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

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.

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…Immunoproteomic analysis (identification of immunoreactive proteins with sera from experimentally infected or naturally infected cattle) has been used to identify additional M. bovis surface proteins that have proven suitable for ELISA development, but that may also be exploitable as possible subunit vaccine candidates. These include the MbovP579 lipoprotein of unknown function (Khan et al., ) and MilA, a putative lipase related to the GDSL family (Wawegama, Browning, Kanci, Marenda, & Markham, ). A similar immunoproteomic study led to the identification of ten M. bovis antigens of which five (PdhA, EF‐Tu, O256, P48 and Lpp) could induce robust IgG1 and IgG2 responses, when used as recombinant proteins formulated with the commercial adjuvant Emulsigen ® together with poly I:C and IDR peptide 1002 (Prysliak & Perez‐Casal, ).…”

In Silico Identification of Diagnostic Candidates from Predicted Lipoproteome of Mycoplasma mycoides subsp. capri

“…All the identified targets MLC_0780, MLC_0810, MLC_0820, MLC_3130 and MLC_8630 are uncharacterized proteins. Recently, an in silico analysis combined with 2D electrophoresis and western blot predicted eight novel uncharacterized antigens to have high immunological value and Mbov_0579 was found to be the best antigenic target for sero diagnosis of M. bovis (Khan et al, 2016).…”

In Silico Identification of Novel Immunogenic Secreted Proteins of Mycoplasma bovis from Secretome Data and Experimental Verification

“…However, to date, few secretory proteins of M. bovis have been identified. However, preliminary studies have demonstrated that they play significant roles in inducing immunity and as diagnostic biomarkers [ 13 , 14 ].…”