Faculty of Veterinary Science, The University of Sydney, 425 Werombi Road, Camden, New South Wales 2570, Australia.
Vet Microbiol. 2011 Feb 24;148(1):35-44. doi: 10.1016/j.vetmic.2010.07.022. Epub 2010 Aug 5.
A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.
一项纵向研究评估了先前开发的直接粪便实时定量 PCR（QPCR）检测方法（Kawaji 等人，2007 年）对感染分枝杆菌副结核亚种（MAP）的绵羊的诊断潜力。在 58 只绵羊中，38 只经口接种 MAP，20 只对照动物在整个试验期间与感染组分开饲养。所有动物在接种前和接种后 4、8 和 13 个月时通过 QPCR、粪便培养和血清 ELISA 进行检测，并在接种后 13 个月进行剖检。在接种后 4 个月时，通过 QPCR 检测到 38 只接种绵羊中有 18 只在粪便中排出 MAP，而此时仅一只绵羊的粪便培养呈阳性。在接种后 8 个月时，通过 QPCR 检测到所有接种绵羊的粪便中均存在 MAP DNA，但通过粪便培养仅从 34%的接种动物中分离出 MAP 生物体。剖检时采集的粪便样本的 QPCR 结果表明，粪便 QPCR 比肠道组织培养更敏感。该 QPCR 检测方法被确认为一种敏感且特异的绵羊 MAP 生前诊断检测方法，规避了粪便培养，后者是一种敏感性较低且耗时较长的检测方法。通过 QPCR 定量粪便中的 MAP DNA 可能提供即时信息，用于估计感染阶段以及从感染动物传播的风险。
