犬猫锡兰钩虫人畜互传与致病的分子特性

Ancylostoma ceylanicum is very common in people and pets (dogs and cats) and can be used as an ideal model organism for the study of ancylostomiaosis. In this project, we propose to study the molecular characterization of zoonotic A. ceylanicum from dogs and cats in transmission and pathogenicity on the basis of our previous research. First, we shall ascertain molecular characterisitcs and transmission risk of zoonotic A. ceylanicum from dogs and cats by amplifying multi-locus genes of rDNA and mtDNA from pet- and human-derived A. ceylanicum, analyzing their genetic polymorphism, and conducting molecular epidemiological investigation of canine and feline hookworm from various geographical locations (such as major cities in China) after establishing molecular detection methods. Second, we shall attempt to dissect molecular basis of host resistance or susceptibility to A. ceylanicum infections in animal models. The differences in transcriptome features, such as transcript abundance, alternative splicing, and gene fusion events in the intestinal tissue of mice or hamster with a various agree of resistance or susceptibility in response to experimental parasitic infection will be quantified using RNAseq technology. Third, we shall screen target genes encoding ancylostome secreted proteins (ASPs) associated with skin invasion, nutrition acquisition, and parasitism establishment in larval and adult A. ceylanicum using genomic tools. They will be cloned, expressed and identified by molecular means so as to ascertain molecular properties of key pathogenic factors. The proposed study will provide technological means and decision basis for rapid detection, prevention and control of the zoonotic hookworm. Furthermore, expertise obtained will possess potential application value for developing pan-Ancylostoma vaccine and novel drug.

锡兰钩虫在人与宠物之间广泛流行，是研究钩虫病理想的生物模型。本项目拟在前期工作的基础上深入研究犬猫锡兰钩虫人畜互传与致病的分子特性，拟对不同地区犬猫和人的锡兰钩虫分离株rDNA和mtDNA进行多位点基因扩增及其遗传多态性分析，建立分子检测方法，对我国主要城市犬猫钩虫进行分子流行病学调查，探明锡兰钩虫人畜互传的分子特性和传播风险；同时以易感鼠(仓鼠）和抗性鼠（小白鼠）作为锡兰钩虫感染模型，采用RNA-seq等技术比较两者感染后生物学特性和肠组织中转录功能的差异，探明宿主抗性与易感的分子基础；进一步以仓鼠作为动物模型，采用组学技术筛选锡兰钩虫幼虫和成虫与钻入皮肤、夺取营养和寄生生活等致病有关的钩虫分泌蛋白（ASPs）靶基因，对其进行克隆表达与分子鉴定，探明关键致病因子的分子特性。上述研究将为人畜共患钩虫的快速检测和防控措施提供技术手段和决策依据，对于抗钩虫疫苗和新药的研制具有潜在应用价值。

锡兰钩虫在人与宠物之间广泛流行，是研究钩虫病理想的生物模型。本项目采用饱和盐水漂浮法对犬猫粪样进行筛查，对所获的钩虫分离株进行形态学和分子鉴定，对犬源锡兰钩虫和猫源管型钩虫线粒体全基因组进行测序，创建新型分子检测方法，对我国广东犬猫钩虫进行分子流行病学调查，对锡兰钩虫与吸血和寄生有关的关键分子如谷胱甘肽-S-转移酶（GST）、血小板抑制剂（HPI）、天冬氨酸蛋白酶抑制剂（API）和金属蛋白酶组织抑制剂（TIMP）等靶基因进行克隆表达与分子鉴定。发现广东地区流浪犬感染的钩虫为锡兰钩虫和犬钩虫，流浪猫感染的钩虫为锡兰钩虫和管型钩虫，表明锡兰钩虫为犬猫的优势虫种，对人类健康具有潜在威胁。首次获得犬源锡兰钩虫和猫源管型钩虫的线粒体全基因组序列,丰富了钩口科线虫的线粒体基因数据库。基于ITS1序列创建了犬猫钩虫的HRM和Multi-ARMS-qPCR分子检测方法，基于ITS1序列3个SNP位点创建了犬猫钩虫的Tm-shift分子检测方法，为钩虫的分子检测、分子流行病学调查、人畜共患风险评估和钩虫病疗效考核提供了新的技术手段。首次对锡兰钩虫GST、HPI、API和TIMP等钩虫分泌蛋白进行亚细胞定位和功能分析，发现Ace-HPI属于CAP超家族成员，在成虫体内高表达，主要分布于头腺和食道，具有抑制血小板聚集的功能，可以刺激外周血淋巴细胞的增殖和细胞因子IL-10和IL-13的大量表达；Ace-GST主要分布在成虫的表皮、肌肉组织和肠道，具有一定的免疫原性；Ace-API免疫定位于分泌腺、假体腔液和皮下组织，具有抑制胃蛋白酶的功能和良好的免疫原性；Ace-TIMP具有对MMP的抑制活性和良好的抗原性。上述发现为钩虫疫苗候选分子或药物靶标的筛选奠定了基础，对于抗钩虫疫苗和新药的研制具有潜在应用价值。