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Updated: Aug 15, 2025
Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae
Published on: February 24, 2023
NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a
Zengjin Hu1,2, Haoran Li1,2, Yuxin Zhao1,3
1Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai, 200241, People's Republic of China.
Background:
Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX's potential as a diagnostic antigen and its role in MS cytoadherence.
Results:
Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin.
Conclusion:
MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.
Insights
Mycoplasma synoviae NADH oxidase (MSNOX) is a surface protein that can be used as a diagnostic antigen and may play a role in bacterial adherence and pathogenesis. Antibodies against MSNOX show potential for diagnosing and treating Mycoplasma synoviae infections.
Area of Science:
- Veterinary Microbiology
- Avian Pathology
- Molecular Pathogenesis
Background:
- Mycoplasma synoviae (MS) causes significant economic losses in poultry due to respiratory and arthritic diseases.
- Membrane-associated proteins are crucial for pathogen cytoadherence and pathogenesis.
- NADH oxidase (NOX) is a potential virulence factor, but its role in MS (MSNOX) is largely unknown.
Purpose of the Study:
- To investigate the potential of MSNOX as a diagnostic antigen.
- To explore the role of MSNOX in Mycoplasma synoviae cytoadherence and pathogenesis.
Main Methods:
- Recombinant MSNOX (rMSNOX) production and characterization.
- Western blot and ELISA assays using MS-positive sera.
- Immunofluorescence, immunogold electron microscopy, and adherence assays.
- Plasminogen (Plg) and fibronectin (Fn) binding assays.
Main Results:
- rMSNOX showed high specificity and immunoreactivity with MS isolates, indicating its potential as a diagnostic antigen.
- MSNOX is localized on the MS membrane and contributes to bacterial adherence to host cells (DF-1).
- rMSNOX binds to plasminogen and fibronectin, suggesting a role as an adhesin.
- Anti-rMSNOX serum demonstrated complement-dependent mycoplasmacidal activity.
Conclusions:
- MSNOX is a surface-exposed, immunogenic protein suitable for Mycoplasma synoviae diagnostics.
- MSNOX plays a significant role in MS cytoadherence and potentially pathogenesis.
- MSNOX is a promising candidate for developing diagnostic tools and potentially a protective vaccine against MS.
