Establishment Of Diagnostic Methods And Studies On Adhesin In Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae, whose appearance was reported in numerous countries and regions, can infect sheep and goats causing contagious pleuropneumonia mainly characterised with cellulose-effusional pneumonia. As for Inner Mongolia and its periphery regions where exist the major breeding farms for sheep and goats in China, the distribution and spread of Mycoplasma ovipneumoniae there tend to be ovwhelming enhanced with the traditional aquaculture model conducted by spread breeders being gradually converted into the large-scale aquaculture model, which will surely result in prodigious economic losses. Therefore, it is urgently needed for us to improve and strengthen the prevention and controls of the Mycoplasma ovipneumoniae, that is, construct the diagnostic methods and research on vaccines are the priciple missions. So this research identified the Mycoplasma ovipneumoniae starins isolated from regions partly in Northern China and conducted the molecular-leveled epidemiological investigation, based on which we also constrct themolecular-leveled fluorescent quantitation PCR detecting method and serological indirect ELISA diagnostic method. Simultaneously, we predicted 3 adhesional proteins, verified their functions and screened out their dominant epitope and functional domain in oder to deeply comprehend the pathogen.The research isolated 226 Mycoplasma ovipneumoniae from most regions of Northern China, identified them with traditional and molecular biological identificational tests and amplified their 16S rDNA,16S-23S rDNA, EFTU gene and HSP70 gene to select 14 strains with local representativeness repectively. Then we sequenced the amplified products and found that the phyletic evolutional tree constructed via EFTU has no apparent difference with that via 16S rDNA, based on which we presumed considering the EFTU gene as the new molecular target to analyse the heredity and evolution relationship and identification for genera of mycoplasma ovipneumoniae strains. In addition, we also presumed using the EFTU gene as the special gene of molecular identification due to its conservatism of different genera. Then we constructed the EvaGreen real time fluorescent quantitation PCR method towards mycoplasma ovipneumoniae pneumonia and it showed better specificity, sensitivity and stability than the narmal PCR method.We also lopped the PI 30, P129 and P71 adhesional proteins into 9 segments reserving their functional domains and mutable points according to the annotation results of gene sequences and the analytical results of bioinformatics softwares. Then we used the 9 segments of protein expressed and purified to immuned mice to screen dominant epitopes. The results showed that the segments of P130-3 and P71-3 could significantly enhance the levels of humoral immunity and cytoimmunity of mice.We constructed the isolation and identification method of tracheal epithelium cell of sheep and the clinical infection model of Mycoplasma ovipneumoniae and verified its adhension capacity for tracheal epithelium cells. Then with the model we had detected the adhension capacity of recombination protein and its antiserum’s inhibition effects on the mycoplasma ovipneumoniae thalli and found that the P130-3 and P129-2 had more obvious effects, which indicated that their functional domains played important roles in the process of adhesion. Considering all the tests results together, the P130-3 was the best lopped protein. So we used the screened P130-3 as the diagnostic antigen to build the indirect ELISA serodiagnosis method and optimized the parameters of reaction conditions. After that we comparaed the detection effects on clinical samples between the EvaGreen real time fluorescent quantitation PCR method and theP 130-3 indirect ELISA method.The research had constructed 2 detection methods towards diseases caused by Mycoplasma ovipneumoniae and built the isolation and identification method of tracheal epithelium cell of sheep and the clinical infection model of mycoplasma ovipneumoniae. We compared the detection effects between 2 detection methods and the adhension capacity and reletive inhibition effects among recombinant protein in the epithelium adhension model. Eventually, we compared the sensitivity of EvaGreen real time fluorescent quantitation PCR method, theP130-3 indirect ELISA method, the commercial passive hemagglutination detection KIT and the isolation and cultivation method of pathogen for the clinical samples detection. The results showed that theP 130-3 indirect ELISA method had the best sensitivity.