Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.
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 × 106, 1 × 107, and 1 × 108CFU g-1) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 107CFU g-1) 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.
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.
Zeli
Z
Tang
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.
Hengchang
H
Sun
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.
TingJin
T
Chen
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.
Zhipeng
Z
Lin
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.
Hongye
H
Jiang
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.
Xinyi
X
Zhou
Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China.
Cunbin
C
Shi
Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China.
Houjun
H
Pan
Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China.
Ouqin
O
Chang
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.
Pengli
P
Ren
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.
Jinyun
J
Yu
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.
Xuerong
X
Li
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. Electronic address: xujin@mail.sysu.edu.cn.
Jin
J
Xu
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. Electronic address: huang66@mail.sysu.edu.cn.
Yan
Y
Huang
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. Electronic address: yuxb@mail.sysu.edu.cn.
Xinbing
X
Yu
D000284:Administration, Oral; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D002347:Carps; D003003:Clonorchiasis / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control / Q000662:veterinary*; D003004:Clonorchis sinensis / Q000737:chemistry; D057056:Cysteine Proteases / Q000378:metabolism*; D004032:Diet / Q000662:veterinary; D019587:Dietary Supplements; D005393:Fish Diseases / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control*; D015801:Helminth Proteins / Q000378:metabolism; D056724:Immunity, Humoral; D018928:Immunity, Mucosal; D030781:Organisms, Genetically Modified; D019936:Probiotics; D011897:Random Allocation; D013171:Spores, Bacterial / Q000235:genetics / Q000276:immunology*; D014612:Vaccines / Q000276:immunology
D015801:Helminth Proteins; D014612:Vaccines; D057056:Cysteine Proteases
Bacillus subtilis spore; Clonorchis sinensis; Cysteine protease; Feed-based immunization; Grass carp