Screening Of Antigens And Establishment Of DIVA Assay For Mycoplasma Bovis

Mycoplasma bovis is a major and successful pathogen of cattle worldwide and can cause respiratory infection,mastitis,and arthritis.Long distance transportation is thought to be involved in the occurrence and spread of this disease in cattle population in China.In order to control this disease,our laboratory has recently developed a protective live attenuated vaccine(M.bovis-150).A comparative genomics of virulent HB0801 and attenuated M.bovis-150 strains revealed a complete absence of large DNA sequence of 14kb in attenuated strain.In this study some genes from the missing fragment were cloned and expressed in ordered to characterize a suitable antigen of M.bovis for DIVA serological assay.The main results were as follow:1.We successfully cloned and expressed two genes(rMbovP730 and rMbovP722)from the missing fragment in M.bovis-150 and subsequently the rMbovP730 was examined with high antigenicity for DIVA.2.Mouse anti-rMbovP730 polyclonal antibodies were produced and evaluated by ELISA.The antibody titer against both recombinant proteins was 2^11*100.3.The polyclonal antibody against rMbovP730 verified the absence of protein encoded by Mbov?730 in M.bovis-150 strain.4.Furthermore,feasibility of rMbovP730 for DIVA assay was tested with immunoblotting and ELISA using serum samples from both experimentally infected and immunized cattle.The results showed that rMbovP730 could differentiate an infected from vaccinated animals(DIVA).5.Therefore,we established an indirect ELISA based on rMbovP730 as a DIVA assay diagnosis.After optimization of all conditions for indirect ELISA,the cut-off OD₆₃₀ value of 0.395 was determined with serum samples from uninfected animals(n=46).The specificity and sensitivity of rMbovP730-based iELISA with antisera from naturally infected animals(n=117)and immunized animals(n=44)were found more than 90%.No reactivity with serum samples from the cattle infected with other common respiratory pathogens further confirmed the specificity of rMbovP730-based iELISA.In conclusion,this study determined rMbovP730-based iELISA as potential DIVA serological assay.