IRTA-St. Carles de la Ràpita, Ctra. Poblenou km 5, 43540 Tarragona, Spain.
Laboratory for Fish and Shellfish Diseases, Wageningen Bioveterinary Research, P.O. Box 65, 8200 AB Lelystad, The Netherlands.
J Invertebr Pathol. 2017 Oct;149:51-55. doi: 10.1016/j.jip.2017.07.004. Epub 2017 Jul 19.
Species belonging to the genus Marteilia are protozoan parasites of bivalves. The species Marteilia refringens, jeopardizing the health of European bivalves, is included on the list of OIE notifiable pathogens. Two genotypes of Marteilia refringens are distinguished: type "O" affecting mainly oysters, and type "M" affecting mainly mussels. Historically, detection of Marteilia species is primarily carried out by histology. In recent years molecular assays are more frequently used for the detection of mollusc pathogens, also in routine monitoring. In the present work, a competitive real-time PCR assay was developed for rapid and sensitive detection of M. refringens and discrimination between "M" and "O" genotypes of M. refringens. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability and efficiency. Subsequent application of the assay on collected bivalves from two geographical locations, the Ebro Delta in Mediterranean Spain and the Rhine-Meuse Delta in the Netherlands resulted in detection of M. refringens type M in Mytilus galloprovincialis and M. refringens type O in Ostrea edulis from Spain. In two O. edulis specimen both M. refringens type O and type M were detected. In the Netherlands M. refringens was not observed in any of the tested Mytilus edulis and O. edulis. The results obtained by real time PCR were in correspondence with the results obtained by histopathology and a substantial agreement with the results obtained by conventional PCR. In conclusion, the developed real time PCR assay facilitates rapid detection and subtyping of M. refringens and could be applied for further studies on epidemiology of the parasite, geographical distribution and host specificity.
属于 Marteilia 属的物种是双壳类动物的原生动物寄生虫。威胁到欧洲双壳类动物健康的 Marteilia refringens 物种被列入 OIE 应报告病原体名单。区分出 Marteilia refringens 的两个基因型：主要影响牡蛎的“O”型和主要影响贻贝的“M”型。历史上，Marteilia 物种的检测主要通过组织学进行。近年来，分子检测方法越来越多地用于检测贝类病原体，也用于常规监测。在本工作中，开发了一种竞争性实时 PCR 检测方法，用于快速、灵敏地检测 M. refringens 并区分 M. refringens 的“M”和“O”基因型。实时 PCR 检测方法具有分析灵敏度和特异性，重复性和效率高。随后将该检测方法应用于两个地理位置（地中海西班牙的埃布罗三角洲和荷兰的莱茵-默兹三角洲）采集的双壳类动物，在西班牙的 Mytilus galloprovincialis 和 Ostrea edulis 中检测到 M. refringens 型 M，在西班牙的 Ostrea edulis 中检测到 M. refringens 型 O。在两个 O. edulis 标本中均检测到 M. refringens 型 O 和 M 型。在荷兰，在测试的 Mytilus edulis 和 O. edulis 中均未观察到 M. refringens。实时 PCR 获得的结果与组织病理学结果相对应，与传统 PCR 获得的结果具有实质性一致性。总之，开发的实时 PCR 检测方法有助于快速检测和 M. refringens 的亚型分类，可用于进一步研究寄生虫的流行病学、地理分布和宿主特异性。
