Kistler Whitney M, Hernandez Sonia M, Gibbs Samantha E J, Ballard Jennifer R, Arnold Sarah L, Johnson Todd, Yabsley Michael J
Daniel B. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia 30602;
J Parasitol. 2013 Dec;99(6):1133-6. doi: 10.1645/13-211.1. Epub 2013 May 3.
Avian hemosporidian parasites are a genetically diverse group of parasites with a near cosmopolitan distribution. Over the past 2 decades, several PCR protocols have been designed to detect these parasites. The majority of these protocols amplify part of or the entire mitochondrial cytochrome b gene. However, many of these protocols co-amplify 2 genera (Haemoproteus and Plasmodium), making it impossible to determine which genus is amplified without post-PCR analysis. A uniform database (MalAvi), containing sequences amplified with the primers HAEMF and HAEMR2, has been developed to increase comparability across studies. We analyzed sequences from the MalAvi database and new sequences and found that digestion with EcoRV could be used to distinguish Haemoproteus from the majority of Plasmodium sequences. In addition, we tested 220 wild birds from Costa Rica and the United States for avian hemosporidians and assessed the ability of EcoRV to distinguish these 2 genera. Thirty-six positive samples were sequenced to confirm the restriction profiles, and we also analyzed 63 new hemosporidian sequences from ongoing studies in the United States for the restriction site. Among these new samples, all of the 85 Haemoproteus (subgenus Parahaemoproteus) and 14 Plasmodium were distinguishable. Overall, 887 of 898 (98.8%) sequences from our studies and the MalAvi database were assigned to the correct genus. Of these samples, all Haemoproteus samples were correctly identified and all but 11 Plasmodium samples were correctly identified by the EcoRV assay. Overall, this restriction enzyme protocol is able to quickly and efficiently classify these 2 genera of avian malarial parasites and would be useful for researchers interested in identifying parasites to genus-level, studies focused on sequence analysis of only a single genus, or for detecting co-infections that would need cloning prior to sequence analysis.
禽血孢子虫寄生虫是一类基因多样的寄生虫，分布几乎遍及全球。在过去20年里，已设计了多种聚合酶链式反应（PCR）方案来检测这些寄生虫。这些方案中的大多数扩增线粒体细胞色素b基因的部分或全部。然而，许多这些方案会共同扩增两个属（血变原虫属和疟原虫属），使得在没有PCR后分析的情况下无法确定扩增的是哪个属。已开发了一个统一的数据库（MalAvi），其中包含用引物HAEMF和HAEMR2扩增的序列，以提高不同研究之间的可比性。我们分析了来自MalAvi数据库的序列和新序列，发现用EcoRV酶切可用于区分血变原虫属与大多数疟原虫属序列。此外，我们检测了来自哥斯达黎加和美国的220只野生鸟类是否感染禽血孢子虫，并评估了EcoRV区分这两个属的能力。对36个阳性样本进行测序以确认酶切图谱，我们还分析了来自美国正在进行的研究中的63个新的血孢子虫序列的酶切位点。在这些新样本中，所有85个血变原虫属（副血变原虫亚属）和14个疟原虫属的样本都是可区分的。总体而言，我们的研究和MalAvi数据库中的898个序列中有887个（98.8%）被正确归为相应的属。在这些样本中，所有血变原虫属样本均被正确鉴定，除11个疟原虫属样本外，其余疟原虫属样本均通过EcoRV检测正确鉴定。总体而言，这种限制酶方案能够快速有效地对这两个属的禽疟原虫寄生虫进行分类，对于有兴趣将寄生虫鉴定到属水平的研究人员、仅专注于单个属的序列分析的研究或对于检测在序列分析前需要克隆的混合感染将是有用的。
