Tang Zeli, Sun Hengchang, Chen TingJin, Lin Zhipeng, Jiang Hongye, Zhou Xinyi, Shi Cunbin, Pan Houjun, Chang Ouqin, Ren Pengli, Yu Jinyun, Li Xuerong, Xu Jin, Huang Yan, Yu Xinbing
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China; Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, Guangdong 510080, China.
Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China.
Fish Shellfish Immunol. 2017 May;64:287-296. doi: 10.1016/j.fsi.2017.03.030. Epub 2017 Mar 18.
Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 10, 1 × 10, and 1 × 10 CFU g) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 10 CFU g) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P < 0.05) higher levels of IgM in samples of serum, bile, mucus of surface and intestinal compared to the control groups. Abundant colonization spores expressing CsCP were found in hindgut that is conducive to absorption and presentation of antigen. Moreover, B. subtilis spores appeared to show no sign of toxicity or damage in grass carp. Our cercariae challenge experiments suggested that oral administration of spores expressing CsCP could develop an effective protection against C. sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish.
华支睾吸虫是一种食源性吸虫。人类可因摄入寄生在草鱼体内的华支睾吸虫囊蚴而感染。为诱导有效的口服免疫反应，枯草芽孢杆菌WB600的孢子被用作载体，将华支睾吸虫半胱氨酸蛋白酶（CsCP）与一种外壳蛋白CotC（B.s-CotC-CP）协同递送至胃肠道。在LB培养基中常规培养8 - 12小时后，含有CotC-CsCP的枯草芽孢杆菌被转移至芽孢形成培养基中。SDS-PAGE、蛋白质免疫印迹和生长曲线表明，重组WB600在37℃连续振荡（250转/分钟）条件下的最佳芽孢形成时间为24 - 30小时。草鱼被投喂添加了三种水平B.s-CotC-CP（1×10、1×10和1×10 CFU/g）的基础颗粒饲料。以商业颗粒饲料或添加仅表达CotC的孢子（1×10 CFU/g）作为对照饲料。我们的结果显示，与对照组相比，经基于饲料的B.s-CotC-CP口服免疫的草鱼产生了强烈的特异性免疫反应，血清、胆汁、体表黏液和肠道黏液样本中的IgM水平显著更高（P < 0.05）。在后肠发现了大量表达CsCP的定殖孢子，这有利于抗原的吸收和呈递。此外，枯草芽孢杆菌孢子在草鱼体内似乎未显示出毒性或损伤迹象。我们的尾蚴攻击实验表明，口服表达CsCP的孢子可在鱼体内对华支睾吸虫产生有效的保护作用。因此，本研究表明基于饲料的重组孢子可引发高水平的黏膜免疫和体液免疫，有望成为预防淡水鱼中华支睾吸虫囊蚴形成的候选疫苗。
