title: 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.
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Zhenhong
authors: Z
authors: Sun
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Ping
authors: P
authors: Fu
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Kai
authors: K
authors: Wei
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Haiyan
authors: H
authors: Zhang
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Yuewei
authors: Y
authors: Zhang
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Jian
authors: J
authors: Xu
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Fei
authors: F
authors: Jiang
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Xu
authors: X
authors: Liu
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Wei
authors: W
authors: Xu
authors: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China.
authors: Wenxue
authors: W
authors: Wu
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D002417:Cattle; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015151:Immunoblotting; D009175:Mycoplasma Infections / Q000276:immunology / Q000382:microbiology; D045682:Mycoplasma bovis / Q000276:immunology* / Q000302:isolation & purification; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000276:immunology*; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit