Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.
Parasit Vectors. 2013 Jun 4;6:160. doi: 10.1186/1756-3305-6-160.
Leishmaniasis is a clinically and epidemiologically diverse zoonotic disease caused by obligatory, intracellular protozoan parasites of the genus Leishmania. Cutaneous leishmaniasis is the most widely distributed form of the disease characterized by skin lesions. Leishmania aethiopica is considered the predominant etiological agent in Ethiopia. The current study was aimed at developing multilocus microsatellite markers for L. aethiopica isolated from human cutaneous leishmaniasis patients in Ethiopia.
L. aethiopica parasites for the study were obtained from Ethiopia and laboratory analysis was conducted at The Ohio State University. DNA was extracted from cultured parasites and an internal transcribed spacer located at the ribosomal region of L. aethiopica genomic DNA was PCR amplified for species identification. Microsatellite markers were identified using multilocus microsatellite typing. We generated an enriched genomic library, and using Primer3 software, designed PCR primers to amplify sequences flanking the detected microsatellites. Subsequent screening of the amplified markers for length variations was performed by gel electrophoresis.Using a variety of molecular methods, 22 different microsatellite markers were identified and tested for typing L. aethiopica strains using a number of clinical isolates. Of the 22 markers tested, 5 were polymorphic and showed distinctive multilocus genotypes, classifying them into four clusters. One marker was found to be specific for L. aethiopica, discriminating it from other species of Leishmania.
Multilocus microsatellite typing using the markers developed in this study could be useful for epidemiological and population genetic studies of strains of L. aethiopica in order to investigate the structure and dynamics of the corresponding natural foci. It could also help to answer specific clinical questions, such as the occurrence of local and diffuse lesions, strain correlates of parasite persistence after subclinical infection and lesion comparisons from patients suffering from L. aethiopica infections.
利什曼病是一种临床和流行病学上具有多样性的人兽共患疾病，由需氧的、内共生的原生动物利什曼原虫属寄生虫引起。皮肤利什曼病是该病最广泛分布的形式，其特征为皮肤损伤。埃塞俄比亚利什曼原虫被认为是埃塞俄比亚的主要病原体。本研究旨在为来自埃塞俄比亚人类皮肤利什曼病患者的利什曼原虫属寄生虫开发多位点微卫星标记。
用于本研究的利什曼原虫寄生虫从埃塞俄比亚获得，实验室分析在俄亥俄州立大学进行。从培养的寄生虫中提取 DNA，并对位于利什曼原虫属基因组 DNA 核糖体区的内部转录间隔区进行 PCR 扩增以进行物种鉴定。使用多位点微卫星分型鉴定微卫星标记。我们生成了一个富集基因组文库，并使用 Primer3 软件设计 PCR 引物以扩增检测到的微卫星侧翼的序列。随后通过凝胶电泳对扩增标记进行长度变异筛选。使用多种分子方法，鉴定了 22 个不同的微卫星标记，并使用多个临床分离株对利什曼原虫属菌株进行了分型测试。在所测试的 22 个标记中，有 5 个具有多态性，表现出独特的多位点基因型，将它们分为四个聚类。一个标记被发现对利什曼原虫属具有特异性，可将其与其他利什曼原虫物种区分开来。
使用本研究中开发的标记进行多位点微卫星分型，可用于利什曼原虫属菌株的流行病学和群体遗传学研究，以调查相应自然病灶的结构和动态。它还可以帮助回答具体的临床问题，例如局部和弥漫性病变的发生、亚临床感染后寄生虫持续存在的菌株相关性以及患有埃塞俄比亚利什曼原虫感染的患者的病变比较。
