Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.

Among 9 organic acids commonly used as food additives in Japan, fumaric acid (FA) exhibited the strongest growth-inhibiting (GI) action on 20 Gram-negative bacterial strains tested. Growth of these 20 strains, including 3 Salmonella serovars, Escherichia coli, E. cloacae, Klebsiella oxytoca, K. pneumoniae, Acinetobacter anitratus, Proteus mirabilis, or Pseudomonas aeruginosa, was inhibited within 160 seconds of contact with 0.3 w/v% solution of FA, although strains of S. Typhimurium exhibited lower susceptibility than the others. Both Bacillus cereus and Staphylococcus aureus were resistant to the GI action of FA and other organic acids. The GI action of FA was remarkably improved by the addition of 0.3-0.1% ascorbic acid, malic acid, tartaric acid or citric acid monohydrate.

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...

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Application of chicken immune sera to identification of Mycoplasma species by the filter immunobinding (FIB) method was investigated. Immune sera against type strains of 15 Mycoplasma species were prepared in chickens by inoculation with concentrated Mycoplasma organisms. Chicken immune sera, when absorbed in a broth medium and diluted in a blocking solution, showed a definite specificity with homologous Mycoplasma antigens by FIB. The identification results of field isolates of Mycoplasma gallisepticum and M. synoviae by FIB using absorbed chicken immune sera were consistent with those obtained by conventional tests based on biochemical properties and growth inhibition using rabbit immune sera. Chicken immune sera were evidenced to be useful for identification of Mycoplasma species when applied to FIB although they were not applicable in growth and metabolism inhibition tests.