Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex

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.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

“…Other proteins are rarely described as surface displayed. Antibodies specific to PdhA, PdhB, and PdhC were detected in sera of animals infected with M. bovis (45). PdhB was shown to be surface localized in Lactobacillus plantarum and interacts with human fibronectin (46).…”

Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection

“…They include exposed surface proteins, such as variable surface proteins (Vsp), the lipoproteins P26, P48-like, and mycoplasma immunogenic lipase A (MilA), and the immunodominant membrane protein pMB67 [12, 13, 14, 15, 16, 17], as well as cytoplasmic proteins, such as heat-shock protein 60 (Hsp60), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the pyruvate dehydrogenase beta (PDHB) subunit, among others [18, 19, 20]. The highly-conserved M. bovis GAPDH was suggested as a potential antigen for diagnosis or vaccines; however, a subunit vaccine based on GAPDH did not protect against M. bovis [21].…”

Status of the development of a vaccine against Mycoplasma bovis

“…Based on the lack of recognition of the M. agalactiae P48 protein by the mAb 10E in Western blot assays and the failure to inhibit binding of the mAb 10E to M. bovis P48 by a rabbit polyclonal antibody against the M. agalactiae P48 protein, the authors concluded that this mAb 10E may be useful in a diagnostic test. Another protein proposed as diagnostic marker for M. bovis infection is the E1 b-subunit of the pyruvate dehydrogenase complex [51].…”