Immunohistochemical detection of mycoplasma gallisepticum antigens in turkey respiratory tissues

An avidin-biotin-immunoperoxidase diagnostic test was developed to facilita te rapid identification of Mycoplasma gallisepticum in respiratory tissues of turkeys. This procedure used polyclonal primary antibodies produced in r abbits. Turkeys were inoculated into the infraorbital sinus and trachea wit h the R strain of M. gallisepticum, Mycoplasma synoviae, Mycoplasma meleagr idis, or Frey's media. The outer walls of the infraorbital sinuses, lungs, and tracheas were collected and fixed in either 10% neutral formalin or pen tanedial methyl glycol at 1, 2, 3, and 4 wk postinoculation. Tissues were s ubdivided and remained in each fixative for 6 or 24 hr. The avidin-biotin-immunoperoxidase diagnostic test was sufficiently sensiti ve to detect M. gallisepticum antigen at 1, 2, 3, and 4 wk postinoculation. Staining of M. gallisepticum was significantly more intense on infraorbita l sinus epithelium than on respiratory epithelium from the trachea or lung. Statistical analysis indicated that the 6-hr fixation time offered better antigen preservation than 24 hr in a fixative. There was no difference in i ntensity of M. gallisepticum antigen staining in tissues fixed in methyl pe ntanedial glycol when compared with tissues fixed in 10% neutral buffered f ormalin. Significant differences in staining intensity were observed betwee n weeks. Specificity of the avidin-biotin-immunoperoxidase test was not complete. No ne of the tissues from the M. meleagridis and control groups showed stainin g. No staining was observed in the ciliated brush border of infraorbital si nus epithelial cells from turkeys infected with M. synoviae. However, weak to moderate staining was observed in several tracheas of turkeys inoculated with M synoviae. Improved specificity of an avidin-biotin-immunoperoxidase diagnostic test to detect M. gallisepticum in respiratory tissues of turke ys probably will require the use of multiple monoclonal antibodies directed against several different epitopes specific to the cell membrane of M. gal lisepticum.