Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes

A genomic library of Mycoplasma synoviae (MS) was generated by using bacter iophage lambda gt11 as a cloning and expression vector. Identification of r ecombinant clones highly specific to MS was achieved by screening the libra ry for expression of MS proteins with polyclonal antiserum that had been pr eadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis of the total cell lysates and imm unoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digox igenin, and used as probes for the specific recognition of several MS strai ns. A high degree of conservation was demonstrated for the MS2/12 and MS2/2 8 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. ga llinarum, M. iners. M. anatis, and M. iowae), thus indicating their high sp ecificity to MS. The sensitivity of the slot blot hybridization method usin g digoxigenin-labeled MS2/12 and MS2/28 probes fur direct detection of MS f rom broth cultures of field isolates was 10(5) colony-forming units/ml. The se results demonstrate the effectiveness of adsorbed antisera for the isola tion of species-specific mycoplasma DNA and the potential for its use as pr obes for the specific and direct detection of MS from broth cultures of fie ld isolates.