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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.

SUBMITTER: Khan FA

PROVIDER: S-EPMC5129939 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Khan Farhan Anwar FA Faisal Muhammad M Chao Jin J Liu Kai K Chen Xi X Zhao Gang G Menghwar Harish H Zhang Hui H Zhu Xifang X Rasheed Muhammad Asif MA He Chenfei C Hu Changmin C Chen Yingyu Y Baranowski Eric E Chen Huanchun H Guo Aizhen A

Oncotarget 20160601 26

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 p ...[more]

PMID: 27281618

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Project description:Mycoplasmas are successful pathogens both in humans as well as in animals. In cattle, Mycoplasma bovis (M. bovis) is known to be responsible for serious health complications, including pneumonia, mastitis, and arthritis. However, M. bovis pathogenesis remains unclear. Secreted proteins of M. bovis could influence infection and modify host defense signaling pathways after they enter their extracellular space in the host micro-environment. Therefore, this study was aimed to compare the secretomes of M. bovis HB0801 virulent (P1) and attenuated (P150) strains and identify potential pathogenesis-related secreted proteins and biomarkers. The cells of P1 and P150 strains were grown in pleuropneumonia-like organism medium to log phase and then transferred to phosphate-buffered saline for 2 h. Then, the supernatant was analyzed by using label-free quantitative proteomics, and 477 potential secreted proteins were identified. Combined with the bioinformatics prediction, we found that 178 proteins were commonly secreted by the P1 and P150 strains, and 49 of them were encoded by mycoplasmal core genes. Additionally, 79 proteins were found to have a different abundance between the P1 and P150 strains. Among these proteins, 34 were more abundant and uniquely expressed in P1, indicating a possible association with the virulence of M. bovis. Three differentially secreted proteins, MbovP0145, MbovP0725, and MbovP0174, as well as one equally secreted protein, MbovP0481, as positive control and one protein of inner membrane, MbovP0310, as negative control were, respectively, cloned, expressed, and evaluated for antigenicity, subcellular location, and the secretion nature with their mouse antisera by western blotting and colony immunoblotting assay. Among them, MbovP0145 was confirmed to be more secreted by P1 than P150 strain, highly reactive with the antisera from naturally infected and P1 experimentally infected cattle but not with the P150 vaccinated calves, indicating its potential as a diagnostic antigen. In conclusion, these findings may represent the most extensive compilation of potentially secreted proteins in mycoplasma species and the largest number of differentially secreted proteins between the virulent and attenuated M. bovis strains to date and provide new insights into M. bovis pathogenesis and diagnosis.

Project description:BACKGROUND:Inexpensive and convenient diagnostic tests for use in clinical work and for the surveillance of infection with Mycoplasma bovis are in demand. The objective of this longitudinal field study was to gain knowledge about the dynamics of antibodies against M. bovis in sera from naturally exposed calves with and without different clinical signs, measured by two different ELISA tests. RESULTS:A total of 83 calves were subject to between one and five blood samples and clinical examinations using a standard protocol during five herd visits to each of four outbreak dairy herds. The blood samples were analysed for the presence of antibodies against M. bovis using the commercial IgG ELISA test BioX K302 (BioX) and an in-house indirect IgG ELISA test (MilA ELISA). Linear mixed models were used to describe and compare the antibody dynamics as measured by the two tests in relation to the disease status and age of the animals. The BioX ELISA response was below the recommended cut-off (37 ODC%) for the entire study period in many of the calves. The estimated mean ODC% increased slowly but did not reach the recommended individual animal cut-off in three of the four herds. The highest estimated ODC% was not reached until the calf was 110-130 days old. The MilA ELISA response rose above the recommended cut-off (135 antibody units (AU)) in almost all calves, and in two herds, the estimated mean was above the individual animal cut-off shortly after the birth of the calf. The highest estimated antibody concentration was reached when the calf was approximately 60 days old. Disease status of the calf was not significantly associated with the results of either test. CONCLUSIONS:We conclude that the BioX ELISA cannot be recommended for use in calves below 3 months of age. The MilA ELISA was able to detect antibodies shortly after birth (i.e. from approximately 3 weeks of age and onwards) and is therefore a more sensitive test for M. bovis exposure in young calves. Neither ELISA seemed able to differentiate between calves with arthritis and/or otitis media, and respiratory disease.

Dataset Information

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

Publications

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

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