Isolation And Identification Of Mycoplasma Bovis And Establish The IELISA Method For Its Antibodies

Mycop Lasma bovis(M.bovis)mainly causes a variety of chronic inflammatory diseases in dairy and beef cattle,including mastitis and bronchopneumonia.In some cases,it has also been found to cause arthritis,conjunctivitis and infection of reproductive organs in cattle,leading to abortion in pregnant cows.Even the transport stress syndrome in cattle has been linked to Mycoplasma bovis.Although the mortality of the disease is not high,the infected cattle carry the pathogen for a long time and are an important source of infection,resulting in sub-health status of cattle.In recent years,Mycoplasma bovis infection has been widely spread in the world.In China,Mycoplasma bovis infection has been reported in all provinces and autonomous regions,which seriously endangers the health and sustainable development of beef cattle breeding and brings significant economic losses.The establishment of a specific,rapid and efficient detection method is of great significance for the rapid diagnosis of the epidemic,the inspection and quarantine of live animals and the epidemiological investigation.Ningxia Hui Autonomous Region is one of the ten pasturing areas in our country,the breeding level of beef cattle and dairy cattle ranks among the best in China.In recent years,epidemiological survey found that the infection rate of Mycoplasma bovis increased year by year in this area.In this study,lung lesions and hilar lymph nodes of diseased and dead cattle with respiratory diseases were collected from some cattle farms and individual cattle farmers in Zhongwei,Ningxia.Pathogen isolation,identification and antibiotic susceptibility test were carried out,and an indirect ELISA antibody detection method was established.The specific results were as following:1.Isolation,identification and drug sensitivity test of Mycoplasma bovis.The pathogen samples were collected from Zhongwei,Ningxia Autonomous Region.Three strains of pathogenic bacteria were successfully isolated using PPLO culture medium.Phylogenetic analysis showed that the isolates were closely related to PG45 and Donetta.The minimum inhibitory concentration(MIC)was used to determine the drug sensitivity of the isolates,and four sensitive antibiotics were screened out,which were levofloxacin,vonnemyline hydrochloride,taimiorcin and lincomycin.2.Prokaryotic expression and purification of Mycoplasma bovis P48 protein.The P48 protein is the main protective antigen of Mycoplasma bovis,with good conservation and high specificity.In this study,specific primers for P48 were designed and the full-length P48 protein gene was amplified using the isolate as a template.The full-length P48 protein gene was linked to the prokaryotic expression vector,transformed into BL21 competent cells,and expressed in Escherichia coli.3 Establishment and preliminary application of indirect ELISA for antibody detection of Mycoplasma bovis.The P48 protein was coated on a 96-well solid phase carrier,and the optimal reaction conditions were determined by checkerboard dot matrix method to establish an indirect ELISA method for the detection of Mycoplasma bovis antibodies.The results showed that the optimal antigen coating concentration was 2.5 μg/m L,and the optimal serum dilution was 1:40.The optimal reaction condition of the tested serum was 37℃ for 120 min.The optimal dilution of secondary antibody was 1:500,and the optimal reaction condition was 37℃ for 120 min.The optimal color development time of TMB was 25 min.When the Cutoff value was 0.5596,the sensitivity and specificity were 93.3%.A total of 100 bovine serum samples from Zhongwei City,Ningxia Autonomous Region were detected by the method established in this study,and 29 were positive and 71 were negative for Mycoplasma bovis antibody.The total coincidence rate with the Canadian Biovet kit was 91.4%,indicating that the established method could be used for the detection of Mycoplasma bovis antibodies in clinical samples.In conclusion,through the isolation and identification of Mycoplasma bovis,drug sensitivity screening,establishment of indirect ELISA antibody detection method and epidemiological analysis,this study provides experimental basis for the diagnosis and control of Mycoplasma bovis in Zhongwei,Ningxia,and provides technical support for the epidemiological investigation of Mycoplasma bovis.