Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection.
A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.|Department of Animal Health, The University of Agriculture, Peshawar, Pakistan.
Farhan Anwar
FA
Khan
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Muhammad
M
Faisal
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Jin
J
Chao
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Kai
K
Liu
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Xi
X
Chen
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Gang
G
Zhao
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Harish
H
Menghwar
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Hui
H
Zhang
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Xifang
X
Zhu
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Muhammad Asif
MA
Rasheed
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Chenfei
C
He
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Changmin
C
Hu
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, International Joint Research and Training Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China.
Yingyu
Y
Chen
INRA, UMR 1225, IHAP, Toulouse, France.|INP-ENVT, UMR 1225, IHAP, Université de Toulouse, Toulouse, France.
Eric
E
Baranowski
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, International Joint Research and Training Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China.
Huanchun
H
Chen
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, International Joint Research and Training Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China.|International Joint Research Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China.
Aizhen
A
Guo
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000942:Antigens, Bacterial / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism*; D002417:Cattle; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008074:Lipoproteins / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009175:Mycoplasma Infections / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology*; D045644:Mycoplasma agalactiae; D045682:Mycoplasma bovis; D040901:Proteomics / Q000379:methods*; D012372:ROC Curve; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D015415:Biomarkers; D008074:Lipoproteins; D011994:Recombinant Proteins
ELISA; Immune response; Immunity; Immunology and Microbiology Section; Mycoplasma bovis; diagnostic biomarker; immunoinformatics; immunoproteomics