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.
Cryptosporidium parvum is an enteric coccidian protozoan which produces an environmentally stable oocyst that is excreted in the feces of infected individuals. There have been ten documented water borne outbreaks in North America. If food or beverages were prepared from contaminated water, that food or beverage would also be a hazard. The objective of this study was to evaluate the survival of Cryptosporidium parvum in beverages. Viability of oocysts, as determined by morphology decreased over 24 h exposure in carbonated beverages. Uptake of vital dyes indicated a loss of >85% of oocyst viability in beer or cola stored at 4C. Loss of viability in tap water, orange juice or infant formula was ± 35%. It is likely that the low pH of the carbonated beverages was involved in the loss of oocyst viability and premature excystation of the sporozoites.
The City of Tampa in the Southwest portion of Florida is examining the development of an advanced treatment facility for supplementing their potable water supply with highly treated reclaimed water. Full-scale microbiological monitoring of a similar treatment train demonstrated that the high-pH chemical treatment process was an effective barrier for removal and inactivation of viruses (99.99%), bacteria (99.9999%) and enteric protozoa (99.9%). A constructed pilot plant and bench-scale studies of the high-pH chemical process demonstrated that the removal of Cryptosporidium oocysts was due to physical removal and no inactivation was shown with up to 180 minutes exposure at a pH of 11.2. Combination of high-pH chemical treatment and chlorination were also ineffective at kill of Cryptosporidium oocysts.
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