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. 2016 Jun 28;7(26):39376-39395.
doi: 10.18632/oncotarget.9799.

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

Farhan Anwar Khan  1   2   3 , Muhammad Faisal  1   2 , Jin Chao  1   2 , Kai Liu  1   2 , Xi Chen  2 , Gang Zhao  1   2 , Harish Menghwar  1   2 , Hui Zhang  1   2 , Xifang Zhu  1   2 , Muhammad Asif Rasheed  1   2 , Chenfei He  1   2 , Changmin Hu  2 , Yingyu Chen  1   4 , Eric Baranowski  5   6 , Huanchun Chen  1   2   4 , Aizhen Guo  1   2   4   7
Affiliations

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

Farhan Anwar Khan et al. Oncotarget. .

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.

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

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. 2D gel electrophoresis and western blot analysis of M. bovis HB0801 whole cell proteins
A. Analysis of HB0801 whole cell proteins with 2-DE. Isoelectric points are indicated at the top and molecular weight markers in kDa on the left. B. Immunoblotting patterns of M. bovis HB0801 whole cell proteins obtained using a pool of sera derived from calves experimentally infected with HB0801. The 21 spots identified by MALDI-TOF MS are indicated on the 2D electrophoresis gel and the PVDF membrane.
Figure 1
Figure 1. 2D gel electrophoresis and western blot analysis of M. bovis HB0801 whole cell proteins
A. Analysis of HB0801 whole cell proteins with 2-DE. Isoelectric points are indicated at the top and molecular weight markers in kDa on the left. B. Immunoblotting patterns of M. bovis HB0801 whole cell proteins obtained using a pool of sera derived from calves experimentally infected with HB0801. The 21 spots identified by MALDI-TOF MS are indicated on the 2D electrophoresis gel and the PVDF membrane.
Figure 2
Figure 2. 2D gel electrophoresis and western blot analysis of M. bovis HB0801 TX114 membrane fractions
A. Resolution of the HB0801 TX114 membrane fraction using 2-DE. Isoelectric points, ranging from pH 3-10, are indicated at the top and molecular weight markers in kDa on the left. B. Antigenic pattern of the M. bovis HB0801 membrane fraction obtained using a serum pool from calves experimentally infected with HB0801. The 17 spots identified by MALDI-TOF MS are indicated on the 2-DE map and the PVDF membrane.
Figure 2
Figure 2. 2D gel electrophoresis and western blot analysis of M. bovis HB0801 TX114 membrane fractions
A. Resolution of the HB0801 TX114 membrane fraction using 2-DE. Isoelectric points, ranging from pH 3-10, are indicated at the top and molecular weight markers in kDa on the left. B. Antigenic pattern of the M. bovis HB0801 membrane fraction obtained using a serum pool from calves experimentally infected with HB0801. The 17 spots identified by MALDI-TOF MS are indicated on the 2-DE map and the PVDF membrane.
Figure 3
Figure 3. Representative SDS-PAGE and Western blotting assay of recombinant proteins
A. SDS-PAGE of recombinant proteins; B. Immune-reactivity of all recombinant proteins with antiserum samples from naturally infected cattle; C. Immunoblotting of recombinant lipoproteins (rMbovP579 and rMbovP739) with antisera from experimental calves 35 dpi (1), experimental calves 35 days post-vaccination (2), and naturally infected cattle (3). Protein markers are indicated by “M” with the respective molecular weights in kDa on the left. Lanes 1-8 in A. and B. indicate proteins rMbovP579, rMbovP739, rMbovP116, rpepA, rpepP, rDLD, rMbovP275, and rTkt, respectively; lanes A. and B. in C. indicate rMbovP579 and rMbovP739, respectively. Serum samples from each group of cattle were diluted at 1:100.
Figure 4
Figure 4. Reactions of M. bovis HB0801 whole cell proteins and recombinant proteins to different antisera measured with iELISA
M. bovis HB0801 whole cell proteins (WCPs) and all recombinant proteins (rMbovP579, rMbovP739, rMbovP116, rMbovP275, rDLD, rpepP, rTkt, and rpepA) were characterized in vivo with antiserum samples from each experimentally infected calf 0-35 dpi A., antisera from each experimentally immunized calf 0-35 days post-vaccination B., and serum samples from each animal naturally infected with M. bovis C.. The Y-axis indicates mean ± SD iELISA OD630 values of serum antibodies from infected, vaccinated, and uninfected (NC) animals against WCPs and all recombinant proteins. Serum antibodies to proteins were diluted as follows: WCPs 1:400, rMbovP579 1:1600, rMbovP739 1:200, and rMbovP116, rMbovP275, rDLD, rpepP, rTkt, and rpepA 1:100.
Figure 5
Figure 5. MbovP579 and MbovP739 expression in M. bovis strains and other mycoplasmas
A1. SDS-PAGE of rMbovP579 (81.6 kDa) and WCPs of mycoplasmas; A2. Western blot analysis with mouse anti-rMbovP579 polyclonal antibodies; B1. SDS-PAGE of rMbovP739 (70.2 kDa) and WCPs of mycoplasma strains; B2. Western blot analysis with mouse anti-rMbovP739 polyclonal antibodies. Protein markers are indicated by “M” with the respective molecular weights in kDa on the left. Lanes 1-9: rMbovP579/rMbovP739, E. coli (BL 21-DE3) WCPs as a negative control, HB0801, M. bovis-150, PG45, PG2, PG3, Y98, and M. arginini, respectively. Serum samples were diluted at 1:100.
Figure 6
Figure 6. ROC analysis of the rMbovP579-based iELISA compared to gold standard tests
Based on the ROC curve, 0.442 was determined as the iELISA cut-off value with a sensitivity of 90.2% and specificity of 97.8%.
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