Abstract
The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.
Highlights
Mycoplasma bovis (M. bovis) is a major but often overlooked pathogen
Taking into account that there are 765 proteins encoded by the genome of M. bovis reference strain PG45, the rate of coverage of proteins separated in the present study was found to be 74.5%
The results indicated that the antigenicity of M. bovis pyruvate dehydrogenase E1 component beta subunit (PDHB) was similar to that of M. agalactiae PDHB but markedly different from that of other pathogens
Summary
Mycoplasma bovis (M. bovis) is a major but often overlooked pathogen. It mainly causes respiratory disease, mastitis, arthritis, keratoconjunctivitis, and otitis. Reported M. bovis immunogenic proteins involved variable surface proteins (Vsps) These membrane-surface antigens can vary in phase and size. [11], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [12] These proteins may be suitable for use as candidate antigens for diagnosis and subunit vaccines against M. bovis. Proteome analysis is a useful complementary method of studying pathogens It facilitates genome annotation and protein identification [13,14]. 19 immunogenic proteins were identified in a strain of M. bovis that had been isolated in China. These proteins were identified using immunoproteomics with four positive sera (Table S1) collected from the disease-affected cattle feedlots in different provinces.
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