Fotouhi-Ardakani Reza, Dabiri Shahriar, Ajdari Soheila, Alimohammadian Mohammad Hossein, AlaeeNovin Elnaz, Taleshi Neda, Parvizi Parviz
Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran.
Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Science, Kerman, Iran.
Infect Genet Evol. 2016 Dec;46:33-41. doi: 10.1016/j.meegid.2016.10.011. Epub 2016 Oct 17.
The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 - KU680871). Four genes had a remarkable number of informative sites (P=0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P=0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D=-2.2, P<0.01), while COII and nagt genes were produced through evolutionary processes for both L. tropica and L. major (Tajima's D=2.85 & 2.91, P<0.01). More different clinical lesions with extensive phylogenetic and evolutionary analyses should be employed to avoid confusion in the diagnosis of leishmaniasis and development of vaccines for eradicating Leishmania parasites.
利什曼原虫属的多态性和遗传多样性存在诸多争议，这取决于许多因素，如核基因和/或线粒体基因、分子工具、利什曼原虫种类、地理起源、利什曼原虫寄生虫的微环境状况以及从临床样本、保虫宿主和媒介中分离利什曼原虫。利用线粒体基因（COII和Cyt b）和核基因（nagt、ITS-rDNA和HSP70）对利什曼原虫种类（硕大利什曼原虫、热带利什曼原虫、塔兰托利什曼原虫、墨西哥利什曼原虫、婴儿利什曼原虫）的遗传变异进行了分析和比较。每个酶基因（COII、Cyt b和nagt）或管家基因（ITS-rDNA、HSP70）的作用被用于准确鉴定利什曼原虫寄生虫。在提取利什曼原虫寄生虫的天然、自然和参考菌株的DNA并进行扩增后，对聚合酶链反应（PCR）产物进行测序，并使用MEGA6和DnaSP5软件进行遗传相似性评估和系统发育分析。在这五个基因的72个序列中，多态性位点的数量与单态性位点相比显著更低。在这72个序列中，利什曼原虫种类的54个新单倍型（五个基因）已提交至GenBank（登录号：KU680818 - KU680871）。除HSP70基因外，其他四个基因具有大量的信息位点（P = 0.00），可能是因为其微卫星区域。nagt基因的非同义（dN）变异比其他表达基因更多（47.4%）。三个表达基因的同义（dS）/dN比值在五种利什曼原虫之间显示出显著差异（P = 0.001）。单倍型多样性的最高和最低水平分别在热带利什曼原虫（81.35%）和硕大利什曼原虫（28.38%）群体中观察到。 Tajima's D指数分析表明，热带利什曼原虫物种中的Cyt b基因显著为负（Tajima's D = -2.2，P < 0.01），而热带利什曼原虫和硕大利什曼原虫的COII和nagt基因是通过进化过程产生的（Tajima's D = 2.85 & 2.91，P < 0.01）。应该采用更多不同的临床病变进行广泛的系统发育和进化分析，以避免在利什曼病诊断和开发根除利什曼原虫寄生虫的疫苗时出现混淆。
