Arai Sakura, Yoshinari Tomoya, Terajima Jun, Hara-Kudo Yukiko, Ohnishi Takahiro
Division of Microbiology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.
Division of Microbiology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.
Parasitol Int. 2020 Apr;75:102048. doi: 10.1016/j.parint.2019.102048. Epub 2019 Dec 28.
Kudoa hexapunctata was taxonomically separated from Kudoa neothunni, but their main host is tuna. K. hexapunctata has been identified as causative agent of foodborne diseases associated with the ingestion of raw Pacific bluefin tuna (PBT) in Japan, but K. neothunni has not. Therefore, it is clinically and epidemiologically important to detect and distinguish these two species. In the present study, we developed a novel duplex polymerase chain reaction (dPCR) targeting the 28S rRNA gene sequences of K. hexapunctata and K. neothunni. The dPCR amplified the desired genetic regions of each species, and the detection limit was 10 copies/reaction. A total of 36 retail tuna samples from different fishing ports were purchased and tested by dPCR. Thirty-one tested positive for K. hexapunctata and four tested positive for K. neothunni. Several retail PBT samples were examined in some of the fishing ports, and among these samples, the detection rates of K. hexapunctata was higher than 85%, and the rates were similar between wild and farmed PBT. The detection rates of K. hexapunctata in wild and farmed retail PBT were 75% and 71%, respectively, in May. However, the rates in June and July were 100% for both. K. hexapunctata and K. neothunni myxospores were not observed in the dPCR-positive samples, except in juvenile PBT, suggesting that the number of parasites was insufficient to cause foodborne disease. Thus, dPCR is a useful method for detecting and distinguishing K. hexapunctata and K. neothunni, and can be used in epidemiological studies of these parasites.
六斑库道虫在分类学上与新金枪鱼库道虫不同，但它们的主要宿主都是金枪鱼。六斑库道虫已被确定为与在日本食用生太平洋蓝鳍金枪鱼（PBT）相关的食源性疾病的病原体，但新金枪鱼库道虫尚未被确定为此病原体。因此，检测和区分这两个物种在临床和流行病学上具有重要意义。在本研究中，我们开发了一种针对六斑库道虫和新金枪鱼库道虫28S rRNA基因序列的新型双重聚合酶链反应（dPCR）。该dPCR扩增了每个物种所需的基因区域，检测限为10拷贝/反应。共购买了36份来自不同渔港的零售金枪鱼样本，并通过dPCR进行检测。31份样本六斑库道虫检测呈阳性，4份样本新金枪鱼库道虫检测呈阳性。在一些渔港对几份零售PBT样本进行了检测，在这些样本中，六斑库道虫的检出率高于85%，野生和养殖PBT的检出率相似。5月份野生和养殖零售PBT中六斑库道虫的检出率分别为75%和71%。然而，6月和7月两者的检出率均为100%。除幼体PBT外，在dPCR阳性样本中未观察到六斑库道虫和新金枪鱼库道虫的粘孢子，这表明寄生虫数量不足以引起食源性疾病。因此，dPCR是检测和区分六斑库道虫和新金枪鱼库道虫的有用方法，可用于这些寄生虫的流行病学研究。
