宠物贾第虫人畜共患基因型传播与致病的分子特性

Giardia duodenalis is an important zoonotic parasite and also very common intestinal protozoa in dogs and cats. Eight genotypes including A-H,have been identified to date, among them A and B are zoonotic genotypes.To study the molecular characteristics of Giardia zoonotic genotype from dogs and cats in transmission and pathogenicity,fecal antigen detection kit for Giardia will be used to screen fecal samples from pets and human at same location, muti-locus conserved and varible genes（SSU rRNA、gdh、tpi、bg、ef-1、ITS、mtDNA）of these isolates will be amplified, their genetic polymorphism (RFLP,SSCP and SNP) will be analysed, populational difference of genotype A and B will be compared between pets and human, new MLG and MLST genotyping standard and method of subtype will be established, and molecular epidemiology of Giardia from dogs and cats will be surveyed in main cities of China, such as Beijing, Shanghai, Guangzhou and so on. By means of comparison of genetic difference of multi-locus genes between isolates from asymptomatic and symptomatic pets, virulence genotypes will be established. On the basis of effect of parasite factor (bg、ef-1）from different isolates on apoptosis and their changes after interacting with host cell,such as Caco-2，HT-29, HeLa, virulence gene and pathogenic factor will be explored. The above-mentioned study will provide technological approaches or decision-making basis for the risk evaluation, prevention and control of Giardia spp., but also the scientific basis for the water quality inspection and drug research of the parasite.

十二指肠贾第虫是一种重要的人畜共患寄生虫，也是犬猫最常见的肠道原虫，迄今已鉴定出8种基因型（A-H）,其中A型和B型属于人畜共患基因型。为了探讨宠物贾第虫人畜共患基因型传播与致病的分子特性，拟采用贾第虫粪抗原检测试剂盒对同一地点犬猫和人的粪样进行筛查，对所获的贾第虫分离株进行多位点保守基因（tpi等）和高变基因（ITS等）的扩增及其遗传多态性（RFLP、SSCP、SNP）分析，比较犬猫和人的贾第虫Ａ型和Ｂ型种群遗传结构的差异，创建新型MLG和MLST分型标准和方法，拟对我国主要城市犬猫贾第虫进行分子流行病学调查；通过系统比较有无症状犬猫的贾第虫分离株多位点基因的遗传差异，创建贾第虫的毒力基因型；根据不同基因亚型分离株与宿主细胞互作后虫体因子（bg、ef-1）的变化及其对细胞凋亡的影响,探明犬猫贾第虫各基因亚型致病的分子特性。上述研究将为实施贾第虫的风险评估和防控措施提供技术手段和决策依据。

蓝氏贾第虫（又名十二指肠贾第虫）是一种重要的人畜共患寄生虫，也是犬猫最常见的肠道原虫，迄今已鉴定出8种基因型（A-H）,其中A型和B型属于人畜共患基因型，C、D型与F型分别为犬、猫特异性基因型。为了探讨宠物贾第虫人畜共患基因型传播与致病的分子特性，本项目采用饱和硫酸锌漂浮法对犬猫粪样进行筛查，对所获的贾第虫分离株进行多位点基因的扩增及其序列分析，比较犬猫贾第虫多种分离株种群遗传结构的差异，创建新型基因分型和PCR检测方法，对广东犬猫贾第虫进行分子流行病学调查，对贾第虫A型分离株的3种α-贾第素和延长因子ef-1α基因进行克隆、原核表达和亚细胞定位。通过犬猫贾第虫tpi、gdh、bg和18S rRNA 的基因扩增及序列分析，发现15株犬源贾第虫和9株猫源贾第虫为AI型，表明犬猫贾第虫对人类健康具有潜在威胁。通过多位点基因分型发现，犬猫贾第虫基因型存在混合感染情况，表明多位点分型在基因分型与检测上更为精确。以β-贾第素基因作为遗传标志，建立了犬猫贾第虫常见基因型的HRM快速分型和犬源贾第虫人畜共患与宿主特异性基因型的PCR-RFLP分型方法，基于贾第虫IGS序列建立了具有良好特异性和敏感性的PCR检测方法，基于蓝氏贾第虫(A型)的TPI基因和钩虫的ITS序列建立了检测犬源贾第虫(A型)和钩虫混合感染的多重PCR方法，为贾第虫的分子检测、分子分类和分子流行病学调查提供了技术手段。首次对A型贾第虫贾第素α-18、-12、-13和延伸因子EF-1α进行了原核表达和亚细胞定位，发现贾第素α-18主要分布在滋养体的鞭毛处，贾第素α-12、-13和EF-1α分布在胞浆内，这不仅为其致病机制研究奠定了基础，而且为抗贾第虫药物靶标的设计提供了科学依据。