Molecular detection of the oyster parasite Mikrocytos mackini, and a preliminary phylogenetic analysis
Molecular detection of the oyster parasite Mikrocytos mackini, and a preliminary phylogenetic analysis
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DOI：
10.3354/dao054219
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发表时间：
2003-04-24
影响因子：
1.4
通讯作者：
Bower, SM
中科院分区：
文献类型：
--
作者：
Carnegie, RB;Meyer, GR;Bower, SM
在加拿大不列颠哥伦比亚省的牡蛎长牡蛎中，原体寄生虫mckrocytos mackini是Denman岛病的病原体，由于它可以感染其他牡蛎物种，并且其生命周期、传播方式和来源尚不清楚，因此受到广泛关注。采用PCR和荧光原位杂交（FISH）方法对mackini进行检测，并与标准的组织病理学诊断进行了验证，并对mackini小亚基核糖体RNA基因（SSU rDNA）进行了初步的系统发育分析。一种专门设计的不扩增宿主DNA的PCR从感染麦氏分枝杆菌的牡蛎和富集的麦氏分枝杆菌细胞分离物中产生了544 bp的SSU rDNA片段，但从未感染的对照牡蛎中却没有。该片段经FISH鉴定为M mackini SSU rDNA。然后设计了一种麦氏杆菌特异性PCR，在不列颠哥伦比亚省丹曼岛的1056只野生牡蛎中检测到的麦氏杆菌感染率比标准组织病理学高3 - 4倍。当在标准样本（即横切面用于组织病理学，左外掌DNA用于PCR）之外处理大体病变时，基于PCR和组织病理学的麦氏小细胞病患病率估计增加（PCR从4.4增加到7.4%，组织病理学从1.2增加到2.1%）。使用组织病理学和组织印迹加PCR，标准样本加观察到的大体病变，代表了一种“全证据”方法，提供了对马基尼氏杆菌真实患病率的最现实估计。最大简约性和进化距离系统发育分析表明，麦基尼可能是一种基础真核生物，尽管它与其他已知的原生物类群没有密切的亲缘关系。
The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M mackini SSU rDNA. A M mackini-specific PCR was then designed which detected 3 to 4x more M mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa.