Diagnosis of mycoplasma mastitis: Validation and development

Microbiological culture of milk samples has been used as a standard diagnosis for mycoplasma mastitis. It is suggested to perform with fresh samples for optimum diagnosis. Submission of fresh samples is often difficult given the logistics of collection and shipping of milk samples from farm to laboratory. Therefore, milk samples are usually chilled or frozen before culture. A study of the effects of storage methods on the recovery of Mycoplasma species from milk samples was performed. The results indicated that holding milk at refrigerated temperatures (5°C) for 5 days and freezing milk samples (-20°C) lowers the number of recovered Mycoplasma species. Moreover the addition of glycerol prior freezing to achieve 10% and 30% (v/v) solutions was found to improve the recovery of Mycoplasma species from frozen milk samples.;Even though a Mycoplasma-like-colony is observed by a standard culture method, the diagnosis can be misinterpreted as Acholeplasma species which is indistinguishable by culture. To validate and suggest techniques used to discriminate between these two genera, a study on the discrimination between Mycoplasma and Acholeplasma using digitonin and nisin disc diffusion assays, and PCR was performed. Findings revealed a high and comparable efficiency of using nisin and digitonin disc diffusion assays and PCR to distinguish Mycoplasma and Acholeplasma species.;Given the fastidious nature of Mycoplasma species, and the time-consuming nature of the standard culture method, a diagnostic test of mycoplasma mastitis that is more sensitive, less time consuming, and can speciate mycoplasma mastitis pathogens would be valuable. The development of real-time PCR assays to detect 3 major mycoplasma mastitis pathogens: M. bovis, M. californicum and M. bovigenitalium was investigated to provide an alternative diagnostic tool for mycoplasma mastitis. The results given by the novel real-time PCR assays showed a perfect agreement with the gold standard, and the results were obtained within 4-5 hours.;The overall goal of studies reported herein was to investigate improvements in the efficiency of the diagnosis of mycoplasma mastitis in dairy cows. Findings indicate that both phenotypic and genotypic diagnostic techniques can be applied, and in conjunction with current standard techniques, will better identify cows with mycoplasma mastitis.