Hijjawi N S, Meloni B P, Ryan U M, Olson M E, Thompson R C A
Western Australian Biomedical Research Institute, Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia, 6150 Australia.
Int J Parasitol. 2002 Dec 19;32(14):1719-26. doi: 10.1016/s0020-7519(02)00199-6.
The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.
本研究描述了安氏隐孢子虫在HCT - 8细胞系中所有生命周期阶段的完整发育过程。体外培养方案与用于微小隐孢子虫所有生命周期阶段成功生长的方案相同（《国际寄生虫学杂志》31 (2001) 1048）。在这些培养条件下，安氏隐孢子虫生长迅速，感染后72小时内完成整个生命周期。安氏隐孢子虫的发育阶段比微小隐孢子虫的大，这使得包括一个以前未被识别的细胞外阶段在内的生命周期阶段更容易识别。从感染牛的粪便中使用激光显微切割技术分离出该细胞外阶段后，进一步证实了其存在。这个阶段大量存在，其中一些可见正在进行接合。从细胞外阶段提取DNA，随后进行聚合酶链反应 - 限制性片段长度多态性分析和18S rDNA测序，证实这是安氏隐孢子虫生命周期中的一个阶段。在体外，总是观察到细胞外阶段在感染了安氏隐孢子虫的HCT - 8细胞上移动。与微小隐孢子虫的比较观察也证实了细胞外阶段的存在。感染微小隐孢子虫牛基因型后5天从体外培养物中回收了细胞外阶段，并且也从感染的小鼠中回收了该阶段。感染72小时后从小鼠中至少纯化出两个形态不同的阶段（阶段一和阶段二）。隐孢子虫生命周期中细胞外发育阶段的存在及其形态特征证实了它与簇虫的关系，并为我们理解隐孢子虫属的分类学和系统发育亲缘关系提供了重要启示。安氏隐孢子虫在细胞培养中的生长现在提供了一种研究其发育、代谢和行为以及测试其对不同治疗剂反应的方法。
