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Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA
Abstract:
Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. A number of serological assays have been developed for diagnosis of M. synoviae infection; however, they lack sensitivity and/or are prone to false-positive reactions. Using a combination of PCR and expression cloning, four overlapping regions (regions 1-4) of the surface antigen MSPB of M. synoviae WVU-1853 were expressed in a bacterial expression system. Immunostaining of the resultant polypeptides with chicken sera raised against different M. synoviae strains, or Mycoplasma gallisepticum S6, suggested that region 4 contained a highly antigenic and species-specific domain (amino acids 212-317) [corrected] of MSPB. A fusion protein of region 4 was expressed in the pMAL expression system and purified from cold-osmotic-shock fluids of Escherichia coli cells for use in an indirect ELISA. The potential of the purified antigen for detection of M. synoviae antibodies was assessed with sera obtained from chickens experimentally infected with different strains of M. synoviae or M. gallisepticum, or from uninoculated chickens. All the sera from M. synoviae-inoculated chickens provided higher absorbance values than those from M. gallisepticum-inoculated or uninoculated chickens. Chickens inoculated with M. synoviae 86079/7NS had detectable increases of serum anti-MSPB immunoglobulins at day 7 after inoculation. These studies have identified the most antigenic region of one of the major species-specific surface proteins of M. synoviae, and shown the potential of this protein as a serodiagnostic reagent.
Insights
Researchers identified a specific region of the Mycoplasma synoviae surface protein MSPB that is highly antigenic and species-specific. This finding shows potential for developing improved diagnostic tests for Mycoplasma synoviae infections in poultry.
Area of Science:
- Veterinary Microbiology
- Immunology
- Molecular Biology
Background:
- Mycoplasma synoviae causes significant respiratory disease and synovitis in poultry.
- Current diagnostic assays for M. synoviae infection often lack sensitivity or yield false positives.
- There is a need for more accurate and reliable diagnostic tools for M. synoviae.
Purpose of the Study:
- To identify and characterize antigenic regions of the M. synoviae surface protein MSPB.
- To develop a potential serodiagnostic reagent for M. synoviae detection.
Main Methods:
- Expressed four overlapping regions of the MSPB surface antigen using PCR and bacterial expression cloning.
- Used immunostaining with chicken sera to identify antigenic and species-specific domains.
- Purified a fusion protein of the identified region (region 4) for use in an indirect ELISA.
- Assessed the diagnostic potential of the purified antigen using sera from experimentally infected chickens.
Main Results:
- Region 4 (amino acids 212-317) of MSPB was identified as a highly antigenic and species-specific domain.
- A fusion protein of region 4 showed potential as a serodiagnostic reagent.
- ELISA using the purified antigen detected M. synoviae antibodies in experimentally infected chickens, with higher absorbance values compared to M. gallisepticum-infected or uninoculated controls.
- Antibody increases were detectable as early as 7 days post-inoculation in chickens infected with a specific M. synoviae strain.
Conclusions:
- The study successfully identified the most antigenic, species-specific region of the M. synoviae MSPB protein.
- The purified antigen derived from this region demonstrates significant potential for use in developing improved serodiagnostic assays for M. synoviae infections.
- This research contributes to better diagnostic strategies for controlling M. synoviae in poultry populations.