中国利什曼原虫种群结构与遗传分化研究

Representatives of the genus Leishmania cause a variety of clinical syndromes ranging from self-healing cutaneous forms (CL) through to mucocutaneous (MCL) and lethal visceral diseases (VL).The leishmaniasis is transmitted via the bite of infected female sandflies. Understanding molecular changes associated with population structure in these parasites can help to understand their epidemiology and spread in humans. In the current context of worldwide (re-)emergence and spreading of leishmaniasis,the relevance of distinction of strains further gains importance.Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. In our previous phylogenetic studies, we provided evidence for the occurrence of a distint lineage in China as yet unclassfied members of Leishmania. That lineage is more closely reated to Sauroleishmania than to L. onovani complex, further indicating that the strains in China may have had a more complex evolutionary history than previously thought. In the present study,we will integrate genotype, clinical manifestations, hosts and vectors, as well as climatic and geographical features into investigating the population genetics and phylogeography of Leishmania strains in China. A more comprehensive sampling is necessary to cover as many as possible endemic foci in China. We will use a panel of standard microsatellite loci to investigate population-level diversity in Leishmania strains isolated from endemic foci in China, and compared their variation to that found in diverse strains of the L. donovani complex isolates from Europe, Africa and Asia. MLMT data will be analysed by using a Bayesian model-based clustering algorithm and reconstructing phylogenetic tree based on genetic distances. Our obejectives are (i) to understand the population structure and genetic differentiation of L. donovani complex strains in China; (ii) to test the hypotheses for the origin of spread of L. donovani complex in China; (iii) to infer the phylogeographic patterns of Leishmania isolates, and reveal the cryptic diversity; and (iv) to test whether specific genetic traits are related to different clinical manifestations of strains infections, such as VL, MCL and CL.The reuslts of this study will facilitate global taxonomic, population genetic and epidemiological studies of the Leishmania parasites. This is crucial for correct diagnosis and prognosis of the diseases as well as for making decisions regarding treatment and control measures.

黑热病对人体危害严重，在全球范围再度回升，经由利什曼原虫引起和白蛉传播。了解这些寄生虫的种群结构与遗传分化有助于解析其流行病学和在人类的传播。中国利什曼原虫异质性高，种群结构和遗传关系仍不清楚。申请者在前期研究中发现中国分布有一独特的谱系，与蜥蜴利什曼原虫形成一支，暗示中国利什曼虫株的异质性和演化历史比先前想象的更复杂。本项目在此基础上，拟广泛采样，整合多位点微卫星分型、临床症状、以及地理特征等数据，联合基于贝叶斯模型的聚类算法和遗传距离等种群遗传学方法重建中国利什曼原虫的演化历史；阐明中国利什曼原虫复合体种群结构和遗传分化；检验其起源和散布方式；探讨遗传型和临床症状及地理起源之间的关系；揭示地理分布、宿主、传播媒介等因素对利什曼原虫的演化和扩布的影响。研究结果将促进利什曼原虫的分类、种群遗传学和流行病学研究，进而为黑热病的正确诊断、预测以及制定治疗和控制措施提供科学依据。

中国利什曼原虫异质性高，种群结构和遗传关系仍不清楚。在本项目的支持下，我们对中国利什曼原虫分离株进行多基因进化分析，支持中国利什曼原虫株组成复杂，透射电镜超微结构比较表明先前发现的Leishmania sp.与报道的蜥蜴利什曼原虫（L. tarentolae）相似（Parasitology Research, 2017）。对荒漠疫区蜥蜴血液寄生的利什曼原虫分子检测和进化分析，发现这些寄生虫没有显著的地理结构和宿主特异性。蜥蜴血液内不仅有蜥蜴类利什曼原虫（如L. gymnodactyli），还有哺乳类利什曼原虫（如L. tropica，L. donovani complex，L. turanica等），蜥蜴很可能是利什曼原虫的保虫宿主（Acta Tropica, 2016; BMC Veterinary Research, 2019）。代表蛇类寄生利什曼原虫分子检测发现花条蛇、阿拉善蝮和棋斑水游蛇除了感染蜥蜴利什曼原虫亚属的种类，还有感染哺乳类的种类（L. donovani complex，L. turanica）（PLoS ONE，2019）。支持中国分布的杜氏利什曼原虫复合体不是从外部引入，具有本地化特点，荒漠疫区的爬行动物可能与利什曼病的流行病学密切相关。此外，我们还开展了（1）肺炎克雷伯菌临床株的分子鉴定，发现khe基因并不是肺炎克雷伯菌的特异性基因，rpoB基因能准确建立物种属、种间及亚种之间的系统进化关系（Antonie van Leeuwenhoek，2016）；（2）杜氏利什曼原虫的4种蛋白的生物信息分析，以寻找疫苗表位和药物靶点（Acta Tropica, 2019）。通过此项目积累了以下关键数据：（1）干旱荒漠区66个样点代表4属13种蜥蜴的600余份血液样品；（2）干旱荒漠区9个样点3种蛇的20余份血液样品；（3）代表中国利什曼原虫的DNA序列：320余条cyt b基因序列，400余条HSP70基因序列，150余条ITS1序列。这些数据代表了我国利什曼原虫多样性的遗传信息，是研究利什曼原虫多样性格局及其群体历史动态的重要数据，也为进一步研究干旱荒漠区爬行动物血液寄生虫的多样性奠定了基础。本项目完成了项目的各项研究目标，共发表标注论文14篇，SCI收录11篇，其中第一标注SCI论文5篇，第二标注SCI论文2篇。