Control of mycoplasmal mastitis requires individual cow milk sampling for culture and identification of Mycoplasma bovis. This sampling is time-consuming and expensive. Currently, some herds sample cows monthly with the dairy herd improvement (DHI) program, but a preservative is added to this milk that kills M. bovis. In this paper, a nested polymerase chain reaction (PCR) procedure that allows for rapid testing of preservative-treated milk is validated. The specificity of the nested PCR assay was confirmed by testing isolated nucleic acids of other organisms phylogenetically related to M. bovis or common to milk. A comparison against blind-passage culture on 53 field milk samples determined its sensitivity. Exposure of seeded milk samples to the procedure resulted in a sensitivity of 5.1 cfu equivalents per milliliter. Analysis of these results proved that the nested PCR assay was as sensitive as traditional culture and can be used on preservative-treated milk.

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A nested PCR method was developed and validated for detection of Mycoplasma bovis in milk samples preserved with bronopol. A previously described, randomly cloned DNA fragment was sequenced. This sequence information was used to develop a set of internal primers for a nested PCR assay. Cationic surfactant purification was used to eliminate milk protein substances that inhibit amplification. Serial diluted seeded milk samples were used to determine a sensitivity of 5.1 CFU equivalents/ml of milk. Specificity of the assay was confirmed testing against 7 other mycoplasma species and 11 other mastitis organisms. A comparison of culture, with blind passage, to the nested PCR reaction was performed on clinical field milk samples from M. bovis affected and M. bovis free herds. The nested PCR method was more sensitive than culture (~5 CFU/ml), specific for M. bovis, effective with preserved milk, and could be completed in less than one day. This may provide a practical, rapid, cost effective procedure to screen for clinical and subclinical M. bovis carriers using routinely obtained preservative-treated milk samples.