| Mycoplasma bovis(M.bovis) is an important pathogenic pathogens,it can cause bovine pneumonia, arthritis, mastitis, corneal conjunctivitis, ear infections, reproductive tract inflammation and even miscarriage and infertility.It can also cause a variety of diseases secondary infection under the environment and other stress factors. Since1961Hale isolated this pathogen from mastitis milk for the first time in the United States, other countries have also found and broken out Mycoplasma bovis disease caused by this pathogen. In different regions of China, led by Hubei province also have broken out Mycoplasma bovis epidemic since2008.At present,the disease with higher morbidity and mortality exists in all of the world. Each year the huge economic losses of the world’s cattle industry were caused by this disease, it seriously affected and constrained the development of the world cattle industry. Therefore, establishing fast and accurate detection methods for the diagnosis and prevention of this disease has important significance.On the basis of Mycoplasma bovis Xinjiang isolates has been isolated and identified, this research respectively started from the point of view of etiology, serology and molecular biology to establish three different rapid test methods in order to diagnose this disease more comprehensively and accurately. Conclusions of this paper are as follows:1.The establishment of M.bovis indirect immuno-histochemical method. The pure culture of M. bovis and the lung from the calve infected with the M. bovis were immuno-histochemically studied by using the chicken anti M.bovis IgY and goat anti-chicken IgG-HRP as the first and second antibody and optimizing the staining conditions to establish M.bovis indirect immunohistochemical detection methods. The method was used to detective the diseased sections of dead calves under optimal conditions, and the result was verified by isolation and PCR methods.The test results are consistent with the separation of isolation and PCR results, so it’s specific and reliable.2.The establishment of M.bovis indirect ELISA method.The M.bovis mycoprotein were cracked into lysate by the ultrasonic as the antigen, the optimal incubation concentration of the antigen was determined200ug/mL by checkerboard titration, and the optimal dilution of serum was1:100. The incubation way of antigen, the best working concentration of the enzyme-labeled secondary antibody, the optimal working time of the first and second antibody were optimized at the same time,and then the indirect ELISA method detecting M.bovis serum antibodies was established.This method was used to detect Brucella abortus and BVDV antiserum had no cross reaction, indicating that the indirect ELISA has good specificity.3.The establishment of based on the Mycoplasma bovis P81gene loop-mediated isothermal amplification method and comparison with the sensitivity of nested PCR. The specific primers were designed according to the GeneBank registered M.bovis-P81gene sequence to establish M.bovis loop-mediated isothermal amplification (LAMP) detection method by optimizing condition.The sensitivity of this method were compared with nested PCR,several other pathogens and clinical samples suspected infection M.bovis were detected by this method. The sensitivity of this method was103times higher than the nested PCR, the detection limit was1.1fg/uL.The test results were negative for several other pathogens,and the coincidence rate of clinical samples test results and isolation was100%. |
