Development of an in vitro culture technique for Cryptococaryon irritans causing the white spot disease of marine fishes

引起海水鱼类白斑病的隐核虫刺激物体外培养技术的开发

• 批准号: 16580145
• 负责人: YOSHINAGA Tomoyoshi
• 金额: $ 2.37万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580145/
• 关键词: White spot disease of marine fishes; Cryptocaryon irritans; in vitro culture; Parasite; fish pathology; ciliate; in vitro culture; 培養; 魚類培養細胞; 繊毛虫; 海産魚; In vitro culture

An in vitro culture technique for Cryptocaryon irritans, the causative agent of white spot disease of marine fishes, was developed. A medium consisting of a fish culture cell layer and an agarose gel layer (Leibovit's L-15, 100 um/ml penicillin, 100iu/m1 streptomycin, 10% fetal calf serum, 0.22% agarose) was developed and used for culture. Thereonts, the infective stage of the parasite, grew to ca. 200 urn long in the media for 4 days, when inserted between the layers, although the parasites gradually decreased in number in the medium. Parasites grown in the medium accomplished normal development when transferred into seawater ; they were encysted, performed cell fissions and released infective theronts. This is the first case, in which the parasite completed its life cycle without host fish.When black mollies were challenged with theronts produced with the above culture technique, 40% of parasites were recovered from the fish ; the recovery rate was comparable to previously reported recovery rates in therents generated using fish host. This indicated that the parasites produced in vitro had infecting ability comparable to those produced in vivo.By using the in vitro culture technique, C. irritans was grown well. However, host fish is still necessary to obtain theronts for the culture. A technique for repeatedly complete the life cycle without host fishes is needed for future developments of vaccines and chemotherapies for the disease.

研究了海鱼白斑病病原菌隐核虫的离体培养技术。配制由鱼培养细胞层和琼脂糖凝胶层（Leibovit’s L-15, 100 um/ml青霉素，100iu/m1链霉素，10%胎牛血清，0.22%琼脂糖）组成的培养基进行培养。因此，当寄生虫在两层之间插入时，寄生虫的感染期在培养基中生长到约200缸长，持续4天，尽管寄生虫在培养基中的数量逐渐减少。在培养基中生长的寄生虫进入海水后发育正常；它们被包裹，进行细胞分裂并释放感染性细胞。这是寄生虫在没有宿主鱼的情况下完成其生命周期的第一例。用上述培养技术生产的幼虫攻毒后，黑鳉鱼体内的寄生虫回收率为40%；回收率与先前报道的使用鱼类宿主产生的昆虫的回收率相当。这表明体外产生的寄生虫具有与体内产生的寄生虫相当的感染能力。采用体外培养技术，可获得良好的体外培养效果。然而，宿主鱼仍然是获得养殖所需的鱼类。在没有寄主鱼类的情况下重复完成生命周期的技术是未来开发该疾病疫苗和化疗的必要条件。