Validation of detection and diagnostic methods for bovine mycoplasma species

Jessica Lowe

Mycoplasma mastitis is often persistent, difficult to cure, and challenging to diagnose. Bacterial culture remains the "gold standard" for mycoplasma identification, but variability regarding carbon dioxide concentrations and incubation periods suitable for growth and identification of isolates promotes under-diagnosis of mycoplasma mastitis. Polymerase chain reaction (PCR) techniques may provide a more sensitive and specific diagnostic tool, but commercial PCR methods are up to 3.5 times the cost of culture techniques. Herein, two studies are presented to address the issues associated with both methods. The first study aimed to determine if mycoplasma mastitis pathogen enumerations differed when incubated under 10% CO2, 5% CO2, or in candle jars (2.7 ± 0.2% CO2) for varying incubation lengths (3, 5, or 7 d). Enumerations of laboratory isolates representing the 3 common mycoplasma mastitis species, M. bovis, M. californicum, and M. bovigenitalium, were not significantly different among CO2 treatments, but were greater after 7 d of incubation compared to 3 d of incubation (all P < 0.05). The results were validated against field isolates derived from clinical mycoplasma mastitis bulk tank milk and quarter milk samples. The percentage of field isolates detected was not significantly affected by CO2 treatment. On average, 57.14% of all field isolates were detected by 3 d of incubation compared to 92.86% on d 7. For dairy producers and diagnostic laboratories, allowing 7 d for incubation increases the sensitivity of the test resulting in fewer false negatives and missed cows with mycoplasma infections. The objective of the second study was to validate a commercial PCR kit (MycoDect, Alstem, LLC, Richmond, CA) for identification of M. bovis in milk. Assay validation included analysis of 40 M. bovis and 40 other Mollicutes isolates. Analysis of the data set resulted in 95% sensitivity and 100% specificity of the assay. The results of this study indicate that the commercially-available conventional PCR assay used in this study can detect M. bovis in milk samples in approximately 4 h at a cost of $8.06 per sample, an improvement from 10 d incubations for M. bovis culture detection and the costs of other PCR detection platforms.

Validation of detection and diagnostic methods for bovine mycoplasma species

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