Whittington Richard J, Whittington Ann-Michele, Waldron Anna, Begg Douglas J, de Silva Kumi, Purdie Auriol C, Plain Karren M
Faculty of Veterinary Science, University of Sydney, NSW, Australia.
J Clin Microbiol. 2013 Dec;51(12):3993-4000. doi: 10.1128/JCM.01373-13. Epub 2013 Sep 18.
Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 μl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.
从临床样本（如粪便）中培养禽分枝杆菌副结核亚种的液体培养法，是反刍动物生前诊断副结核病最敏感的检测方法。在澳大利亚、新西兰、美国和其他一些国家，配备改良型Bactec 12B培养基（碧迪公司）的Bactec 460系统一直是最常用的液体培养系统，但该系统已于2012年停产。在本研究中，开发了一种新的液体培养基M7H9C。它由添加了酪蛋白胨、白蛋白、葡萄糖、过氧化氢酶、蛋黄、分枝杆菌素J和抗生素混合物的Middlebrook 7H9培养基基础液组成。我们发现，聚氧乙烯硬脂酸酯（POES）对于在Bactec 12B或M7H9C培养基中培养禽分枝杆菌副结核亚种并非必需。使用禽分枝杆菌副结核亚种C株和S株的纯培养物确定的两种培养基的检测限均为每50 μl接种物7个杆菌。使用来自绵羊和牛的784份粪便和组织样本对新培养基进行了验证，其中超过25%的样本含有活的禽分枝杆菌副结核亚种。两种培养基对临床样本的检测结果存在差异，主要与每克含有少于10个活菌的样本有关，但这些结果相对不常见，且两种培养基的性能没有显著差异。M7H9C培养基的成本不到Bactec 12B培养基的一半，培养期间无需定期检查，但在预定的培养期后，每种培养物都必须进行IS900 PCR确证试验。
