ABSTRACT: Malacosporeans are a group of endoparasitic cnidarians (Myxozoa) that use freshwater bryozoans and fish as final and intermediate hosts, respectively. The malacosporean Tetracapsuloides bryosalmonae causes proliferative kidney disease (PKD), an emerging disease in aquaculture and wild fish populations, including threatened salmonids in Europe and the USA. Mixed infections of malacosporeans are often encountered, and a monitoring tool for screening of multiple malacosporean species in either their fish or bryozoan hosts is therefore desirable. We describe an inexpensive method that combines PCR amplification of the partial 18S rRNA gene (~260 bp) and a single-step restriction fragment length polymorphism (RFLP) method for identification of 10 malacosporean lineages and species. We demonstrate and test this methodology on a set of DNA extracted from malacosporeans infecting fish kidney and tissues sampled from bryozoan colonies and compare the results with Sanger sequencing of the same parasite DNA isolates. The PCR-RFLP and Sanger sequencing methods agreed in 100% of cases. The PCR-RFLP method offers a number of opportunities, including screening large panels of host tissue samples to gain insights into infection patterns, characterizing mixed infections, and confirming highly pathogenic T. bryosalmonae infections. The method can also be further refined as new sequence data become available for malacosporeans.

中文翻译：

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PCR-RFLP：一种有针对性的方法，可揭示宿主的特定疟原虫感染谱（刺孢：粘液虫：疟原虫）。

摘要：疟原虫是一群内寄生刺胞（Myxozoa），它们分别使用淡水苔藓动物和鱼类作为最终宿主和中间宿主。疟原虫四囊藻类苔藓沙门菌引起增生性肾脏疾病（PKD），这是水产养殖和野生鱼类种群中的一种新兴疾病，包括欧洲和美国的濒危鲑鱼。疟疾孢子的混合感染是经常遇到的，因此需要一种监测工具来筛选鱼类或苔藓动物宿主中的多种疟疾孢子。我们描述了一种便宜的方法，该方法结合了部分18S rRNA基因（〜260 bp）的PCR扩增和单步限制性片段长度多态性（RFLP）方法来鉴定10个疟原虫谱系和物种。我们论证并测试了从感染疟疾鱼肾的疟原虫中提取的一组DNA和从苔藓菌落采样的组织中提取的DNA的方法，并与相同寄生虫DNA分离物的Sanger测序结果进行了比较。PCR-RFLP和Sanger测序方法在100％的病例中是一致的。PCR-RFLP方法提供了许多机会，包括筛选大量宿主组织样品以深入了解感染模式，鉴定混合感染并确认高致病性T. bryosalmonae感染。随着新序列数据可用于疟疾孢子菌，该方法也可以进一步完善。