Levy Michael G, Poore Matthew F, Colorni Angelo, Noga Edward J, Vandersea Mark W, Litaker R Wayne
College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606-1499, USA.
Dis Aquat Organ. 2007 Jan 18;73(3):219-26. doi: 10.3354/dao073219.
Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.
淀粉卵甲藻病由双鞭毛虫外寄生虫眼点淀粉卵甲藻引起，是影响暖水和温带水域海洋鱼类的最严重疾病之一。目前的诊断方法完全依赖于在受感染鱼类的皮肤或鳃上通过显微镜识别寄生虫。然而，亚临床感染通常未被发现，同时尚未设计出检测自由游动的感染性（动孢子）阶段的方法。针对该寄生虫的核糖体DNA区域，我们开发了一种灵敏且特异的PCR检测方法，该方法能够从寄生虫生命周期的三个阶段（滋养体、包囊体、动孢子）中的任何一个阶段检测到少至单个细胞。该检测方法在简单的人工海水培养基和含有浮游生物群落组合的天然海水中表现同样良好。该检测方法也不受鳃组织的抑制。对从红海（以色列）、地中海东部（以色列）、亚得里亚海（意大利）、墨西哥湾（佛罗里达）的鱼类以及来源不明的鱼类中获得的5株眼点淀粉卵甲藻分离株的内部转录间隔区进行序列分析，结果显示差异不显著，表明所有分离株均为同一物种。然而，其中3株在细胞培养中繁殖的分离株在行为和形态上存在差异，并且这些差异在至少2年的培养过程中保持一致。因此，我们的研究结果并未排除不同菌株实际上是“亚种”或更低分类单元的可能性，它们在致病和免疫原性特征、环境耐受性及其他特征方面也可能存在差异。
