| Mycoplasma ovipneumoniae(MO)is one of the most important pathogens causeing interstitial pneumoniae of sheep or goats. It was the first time to isolate the pathogen from the sheep suffering from interstitial ovipneumoniae in the world in 1963. It was isolated from the lungs of sheep suffering from pneumoniae in China in 1979, and then spread gradually in the country and cause huge economic losses. On the basis of the consolidated pre-analytical study, the system preparation of inactivated MO vaccine, comparison of different antibody levels of inactivated MO vaccine caused by different adjuvants, evaluation of two protective immunities in sheep which obtained many antigens, challenge protection test and other key technologies on Vaccine’s development have been carried out. The research contents and results are as follows:1. Development and test of inactivated Mycoplasma ovipneumoniae vaccineFirtstly, culture Mo-1411, Mo-1421 isolated from Xinjiang was implied to enlarge development scale, and the concentration of amplification cultures of the MO was made by using the membrane separation device in order to achieve inactivated antigens with formaldehyde. And the next step was to use ISA 206 Duplex adjuvant trialing of inactivated to develop MO oil adjuvant vaccine. 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℃. What’s more, animals did not have any adverse reactions after vaccination and the injection site were normal.And what should be taken seriously is that there were no festering and swelling phenomenon. The experiment indicated that the vaccine was safe and reliable for large-scale production and provided technical supports in the development of MO vaccine.2. Comparison of Two imported adjuvants on antibody levels in mice immunized with inactivated MO vaccineTwo imported oil adjuvants, ISA201 and ISA206 were implied to prepare inactivated MO vaccine separately, with which to immunize mice of different doses. And then serum antibodies of Ig G against MO were detected by the indirect hemagglutination of MO after the 2nd immunization from the 1st to the 4th week. The result showed that the ISA 206 adjuvant vaccine 0.2ml dose group at 14 d antibody levels reached a high level. It is concluded that the ISA 206 adjuvant vaccine 0.2 ml dose could induce the body to produce highest-titer antibodies withthe fastest speed and a long maintaining, which are better than the other three groups. ISA 206 adjuvant vaccine 0.2ml dose group at 14 d antibody levels reached 1: 128. And antibody titers began to fall after 28 days. Comparative results show that ISA 206 adjuvant-induced antibodies produced more qualified MO than ISA201 adjuvants, which can be used as a preferred inactivated MO vaccine adjuvant.3. Experimental study on inactived MO Vaccine and Evaluation of Protective Immunity in sheepThis study was designed to detect the protective immunity of MO vaccine in sheep. 60 herds of 10-15-day-old lambs selected randomly, were divided into three immunized groups, A, B and C with 20 lambs each group, and they were immunized the first time when they were 10-15 days old, and the second after 14 days. Vaccine produced by Taifeng 3 ml for each one in group A, vaccine produced by our laboratory 3 ml for each one in group B and vaccine produced by our laboratory 2 ml for each one in group C. Determine the serumal antibody titers of the three groups after sheep were immunized at the 14 th,21th,28 th,35th,42 th days, and select randomly three lambs with the same day-old from immunized and non-immunized ones respectively to do the challenge protection test. Each lamb was challenged with a broth culture of MO by nasal mucosal route(drip)for 1 ml and tracheal for 2 ml(8.5 × 107 CFU / ml). Body temperature and weight and other clinical signs were observed and recorded after challenging. The sheep were Monitored for clinical signs and serological responses then mortemed for 28 days after challenging. The results showed that the value of serumal antibodies against MO in group A was the highest((5.8±1.31)log2)by 28 days and went down after 35 days. All of the three challenge protective sheep kept good mental state except for fluctuation of body temperature before and after poison attack but in the normal range. Relatively high antibody titers((4.33 ± 0.57) log2) could be produced after poison attack for 14 days and highest antibody titers((6.33 ± 1.52) log2) for 28 days. Slight change of the lungs could be observed from Anatomy. Blood stasis in the alveoli, inflammatory cells around the bronchi and the wider of the interstitial lungs could be observed from Histopathology. MO hasn’t been gotten from the lungs through separation and recovery. The value of serumal antibodies against MO in group B was the highest((5.7 ± 1.49) log2) by 21 days, which would maintain until 35 th days. The value of serumal antibodies against MO in group C was the highest((6.7 ± 0.95) log2) by 28 days, until the 35 th day dropped for two titers. All of the three groups of sheep in poison attack kept good mental state and body temperature. Relatively high antibody titers((4.33 ± 0.57) log2) could be produced after poison attack for 14 days and highest antibody titers((6.66 ± 0.57) log2) for 28 days. Slight change of the lungs could be observed from Anatomy. Lymphocytes around the bronchi and hyperplasia of partial regions of the Interstitial lungs could be observed from Histopathology. MO hasn’t been gotten from the lungs through separation and recovery. Body temperature raised and mild symptoms of cough and pants have happened to the three unimmunized poison-attacked sheep before and after poison attack. Relatively high antibody titers((4.33 ± 0.57) log2) could be produced after poison attack for 21 days and highest antibody titers((5.33 ± 0.57) log2) for 28 days. There were white nodules in lungs sheep whose size was similar to a rice or soy and hilar lymph nodes enlargement. Partial regions of the liver appeared pathological changes. It is similar to the cases of natural infection. Lymphocytes around the bronchi, the width of the interstitial lungs, blood stasis in the bronchi, dropped epithelial cells of the bronchi and other pathological changes in lung tissues could be observed from Histopathology. MO has been gotten from the pathological lungs of two sheep through separation and recovery. The results showed that the difference of antibodies level produced by Mycoplasma ovipneumoniae inactived vaccine from our study and Taifeng is not significant. But they could be used to prevent sheep mycoplasma pneu Monia and induce the body producing immune response which could resist to the pathological effects caused by lung tissues infected MO. |
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