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Mycoplasma bovis, the main causative agent of mycoplasmal mastitis, arthritis and pneumonia in cattle, causes considerable economic losses. Veterinary hygiene measures would be most effective if introduced at an early stage, especially the culling of cows shedding the pathogen for the control of mastitis. It is therefore crucial to ensure that diagnostic methods are available which can perform rapid and specific detection of the agent at acceptable costs.Six different detection methods have been compared and evaluated in terms of performance parameters and suitability for routine diagnosis. Conventional M. bovis isolation and identification from culture is the only technique used for routine diagnosis at present. However, this process is rather laborious and timeconsuming, and final results are available only after several days. Enzymelinked immunosorbent assay (ELISA) techniques can be used to screen for M. bovis antibodies or antigens in clinically-diseased animals. Detection of the agent in subclinical cases was accomplished in pre-incubated samples by an antigen capture ELISA involving a monoclonal antibody.Whole-cell protein patterns generated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to identify and classify field isolates. Nucleic acid hybridisations using probes of defined specificity were conducted both as filter dot blot assay and in solution with ribosomal ribonucleic acid as the target. The latter was found to be potentially suitable for the screening of biological samples, although problems due to high background and reduced specificity remained. Finally, the presence of M. bovis cells in culture supernatant and in milk samples was demonstrated using the polymerase chain reaction. This procedure is potentially superior to all others currently available, due to its high sensitivity, specificity and speed. However, a number of practical problems must be solved prior to full-scale introduction of this technique for routine diagnosis.

Two methods are suggested that allow direct detection of Mycoplasma bovis from clinical samples, i.e. milk and nasal swabs, respectively. Milk samples were trypsinized in the presence of Triton X-100 and passed through a DNA-binding filter membrane, from which DNA was extracted and subjected to PCR. The detection limit was 500 cfu ml-1 on agarose gels and 50 cfu ml-1 after Southern hybridization so that the method can be used to monitor low-titre samples from animals at the subclinical stage. Results became available within 24 h, thus rendering the procedure more rapid than ELISA and culture techniques. Six other methods designed for milk or other complex samples were tested in this study, but found unsatisfactory. Rapid and specific detection of the pathogen by PCR from nasal mucus treated with lysis buffer and proteinase K was demonstrated for swabs taken from experimentally infected calves. Both methods represent useful tools for effective livestock monitoring and single-animal diagnosis.

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