水牛抗血吸虫再感染及其机制研究

Buffalo is one of the most important reservoir hosts in the Schistosomiasis japonicum endemic area of China. Due to their involvement in human schistosomiasis transmission, water buffaloes are of significant public health concern. In our previous studies, we found that the prevalence of S. japonicum infections in juvenile buffaloes was significantly higher than that in adults. In artificial infecting experiments, it was also observed that the worms in yang buffaloes were gradually cleared in the period following S. japonicum infection and the infected/cured buffaloes have resistance to post-treatment reinfection. In this project, the level and molecular mechanisms of the resistance to post-treatment reinfection in buffaloes will be studied. The process and level will be illuminated through investigating the diffences of the feces egg number, egg hatching, worm burden and liver egg burden during the inducement course of reinfection group and between the reinfection group and control group (first infection). Than, the differences in morphology, gene expression and apoptosis of parasite,specific antibody isotype responses, cytokines，target antigens and immune response types of the host will be compared between reinfection and control groups. The results obtained here will illustrate the molecular mechanisms of the resistance to post-treatment reinfection in buffaloes, provide new idea for development of anti-schistosomiasis vaccine and screening of the new drug targets, as well as offering scientific basis for making prevention tactics of schistosomiasis.

水牛是我国血吸虫病疫区最重要的保虫宿主，水牛血吸虫病防治在我国血吸虫病防控上具有重要意义。疫区幼年水牛血吸虫感染率显著高于成年水牛，人工感染实验证明幼年水牛感染血吸虫的后期虫体可以逐渐消亡，且感染/治疗的水牛对血吸虫再感染具有抗性。本项研究拟通过感染/治疗方式构建水牛抗再感染模型，详细观察再感染组诱导过程中以及再感染组和对照组之间粪便虫卵数、粪便毛蚴孵化数、解剖收集的虫体数等方面的差异，阐明水牛抗再感染现象产生过程，量化抗再感染程度；从再感染组和对照组血吸虫在形态学、基因表达、虫体细胞凋亡等方面系统分析再感染血吸虫“受伤”程度和原因；分析再感染组和对照组水牛抗血吸虫特异性同型抗体、细胞因子、靶抗原、免疫反应类型等方面的差异，揭示水牛抗抗再感染相关的免疫机制。本研究可为研制抗血吸虫疫苗、寻找治疗药物靶标、制定血吸虫防治对策提供科学依据。

水牛是血吸虫的重要保虫宿主，探讨水牛抗血吸虫再感染和机制具有重要理论和应用价值。本项研究对水牛抗血吸虫再感染程度、再感染组和初次感染组虫体的形态学、基因表达和蛋白表达、再感染和初次感染水牛免疫差异等开展了研究。通过人工感染实验观察到水牛具有极强的抗再感染能力，第三次感染和初次感染相比，虫荷数可减少97%以上、每克肝脏虫卵和每克直肠虫卵数均可减少87%以上；再感染组虫体长度、内部器官、子宫中虫卵数均小于初次感染组虫体。应用 RNA-Seq技术在再感染组与初次感染组虫体中鉴定出13605个基因，其中差异表达基因112个；与氧化还原、钙依赖的磷脂结合、脂结合和钙离子结合等功能相关的基因表达下调；与代谢进程、生物学合成功能、铁离子结合、血红素结合、四吡咯结合等功能相关的基因表达上调。应用TMT-based proteomics技术鉴定到再感染与初次感染虫体差异表达蛋白14个，主要涉及氧化还原进程、TOR信号通路和蛋白质代谢进程。再感染水牛在第三次感染后产生的抗体和抗体亚型与初次感染产生的抗体和抗体亚型一致，总量甚至低于初次感染；第三次感染前和后血清中IL-4、IL-10和IFN-水平显著高于初次感染，淋巴细胞数量和比例显著增加。水牛提前18天预服吡喹酮使虫体发育率减少87%；建立家畜血吸虫感染PCR诊断技术可以早期诊断，敏感性和特异性均达92%以上。我们的研究结果证实通过两次感染/治疗可以诱导非常强烈的抗再感染能力、再感染组中血吸虫的关键基因表达抑制和水牛再感染时的细胞免疫可能是抗再感染的关键机制，这对今后开展抗血吸虫疫苗、疫苗免疫程序和寻找治疗药物靶标等研究具有参考意义；本研究发现的吡喹酮预防作用和建立的分子诊断技术，在今后血吸虫病消除进程中具有实用价值。