Parker Alysia M, House John K, Hazelton Mark S, Bosward Katrina L, Sheehy Paul A
The University of Sydney, Sydney School of Veterinary Science, Faculty of Science, Camden, New South Wales Australia.
PLoS One. 2017 Mar 6;12(3):e0173422. doi: 10.1371/journal.pone.0173422. eCollection 2017.
Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.
支原体属是奶牛乳腺炎、关节炎和肺炎的主要病因，并且与奶牛的生殖障碍有关。虽然培养是传统的鉴定方法，但聚合酶链反应（PCR）的应用已变得更为普遍。一些研究人员已开发出用于检测牛奶中牛支原体的PCR方案，但很少有研究评估其他样本类型或其他重要的支原体物种。因此，本研究的目的是开发一种多重PCR检测方法，以检测牛支原体、加利福尼亚支原体和牛生殖支原体，并针对牛奶、精液和拭子样本的传统培养方法评估其分析性能。测定了PCR的特异性，并评估了加标牛奶、精液和拭子中的检测限。然后将该PCR方法与来自个体牛奶、储奶罐牛奶（BTM）、精液和拭子（阴道、包皮、鼻和眼）的474份现场样本的培养结果进行比较。特异性分析对所有牛支原体、加利福尼亚支原体和牛生殖支原体分离株均产生了适当的扩增。其他任何柔膜菌纲或真细菌分离株均未出现扩增。PCR的检测限在牛奶中最佳，其次是精液和拭子。当样本中同时存在所有三种支原体物种时，检测限会增加。在比较培养和PCR时，总体上培养和PCR阳性样本的比例没有显著差异。培养能够检测出明显更多的阳性拭子样本。精液、个体牛奶或BTM样本未发现显著差异。PCR鉴定出5个含有两种物种的样本。培养后进行16S - 23S rRNA测序无法鉴定出不止一种物种。因此，鉴定牛支原体、加利福尼亚支原体和牛生殖支原体的最佳方法可能取决于所分析的样本类型，以及是否需要鉴定多种目标物种。
