Itoïz Sarah, Perennou Morgan, Mouronvalle Clara, Derelle Evelyne, Le Goïc Nelly, Bidault Adeline, de Montaudouin Xavier, Arzul Isabelle, Soudant Philippe, Chambouvet Aurélie
Univ Brest, CNRS, IRD, Ifremer, LEMAR, F-29280 Plouzané, France.
Univ Brest, CNRS, IRD, Ifremer, LEMAR, F-29280 Plouzané, France; EPHE, PSL Research University, UPVD, CNRS, USR 3278 CRIOBE, Perpignan F-66360, France.
J Invertebr Pathol. 2021 Sep;184:107603. doi: 10.1016/j.jip.2021.107603. Epub 2021 May 7.
The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray's Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.
致病因子奥尔森派琴虫（Perkinsus olseni）因其在亚洲、欧洲、澳大利亚和南美洲广泛的地理分布，被全球公认为贝类生产的主要威胁。另一个物种切萨皮克派琴虫（Perkinsus chesapeaki），此前从未被发现与重大死亡事件有关，最近在法国海岸被检测到，它与奥尔森派琴虫一起偶尔感染蛤蚌种群。识别影响菲律宾蛤仔（Ruditapes philippinarum）的潜在隐性感染对于制定适当的宿主资源管理策略至关重要。在此，我们开发了一种基于双重实时定量PCR的分子方法，用于同时检测这两种寄生虫——奥尔森派琴虫和切萨皮克派琴虫——在蛤蚌的不同组织中：鳃、消化腺、足部、外套膜、闭壳肌和软体动物的其余部分。我们首先检查了宿主组织样本中可能存在的PCR抑制剂。qPCR反应受到抑制，这取决于宿主器官的性质。外套膜和软体动物的其余部分在宿主gDNA浓度阈值为2 ng.µL时具有很高的抑制作用，闭壳肌和足部在5 ng.µL时具有中等抑制作用，而鳃和消化腺即使在宿主gDNA最高浓度为20 ng.µL时也未显示对qPCR反应有任何抑制作用。然后，以鳃为模板，与世界动物卫生组织推荐的雷氏硫乙醇酸盐流体培养基方法相比，检查了该分子技术的适用性。双重qPCR方法带来了新的见解，并揭示了原位组织样本中奥尔森派琴虫和切萨皮克派琴虫同时存在的隐性感染，这与RFTM诊断不同。这种双重qPCR方法的开发是监测原位共感染的基础性工作，将有助于制定优化的资源管理和保护策略，以应对新出现的疾病。
