AFLP Polymorphisms Allow High Resolution Genetic Analysis of American Tegumentary Leishmaniasis Agents Circulating in Panama and Other Members of the Leishmania Genus

American Tegumentary Leishmaniasis is caused by parasites of the genus Leishmania, and causes significant health problems throughout the Americas. In Panama, Leishmania parasites are endemic, causing thousands of new cases every year, mostly of the cutaneous form. In the last years, the burden of the disease has increased, coincident with increasing disturbances in its natural sylvatic environments. The study of genetic variation in parasites is important for a better understanding of the biology, population genetics, and ultimately the evolution and epidemiology of these organisms. Very few attempts have been made to characterize genetic polymorphisms of parasites isolated from Panamanian patients of cutaneous leishmaniasis. Here we present data on the genetic variability of local isolates of Leishmania, as well as specimens from several other species, by means of Amplified Fragment Length Polymorphisms (AFLP), a technique seldom used to study genetic makeup of parasites. We demonstrate that this technique allows detection of very high levels of genetic variability in local isolates of Leishmania panamensis in a highly reproducible manner. The analysis of AFLP fingerprints generated by unique selective primer combinations in L. panamensis suggests a predominant clonal mode of reproduction. Using fluorescently labeled primers, many taxon-specific fragments were identified which may show potential as species diagnostic fragments. The AFLP permitted a high resolution genetic analysis of the Leishmania genus, clearly separating certain groups among L. panamensis specimens and highly related species such as L. panamensis and L. guyanensis. The phylogenetic networks reconstructed from our AFLP data are congruent with established taxonomy for the genus Leishmania, even when using single selective primer combinations. Results of this study demonstrate that AFLP polymorphisms can be informative for genetic characterization in Leishmania parasites, at both intra and inter-specific levels.美洲皮肤利什曼病（American Tegumentary Leishmaniasis）由利什曼属（Leishmania）寄生虫引发，在整个美洲地区造成严重的健康负担。在巴拿马，利什曼寄生虫呈地方性流行态势，每年新增数千例病例，且以皮肤型利什曼病为主。近年来，该疾病的疾病负担持续上升，这与其自然野生栖息环境遭到日益加剧的破坏高度契合。对寄生虫遗传变异的研究，有助于更深入地理解这类生物的生物学特性、群体遗传学特征，乃至其进化历程与流行病学规律。目前针对从巴拿马皮肤利什曼病患者体内分离的寄生虫开展遗传多态性特征解析的研究寥寥无几。本研究通过扩增片段长度多态性（Amplified Fragment Length Polymorphisms, AFLP）技术——一种极少用于解析寄生虫遗传组成的分子生物学方法——，获取了巴拿马本地利什曼分离株以及其他数个物种种株的遗传变异数据。本研究证实，该技术可在极高重复性的前提下，检测出巴拿马利什曼（Leishmania panamensis）本地分离株中极高水平的遗传变异。通过对巴拿马利什曼中特异性选择性引物组合所产生的AFLP指纹图谱进行分析，结果提示其繁殖方式以克隆增殖为主。使用荧光标记引物时，本研究鉴定出多个类群特异性片段，这些片段有望作为物种诊断标志物。AFLP技术可实现对利什曼属的高分辨率遗传分析，能够清晰区分巴拿马利什曼样本中的多个类群，以及巴拿马利什曼与圭亚那利什曼（L. guyanensis）这类亲缘关系极近的物种。基于本研究的AFLP数据构建的系统发育网络，与利什曼属的既定分类学体系相一致，即便仅使用单一选择性引物组合亦是如此。本研究结果表明，AFLP多态性可用于利什曼寄生虫的种内与种间遗传鉴定分析，且具备良好的信息价值。