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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.
Highlights
Mycoplasma bovis (M. bovis) belongs to Mollicutes, a class of simple self-replicating bacteria characterized by very small genomes (580 to 2200 kb) and the lack of a cell wall
In an indirect ELISA (iELISA) based on the M. bovis whole cell proteins (WCPs), 40% (8/20) of experimentally infected calves 14 days postinfection and all infected calves 21 dpi were positive for antibody responses, whereas none of the vaccinated www.impactjournals.com/oncotarget
Mycoplasma bovis infection is a major threat to the worldwide cattle industry
Summary
Mycoplasma bovis (M. bovis) belongs to Mollicutes, a class of simple self-replicating bacteria characterized by very small genomes (580 to 2200 kb) and the lack of a cell wall. Highly conserved, sensitive, and specific antigenic membrane molecules or MAPs may be the most promising targets for establishing novel serodiagnostics Proteomics approaches, such as dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS), have increasingly been used in immunoproteomics studies of several mycoplasma species [20, 24, 25, 26, 27]. Immunoinformatics, a combination of immunology and informatics, has helped elucidate the networks involved in defense system activation [29] Such methods have been used to successfully identify antigenic epitopes in several pathogens, such as Mycobacterium tuberculosis and Neisseria meningitidis [30, 31], and to select potential candidates for serodiagnostics and vaccine development [32, 33]. An indirect ELISA (iELISA) based on recombinant MbovP579 (rMbovP579) was able to effectively detect the M. bovis-specific IgG in animals with acute or chronic infections
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