| 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 mastitis milk in1961for the first time, and first reported with bovine respiratory disease in1976. China was isolated from the lungs of calves suffering from pneumonia to Mb in2008for the first time, and thereafter 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 Antibody Dynamic Analysis of T cell subsets, T lymphocyte immune characteristics as well as cytokines such as basic research, and aims rich Mycoplasma bovis immunogenicity of content, fill Mycoplasma bovis for screening of dominant strains on specific immune and non-specific immune blank theoretical reference for the subsequent Mb immune mechanisms and pathogenesis of the theoretical basis for research and development of diagnostic kits, vaccine development, and further study.1.Mycoplasma bovis strain recovery, proliferation, PCR identification and concentration determinationThis test was used the improvement mycoplasma medium for expand cultivation of six Mb isolates which isolated from all over the country, PCR identification by specific primers which based on conserved genes of Mb,and concentrations of pure cultures were measured by ColorChange Units(CCU). The study found that the improved liquid medium was suitable for the growth of Mb and shorter culture time was consumed,the color of liquid medium began to change in24h and reached the logarithmic growth phase in48h,the concentrations of the pure cultures of six isolates were all above109CCU/ml.2.Mycoplasma bovis-inoculated mice body Antibody Dynamic ResearchIn order to determine the three strains of Mycoplasma bo vis dynamic changes in the level of antibody in mice, selective advantage strains, the test by ELISA, measured different time points after the body’s antibodies in mice inoculated mice, and to analyze the growth and decline of law. The results showed that the three strains of Mycoplasma bovis vaccination can cause the growth of the body’s antibody levels in mice, and to regularly decline, which in the first week after challenge, W70group antibody levels were significantly higher than in1738and Q3group (P<0.01); after challenge, the second week, the W70group of antibody levels> Q3antibody level>1738antibody levels, three groups with each other difference between antibody levels are extremely significant (P<0.01); challenge three weeks after antibody levels begin to decline, significant difference. Although the three strains in the first week after vaccination to produce antibodies, but the1738strain is relatively W70, the Q3two strains in the latter part of the antibody levels slowly increase the speed, the the antibody peak level is relatively obvious. W70, Q3two strains in the second week after vaccination antibody levels peak, followed by antibody levels gradually decreased, and finally tends to a constant change. W70plants produce antibodies more obvious, not only to produce faster and higher titers of antibodies produced can be used as back-up vaccine production reference strains.3.Mycoplasma bovis-inoculated mice of T lymphocyte subsetsBy observing infected with Mycoplasma bovis mouse CD4+/CD8+values change, provide the basis to further investigate the immune mechanism. In this test,120Balb/C mice were randomly divided into four groups, different from strains (W70strain, the Q3strain,1738strains, the control group) were inoculated Od,7d,14d,21d,28d,35d collected peripheral blood, and flow cytometry detected T cell subsets, calculate the CD4+/CD8+value. The results showed that mice infected with Mycoplasma bovis CD4+cell number increase in CD8+cells decreased CD4+/CD8+ratio began to decline in the first two weeks after reaching the maximum, W70strain in the second week after infection, reaches the maximum value, a significant difference with the other three groups, no significant difference between the other two groups. The CD4+/CD8+ratio increased significantly vaccination of cattle mycoplasma strains, and promote the body’s peripheral lymphocyte proliferation, enhanced cellular immune function. As well as our understanding of Mycoplasma bovis in animals machine body of the CD4+, CD8+lymphocyte antigen recognition and cellular immune mechanism and the role of theoretical reference.4.Mycoplasma bovis-inoculated mice body T-lymphocyte immune characteristicsE-rosette test for detection of the cells immune status of the Balb/C mice. The test results showed that each group after vaccination could increase the rate of rosette formation, which in14days after inoculation, W70strain group rosette formation rate was the highest bit is22%, the minimum is35d, rosette formation rate of13%, indicating that the the the three Mb strains W70strain more to stimulate the body’s immune response, enhance immune function, and promote the proliferation and differentiation of lymphocytes.5.Mycoplasma bovis-inoculated mice of cytokines changeIn order to explore the interleukin-1(IL-1), interleukin-6(IL-6), tumor necrosis factor-alpha (TNF-alpha) in Balb/C mice expression and disease severity ofrelationship, the test selection the three mycoplasma bovis strain was inoculated into BALB/C mice were collected at different times clotting serum was separated, detected by ELISA in3groups of IL-1, IL-6, TNF-alpha cytokine content and explorethe body expression. The results showed that Mycoplasma bovis infected mice serum in IL-1, IL-6and TNF-alpha level was significantly higher, statistically significant difference with control group; serum in IL-1, IL-6and TNF-alpha level increased, indicating that the cytokines IL-1, IL-6and TNF-alpha involved in the occurrence of bovine mycoplasma pneumonia, development, and closely related to the degree of infection and disease.6.Mycoplasma bovis inoculated mice organ indexBy studying of Mycoplasma bovis organs of the mice Index (lungs index, dirty index and thymus index), to investigate Mycoplasma bovis body site of infection and immune organs. The results show that, compared with the control group, in of Mb to infection BABL/c mice, each test group of mice spleen index and thymus index change and the control group was extremely significant (P<0.01). But the difference between the experimental group is not significant, so we can easily come Mb strains of mice infected with the body, though the role of the different levels of immune clearance, reflect the various virulence of, but can not accurately determine the strength of the immunogenicity requires a combination of antibody and cellular immune test to be integrated concluded.Experimental studies have shown that Balb/C mice inoculated with Mycoplasma bovis can increase the organs of the mice index to improve humoral immunity, enhance cell-mediated immunity, promote the production of inflammatory cytokines, indirect improve the body’s non-specific immune function of mice,enhance the immune system of mice. |
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