结肠小袋纤毛虫猪-人共患传播的遗传特征

Pigs infected with Balantidium coli were regarded as the major reservoir for human infections.The results of previous animal infection tests and clinical cases indicated that there are host-adaption phenomenon in B.coli,and the abilities of infection and virolence are different in various hosts. However,molecular genetic features of its host adaption and cross transmission between pigs and humans remains unknown.In addition, B.coli lacks reliable genetic markers for its subtyping and tracking sources of infections or contaminations. In this study, B. coli from both pigs and humans will be selected for their genomic sequencing using Illumina Hiseq platform of next generation sequencing(NGS), and then comparative genomic technology, MLST and population genetic structure analysis will be used for screening the right genetic loci associated with its host adaption and uncovering the genetic mechanism of these loci variation. The cross transmission ability and pathogenicity of common subtype between pigs and humans will be confirmed through animal cross transmission tests. Finally,these findings combined with monitoring results of feces from both keepers in the B.coli-positive pig farms and practioners in pig slaughterhouses will explore and reveal genetic features of cross transmission of B. coli between humans and pigs. It will provide a powerful tool for classification and tracking sources of B.coli infection in humans, and helpful for understand knowledge of biology and control balantidiosis of human and other hosts.

猪是人感染结肠小袋纤毛虫最重要的天然宿主。本课题组前期动物感染试验和临床病例研究结果提示，结肠小袋纤毛虫存在宿主适应性，对不同宿主的感染性和致病性存在差异。但是目前其宿主适应性和人兽互传的遗传特征不明确，亚型分析和病例溯源也缺乏有效的标记基因。本研究拟通过二代高通量测序Illumina Hiseq技术平台获得猪源和人源结肠小袋纤毛虫基因组序列；采用比较基因组学、MLST分型和群体遗传结构分析等技术和方法，寻找与结肠小袋纤毛虫宿主适应性相关的基因位点，解析其宿主适应性相关基因位点发生改变的遗传机制；用动物交叉感染试验验证猪-人共同亚型的感染性和致病性，结合阳性猪场饲养员和生猪屠宰场一线工作人员结肠小袋纤毛虫感染情况跟踪监测结果，探讨和揭示结肠小袋纤毛虫猪-人共患传播的遗传特征，为人感染结肠小袋纤毛病例溯源和分类提供有力的分子工具，为预警和防控结肠小袋纤毛虫病暴发提供重要理论依据和技术支持。

本研究通过高通量三代测序平台PacBio Sequel和二代测序平台Illumina Hiseq对小袋虫基因组进行测序和从头拼接组装，并对其进行基因注释和比较基因组学分析。结果显示，小袋虫基因组大小为50.96 Mb，基因组中有12,366个基因，其中小袋虫特有的基因有4461个，扩张的基因有510个。这些特有和扩张的基因主要与细胞内吞、膜泡运输、泛素化蛋白水解作用、胞外囊泡、钙信号通路和碳水化合物利用等功能密切相关。注释到18种粘多糖降解酶基因，与小袋虫滋养体入侵和损伤肠粘膜引起肠炎有关。从小袋虫基因组中筛选了6个合适的遗传标记位点，建立了小袋虫MLST分型方法；小袋虫6个标记位点中BC-MS5位于基因编码区和3’-UTR区域，具有较高的分辨率和稳定的序列变异特征，是小袋虫基因型分析和感染溯源研究理想的遗传标记位点。基于BC-MS5序列将猪源、豚鼠源、人源、羊源和鹿源小袋虫分为5个基因型（MS5Ⅰ~Ⅴ），基于ITS1/2序列将不同宿主源小袋虫分为2个基因型(ITSA和ITSB)，其中ITSA/MS5Ⅱ亚型能感染豚鼠、猪、湖羊、鹿和人，ITSB/MS5Ⅲ亚型是猪群感染的优势种群。小袋虫ITS1/2基因型B在猪群和豚鼠群中存在连锁不平衡，形成克隆群结构，而ITS1/2基因型A在猪和豚鼠群中存在基因重组，形成流行群结构，可能是猪源小袋虫优势种群ITSB/MS5Ⅲ和人兽共患亚型ITSA/MS5Ⅱ形成的遗传机制。初步的研究数据显示，小袋虫宿主适应性不明显。本研究为小袋虫感染溯源和分子流行病学研究提供有力的分子检测工具，为了解小袋虫感染性和致病性等生物学特性提供重要的理论依据。