A Filter Immunobinding Technique for the Rapid Detection and Simultaneous Identification of Avian and Bovine Mycoplasmas.

Abstract

ABSTRACT. A filter immunobinding (FIB) method was developed for the detection and identification of mycoplasmas. Type strains of a total of 18 avian and bovine mycoplasma species propagated in broth media were diluted and immobilized on a nitrocellulose membrane as antigens for investigating the specificity with rabbit hyperimmune sera. Non-specific FIB reactions were easily eliminated by the procedure of absorbing rabbit hyperimmune sera in the broth. Absorbed rabbit hyperimmune sera exhibited clear species-specificity with mycoplasma antigens by the FIB. These specific reactions always agreed with the results of identification by tests of biochemical properties and growth inhibition for the isolates of M. bovirhinis, M. bovis, M. columbinum, M. columborale, M. gallisepticum and M. synoviae. Some bovine mycoplasma species, which were impossible to identify by growth inhibition test, because of their strong production of film and spots on the agar, specifically reacted with absorbed rabbit hyperimmune sera against M. bovis in the FIB. The detection limit of mycoplasmas by this method was about 10 4 -10 5 colony-forming units/ml, which is lower than that of colony determination on agar. The FIB seems to be a useful technique for rapid detection and simultaneous identification of mycoplasmas. canadense 275C, and M. verecundum 107. After these mycoplasma strains were cultured for 2-3 days at 37°C in modified Hayflick broth supplemented with 0.02% of β-NADH and either 0.5% glucose or 1% arginine by weight, they were diluted 10-fold in Tris-buffered saline (TBS, pH7.4), and small aliquots of the broth culture of each mycoplasma were kept frozen in vials at 30°C until use as antigens in the FIB test.Rabbit antisera: Rabbit antisera against the mycoplasma strains listed above were obtained by immunizing rabbits with concentrated mycoplasma antigens which were harvested by centrifugation of a large volume of the broth culture of these mycoplasma strains and by washing the precipitates 3 times in phosphate buffered saline (PBS, pH7.2). Rabbit antisera were diluted with the blocking solution and used as unabsorbed antisera. Absorbed antisera were prepared by adding nine volumes of the broth to 1 volume of each rabbit antiserum, allowing the solution to stand at room temperature for 2 hr, and then leaving overnight at 4°C. This was followed by centrifugation ofThe most reliable diagnosis of Mycoplasmal infection is the isolation and identification of the pathogen from the disease. As various species of non-pathogenic mycoplasmas are frequently cohabitants and grow together with pathogenic organisms on the primary culture media at isolation, the isolates subsequently need to be identified. Methods such as the test of biochemical properties and immunological techniques including growth inhibition [1,3], metabolism inhibition [9], immunofluorescence [4,5,11] and immunoperoxidase [6,10], require time to make a definitive identification, because mycoplasmas take a few days to grow in a broth or on an agar. Thus, a rapid diagn...

Detection of Mycoplasma agassizii in the Texas Tortoise (Gopherus berlandieri)

“…Polyclonal rabbit antiserum to M. agassizii PS6 was commercially prepared (Pel-Freez Biologicals, Rogers, Arkansas). An immunobinding assay (Kotani and McGarrity, 1985;Brown et al, 1990;Takahata et al, 1997;Poumarat, 1998) was performed as previously described (Brown et al, 1990).…”