Slifko T R, Friedman D, Rose J B, Jakubowski W
Department of Marine Science, University of South Florida, St. Petersburg, USA.
Appl Environ Microbiol. 1997 Sep;63(9):3669-75. doi: 10.1128/aem.63.9.3669-3675.1997.
Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.
当前用于检测水中隐孢子虫卵囊的检测方法通常无法评估其活力或感染性。本研究开发了一种方法，通过使用培养的HCT-8细胞作为微小隐孢子虫生殖阶段的宿主，对感染性卵囊进行低水平检测。通过在间接抗体检测中用针对生殖阶段的一抗和异硫氰酸荧光素偶联的二抗标记寄生虫的细胞内发育阶段，来检测感染灶。完整的检测方法被命名为病灶检测法（FDM）。通过落射荧光显微镜对感染灶（表明从一个活卵囊中释放的四个子孢子中至少有一个感染了细胞）进行计数，并在诺马斯基微分干涉相差显微镜下进行确认。时间序列实验表明，宿主HCT-8细胞中的自身感染生命周期在孵育12小时后开始。通过稀释研究，检测到低至一个感染性卵囊的水平。细胞培养FDM与其他活力检测方法相比效果良好。活体染色和脱囊实验表明，活力低于1%（通过活体染料）且脱囊后子孢子产量低的卵囊群体无法感染宿主细胞。到目前为止，水行业一直依赖一种（根据信息收集规定的）卵囊检测方法，该方法无法确定活力。所述细胞培养方法的可量化结果证明了两个重要应用：（i）一种可与美国环境保护局目前规定的检测方法结合使用的感染性检测方法，以及（ii）一种在存活和消毒研究中评估卵囊感染性的方法。
