Université de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants Anses VetAgro Sup, F-69280, Marcy l'Etoile, France.
J Microbiol Methods. 2012 Aug;90(2):73-9. doi: 10.1016/j.mimet.2012.04.020. Epub 2012 May 3.
Contagious agalactia is an important disease worldwide that affects small ruminants. Clinical manifestations vary from mastitis, pneumonia, arthritis and keratoconjunctivitis to septicemia. Four mycoplasmal etiological agents have been identified: Mycoplasma (M.) agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. The current procedure for direct diagnosis, recommended by the World Organization for Animal Health, involves the isolation of one or several causative agents from clinical specimens and further time-consuming identification steps. The present study reports the development of a new multiplex real-time PCR (including an internal positive control) that detects all four pathogens simultaneously and distinguishes M. agalactiae from the others. First, intra- and inter-species polymorphisms of the two target house-keeping genes, namely polC and fusA, were analyzed to design primers and probes adapted to the diversity of currently circulating strains. The specificity and the sensitivity of the assay were then challenged and the limit of detection was found to be as low as 6 to 12 copies of the target genes. The assay requires further assessment on clinical specimens but its performances (notably low intra- and inter-assay variability) are already very promising for use in large-scale diagnosis and prophylactic surveys of contagious agalactia.
传染性乳腺炎是一种全球性的重要疾病，影响小反刍动物。临床表现从乳腺炎、肺炎、关节炎和角膜炎到败血症不等。已经确定了四种支原体病因：无乳支原体、山羊支原体亚种、山羊支原体亚种和腐败支原体。目前世界动物卫生组织推荐的直接诊断程序包括从临床标本中分离一种或多种病原体，以及进一步耗时的鉴定步骤。本研究报告了一种新的多重实时 PCR（包括内部阳性对照）的开发，该方法可同时检测所有四种病原体，并将无乳支原体与其他病原体区分开来。首先，分析了两个靶管家基因 polC 和 fusA 的种内和种间多态性，以设计适应当前流行株多样性的引物和探针。然后挑战了该检测方法的特异性和敏感性，发现检测限低至靶基因的 6 到 12 个拷贝。该检测方法需要进一步在临床标本上进行评估，但它的性能（尤其是低的内和间检测变异性）已经非常有希望用于大规模的传染性乳腺炎诊断和预防性调查。
