A Comparison of Flow Cytometry and Other Techniques Used for Diagnosing Mycoplasma Infections

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Mycoplasma isolates from chickens are usually identified by a fluorescent antibody (FA) technique that requires 7 to 10 days from sampling to the completion of identification. The FA procedure uses Mycoplasma-positive antibodies conjugated with fluoroisothiocyanate (FITC) to identify the colonies of the organism on agar. This paper describes a flow cytometric procedure that uses the same antibody-FITC conjugate and can be completed in 3 or 4 days. Broilers and egg-type hens inoculated with Mycoplasma gallisepticum and M. synoviae were swabbed from the choanal cleft and cultures were grown in broth. Broth aliquots were briefly incubated with polyclonal antisera to M. gallisepticum and M. synoviae conjugated with FITC, the same reagents routinely used in the FA technique. The sample was then analyzed by flow cytometry for the association of the fluorescent label and the organism. Samples from inoculated chickens incubated with the homologous antibody-FITC had greater fluorescent intensity than did those incubated with the heterologous antibody-FITC. Diagnosis by flow cytometry was consistent with that by the FA technique except that in one trial one sample was incorrectly identified by flow cytometry as positive for M. gallisepticum, although it was not inoculated with M. gallisepticum. Also, flow cytometry did not correctly identify M. synoviae organisms as readily when broilers were inoculated with both M. gallisepticum and M. synoviae.