Istituto Zooprofilattico Sperimentale delle Venezie, Sezione di Verona, Via San Giacomo 5, 37135 Verona, Italy.
J Microbiol Methods. 2011 Mar;84(3):413-7. doi: 10.1016/j.mimet.2011.01.019. Epub 2011 Jan 21.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10(4)-10(-1)) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10(2) and 10(3)MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6weeks by real-time PCR.
分支杆菌副结核亚种（MAP）是副结核病的病原体，这是一种反刍动物的慢性肉芽肿性肠道疾病。通过粪便培养检测 MAP 可提供感染的明确诊断。与固体培养基相比，用于 MAP 培养的自动化液体系统更灵敏、更快速，但它们昂贵且需要专门的设备。在这项研究中，使用改良的 Middlebrook 7H9 液体培养基（7H9+）的非自动化培养方法与 Herrold 的固体培养基（HEYM）和直接实时 PCR 进行了比较，用于奶牛粪便。通过实时 PCR 每周监测 7H9+中的 MAP 生长，直到接种后第 12 周。使用从健康奶牛粪便中提取的粪便样本，用 MAP 生物的十倍稀释液（10(4)-10(-1))和连续稀释 1 至 10 的天然 MAP 感染粪便对三种方法的分析灵敏度进行了评估。固体培养和直接实时 PCR 的检测限分别为 10(2)和 10(3)MAP/g。相比之下，7H9+培养物的检测下限低至 1MAP/g。与 HEYM 培养相比，检测病原体的时间明显缩短。此外，这三种方法还应用于从高流行率和低流行率牛群中采集的环境粪便样本。在低流行率牛群中，7H9+培养显示出最高的敏感性，并提供了比 HEYM 更快的结果。在高流行率牛群中，三种方法的敏感性相同，实时 PCR 的周转时间最短。总之，使用 7H9+从牛粪便中检测 MAP 可最大限度地提高诊断灵敏度，缩短周转时间，因此可以替代固体培养基培养。因此，我们提出了一种基于以下两种方法评估 MAP 粪便排泄的两步方案：1）对所有样本进行直接实时 PCR；2）将阴性样本接种到 7H9+中，并在第 3 周和必要时在第 6 周进行实时 PCR 分析。
