogenesis of M.bovis is not clear,The previous study has attenuated a virulent M.bovis strain and this study was aimed to screen an attenuated strain with good immunogenicity from the different passages of the strains for vaccine development.1. The screening of attenuated strain for vaccine in calvesThis lab previously found that M.bovis HB0801 strain was significantly attenuated after 150 passages (P150) of in vitro successive passaging. This study compared the P150 and P180 (the 180 passage) strains in virulence and immunogenicity in calf experiments. A total of 20 calves were allotted to four groups to evaluate protective efficacy of these two attenuated strains against M. Bovis infectioa P150 and P180 were immunized at dose of 109CFU per calf via nasal cavities and each strain for 5 calves, respectively. The rest 10 calves were not immunized as positive group control (PC group:no immunization but challenge) and negative group (NC group:no vaccination and challenge), each group had 5 calves. The calves except for NC group were challenged 46 days later with a 10 mL fresh broth culture contained 1010CFU of M. bovis on consecutive three days via trachea, the NC group as the control was mock-challenged 10 mL fresh broth for consecutive three days. The calves were monitored for 3 weeks after the challenge and then euthanized for post-mortem examination. As a result, P150 and P180 immunization provided excellent protection for the calves against wild type M. bovis challenge. Vaccinated calves showed no obviously clinical signs while all unvaccinated calves in PC group developed symptoms of pneumonia. In unvaccinated calves in PC group, there was a significant decrease in body weight gain, significant increase in lung lesions, rectal temperatures and leukocyte count compared to the vaccinated calves in both P150 and P180 groups. These two strains also reduced the nasal shedding and colonization of M. bovis in respiratory tract.2. The virulence stability evaluation of P150 and P180 in cattleTo study P150 and P180’s virulence stability in cattle, the consecutive passages in calves were performed. Each passage was divided into three groups named P150, P180 and NC groups, and each group included two calves. First passage injected 50 ml culture (equal to 5×1010 CFU) of corresponding strains via chest, the control group mock-injected same volume of sterile PPLO medium via chest. As mentioned earlier, clinical signs were observed post injection and postmortem gross pathology was checked at 7 days after injection. The filtered lung homogenates of last time were used as next passage injection, the dose of injection was 30mL, Meanwhile the M.bovis lung load was counted and other bacteria were isolated. Since the third passage, the route of injection was switched to trachea injection. Results showed that calves did not present typical signs of M.bovis after injection of attenuated strains and there were no typical pathological changes of M.bovis pneumonia postmortem, the quantity of M.bovis gradually reduced in lungs tissue with the increase of passages. There was no M.bovis isolated from lungs after the fourth passage. Therefore, the virulence of attenuated strains P150 and P180 was not changed in calf body after four consecutive passages.3. The detection of minimum vaccination dose of the attenuated MbovisThe 15 five months health calves were divided into five groups each three calves. The experimental groups were administered P150 at four different doses of106CFU,107 CFU,108CFU,109CFU groups, while the control group was mock-injected equal amount of PPLO medium. After 55 days post immunization, the calves were challenged with virulent strain of 3><1010CFU/per calf, and clinical symptoms were observed as described previously post challenge. The experiment results indicated that the minimum dosage of 108 could protect calves against challenge of 3x1010CFU wildtype M.bovis infection.4. The experiment of single dose for repeat inoculation of attenuated Mbovis strainThe 6 five months health calves were divided into two groups, the immunization group and control group, each group three calves. The immunization group was immunized 10 ml diluted freeze-dried attenuated strain P150 at dose of 108 CFU/calf, the control group mock-immunized 10 ml PPLO medium. The immunization for consecutive three times at an interval of 14 day, and clinical sings and body temperature changes were observed. The results showed that three consecutive immunization of P150 did not cause clinical signs and exert negative influence on growth.5. The safety experiment for pregnant cows for attenuated vaccine of M.bovisThe 5 pregnant cows about 4 to 7 months were divided into two groups,2 in immunization group, while 3 in control group. The immunization group was immunized 10 ml diluted P150 at 108 CFU/cow, the control group immunized 10 ml of sterile PPLO medium. The clinical signs and reproduction was observed after vaccination. The results showed that immunized P150 was safe for pregnancy cows and did not affect the birth of calves and growth of newborn calves. |
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