Oncotarget

Research Papers: Immunology:

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

Farhan Anwar Khan, Muhammad Faisal, Jin Chao, Kai Liu, Xi Chen, Gang Zhao, Harish Menghwar, Hui Zhang, Xifang Zhu, Muhammad Asif Rasheed, Chenfei He, Changmin Hu, Yingyu Chen, Eric Baranowski, Huanchun Chen and Aizhen Guo _

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Oncotarget. 2016; 7:39376-39395. https://doi.org/10.18632/oncotarget.9799

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Abstract

Farhan Anwar Khan1,2,3,*, Muhammad Faisal1,2,*, Jin Chao1,2, Kai Liu1,2, Xi Chen2, Gang Zhao1,2, Harish Menghwar1,2, Hui Zhang1,2, Xifang Zhu1,2, Muhammad Asif Rasheed1,2, Chenfei He1,2, Changmin Hu2, Yingyu Chen1,4, Eric Baranowski6,7, Huanchun Chen1,2,4 and Aizhen Guo1,2,4,5

1 The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China

2 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China

3 Department of Animal Health, The University of Agriculture, Peshawar, Pakistan

4 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

5 International Joint Research Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China

6 INRA, UMR 1225, IHAP, Toulouse, France

7 INP-ENVT, UMR 1225, IHAP, Université de Toulouse, Toulouse, France

* These authors have contributed equally to this work

Correspondence to:

Aizhen Guo, email:

Keywords: diagnostic biomarker, ELISA, immunoproteomics, immunoinformatics, Mycoplasma bovis, Immunology and Microbiology Section, Immune response, Immunity

Received: April 27, 2016 Accepted: May 23, 2016 Published: June 02, 2016

Abstract

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.


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