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While the risk from pathogenic microorganisms in foods has been recognized for hundreds of years, bacterial agents are generally implicated as the contaminants. Although many outbreaks of gastroenteritis caused by protozoan pathogens have occurred, it is only in the last 3 years that attention has focused on protozoan association with foodborne transmission. Recognized as waterborne parasites, Giardia, Cryptosporidium, and Cyclospora have now been associated with several foodborne outbreaks. The oocysts and cysts of these organisms can persist and survive for long periods of time both in water and on foods. While Cyclospora oocysts require a maturation period, Cryptosporidium oocysts and Giardia cysts are immediately infectious upon excretion from the previous host. As a result, these parasites have emerged as public health risks and have become a concern to the food industry. More than 200 cases of foodborne giardiasis (seven outbreaks) were reported from 1979 to 1990. Four foodborne Cryptosporidium outbreaks (with a total of 252 cases) have been documented since 1993. Cyclospora caused a series of sporadic outbreaks of cyclosporasis throughout North America that have affected over 3,038 people since 1995. Control and prevention of protozoan foodborne disease depends upon our ability to prevent, remove, or kill protozoan contaminants. This review will address the biology, foodborne and waterborne transmission, survival, and methods for detection and control of Giardia, Cryptosporidium, and Cyclospora.

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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.

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Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.

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