This study identifies several novel susceptibility genes for MMD. The association with homocysteine metabolism and the immune system enrichment of susceptibility gene expression suggest that therapeutic interventions targeting these pathways may be effective approaches for MMD treatment.

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Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX–expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX– for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca2+-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca2+ at 22–65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNaseΔ181–342 without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.

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.