AN IN-VITRO METHOD FOR DETECTING INFECTIOUS CRYPTOSPORIDIUM OOCYSTS WITH CELL-CULTURE

Current assay methods to detect Cryptosporidium oocysts in water are g enerally not able to evaluate viability or infectivity. A method was d eveloped for low-level detection of infective oocysts by using HCT-8 c ells in culture as hosts to C. parvum reproductive stages. The infecti ve foci were detected by labeling intracellular developmental stages o f the parasite in an indirect-antibody assay with a primary antibody s pecific for reproductive stages and a secondary fluorescein isothiocya nate-conjugated antibody. The complete assay was named the focus detec tion method (FDM). The infectious foci (indicating that at least one o f the four sporozoites released from a viable oocyst had infected a ce ll) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series ex periments demonstrated that the autoreinfective life cycle in host HCT -8 cells began after 12 h of incubation. Through dilution studies, lev els as Low as one infectious oocyst were detected. The cell culture FD M compared well to other viability assays. Vital stains and excystatio n demonstrated that oocyst populations less than 1% viable (by vital d yes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an ooc yst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cel l 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, an d (ii) a method to evaluate oocyst infectivity in survival and disinfe ction studies.