Abstract
Research was carried out to improve the diagnosis of mycoplasma infections, with special reference to Mycoplasma bovis. The primary isolation procedures were reviewed and a range of antibiotics was tested to be included in E medium. The drugs selected were Carbenicillin, Vancomycin and Fosfomycin. Isolation of Mycoplasmatales from the upper respiratory tract of apparently healthy calves was done by means of nasal swabs from animals in 7 farms situated in Rio de Janeiro State, Brazil. Mycoplasma bovis was isolated from 4.9% of animals while Acholeplasma laidlawii represented 9.0% of the isolates. These findings proved that Mycoplasma bovis is present in the southeast region of Brazil. An Enzyme-Linked Immunosorbent Assay (ELISA) using antigen-coated microtitre plates and horseradish peroxidase-labelled antibovine IgG was developed for detection of Mycoplasma bovis antibodies in bovine serum. The results obtained from 1,300 bovine field sera showed that the indirect ELISA was more sensitive and more specific than Complement Fixation (CF) and Disc Film Inhibition (DFI) tests for serodiagnosis of Mycoplasma bovis infections, the results correlating better with DFITest. The same indirect ELISA was adapted for visual reading and the results obtained from 546 bovine field sera collected in Brazil were compared with those obtained spectrophotometrically. There was a very good correlation between the two methods of reading, proving that' the ELISA can be used in the field as a screening test without the need of sophisticated equipment. Again the presence of Mycoplasma bovis in the southeast region of Brazil was proved by the detection of positive samples tested by both methods. An indirect and a sandwich ELISA were adapted for the identification of Mycoplasmatales isolated from field materials by means of antigen detection in E and Moredun broth. Hyperimmune sera raised in chicken and rabbits against Mycoplasmatales, as well as positive bovine sera for Mycoplasma bovis, were used to perform the tests. The best results were found with bovine sera although the tests did not show high specificity. Attempts were made to treat antigen in different ways without great improvements in the results. The ELISA did not appear to be quicker, easier or more specific than DFIT for Mycoplasma bovis identification. The indirect ELISA was employed for Mycoplasma bovis antibody detection in field milk samples. Although promising results were found, the sensitivity and specificity of the immunosorbent assay could not be assessed fully because of the lack of good positive and negative reference samples. However, it seemed likely that this ELISA will be of great value if milk samples collected from cows suffering from clinical mastitis were available for testing.