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Cryptosporidium cell culture infectivity assay design

机译：隐孢子虫细胞培养物感染性测定设计

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Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.
机译：引起胃肠道疾病隐孢子虫病的隐孢子虫属的成员仍然代表着全世界水传播疾病的重要原因。尽管已投入大量精力开发寄生虫培养技术，但体外生长受到许多因素的阻碍，这些因素包括感染力水平低，生命周期开发延迟和同步性差。在这项研究中，我们研究了影响囊肿子孢子与靶宿主细胞之间接触时间的因素，以及其对感染建立的后续影响。我们证明兴奋速率对感染的建立有影响，并且在我们的标准测定形式中，当子孢子从卵囊中释放出来以成功建立感染时，大多数子孢子离细胞单层不够近。但是，这可以通过将卵囊离心到细胞单层上而轻易克服，导致子孢子附着和随后的感染增加大约4倍。我们进一步证明，可以对准分子程序进行调整以控制准分子率以匹配分析的最终目的，并且准分子率可以影响数据解释。最后，在考虑用于发育分析和阶段特异性基因表达的体外培养实验的设计时，在子孢子附着后添加离心和洗涤步骤可能是合适的，因为这似乎增加了早期发育阶段的同步性。

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