Specific immunogenic proteins of Mycoplasma synoviae (MS) strain WVU-1853 were purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western transfer using anti-MS, anti-M. gallisepticum, and non-immune chicken sera. A cluster of prominent immunoreactive proteins with molecular masses from 46 to 52 kDa (p46-52) and less reactive single proteins of approximately 22 and 92 kDa were shown to partition into the detergent phase of Triton X-114 MS lysates, suggesting that these amphiphilic polypeptides are integral membrane proteins. Monoclonal antibodies, produced by immunizing mice with MS whole cell proteins and shown to bind species-specific determinants, reacted strongly with p46-52 and less intensely with the 22-kDa protein, but they did not react with the 92-kDa protein. A protein fraction extracted from the Triton X-114 detergent phase and further purified by ion-exchange chromatography was found to be highly enriched in p46-52 and was used as antigen in a prototype enzyme-linked immunosorbent assay (ELISA) to detect MS antibodies. Seventy serum samples were taken variously from specific-pathogen-free chickens experimentally infected with MS or MG and from commercial broiler breeder chickens vaccinated for MS and MG. All samples were tested by both rapid serum agglutination and a prototype ELISA described herein; the two tests were strongly correlated (r = 0.776). The results indicate that the ELISA antigen used--the immunodominant cell surface proteins in the p46-52 cluster--appears to be a good candidate for use in the development of improved rapid diagnostics of MS infections in birds.

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~ ~~~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.lmmunostaining 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 178-213) 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. synoviaeinoculated chickens provided higher absorbance values than those from M. gallisepticum-inoculated or uninoculated chickens. Chickens inoculated with M. synoviae 86079nNS had detectable increases of serum anti-MSPB immunoglobulins a t 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.