Studies On Some Biological Characteristics Of Mycoplasma Bovis NM2012 Strain

In order to develop a more effective,safe and stable Mycoplasma Bovis(M.boivis)vaccine against epidemic strains in China,In this study,the genetic stability of M.bovis NM2012 strain and the pathogenicity to dairy cows and immunogenicity were studied.The indirect ELISA for detection of the mouse antibody against M.boivis was established.Two pairs of primers were designed according to the whole genome sequence of M.bovis NM2012 strain registered in GenBank.Using the DNA,which was extracted from M.boivis NM2012 strain of 6,12 and 20 generation strain respectively,as a template,whole vpmaX and GAPDH genes were amplificated.The obtained fragment was ligated with the pMD-19T vector and cloned.The positive clones,which were identified by bacteria PCR and plasmid PCR,were sequenced by HuaDa company.The gene sequence and deduced amino acid sequence were compared with the vpmaX and GAPDH genes of M.bovis NM2012 registered in GenBank.The results showed that the nucleotide and deduced amino acid homology of the vpmaX gene were 100%all,The nucleotide sequence of GAPDH gene and deduced amino acid homology were 99.9%～100%and 99.7%～100%respectively,which indicated that the genetic characteristics of vpmaX and GAPDH genes of M.bovis NM2012 strain were stable.In order to understand the pathogenicity of M.boivis NM2012 strain,Holstein calves were artificially infected by endotracheal inoculation with F6 of M.bovis NM2012 strain.Clinical symptoms were observed and the change of white blood cell(WBC)and M.bovis specific antibody IgG was detected after challenge.At the 14th day and 27th day after challenge,tested calve were killed in two lots.The pathological changes of the killed calves were observed by naked eyes and histologically analyzed.The results showed that M.bovis NM2012 strain exhibited very strong pathogenicity to Holstein calves.In this study,an indirect ELISA method for the detection of murine anty-M.bovis antibodies was established and the immunogenicity of M.bovis NM2012 strain was studied.Whole cell protein lysates were used as the coated antigen,The optimal concentrations of antigen and serum were determined by checkerboard titration and the indirect ELISA was developed.It was found that the reproducibility and specificity of the method were good.The antibody level of anty-M.bovis in mice at different time after inoculation of M.bovis NM2012 strain was detected by this method.The results showed that three generations of M.bovis NM2012 strain were all able to induce rapid production of anty-M.bovis antibodies in mice and the antibody maintained at a high level for a long time,which indicated that the M.bovis NM2012 strain had good immunogenicity.The results showed that the M.bovis NM2012 strain had good genetic stability,strong pathogenicity and better immunogenicity.The strain could be used as a vaccine strain for producing inactivated vaccine of M.Bovis.