Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.Cryptosporidium parvum is now recognized as a frequent cause of waterborne disease in humans (1,10,16,17,20). A primary means of parasite transmission is via drinking water, through the use of untreated surface water, contaminated distribution systems, or water treatment facilities employing only chlorine disinfection protocols. Significant morbidity and mortality have been associated with outbreaks of this parasite, particularly in immunocompromised individuals and in children (10).An ongoing challenge of detection and disinfection of Cryptosporidium spp. is the difficulty in determining whether a parasite is viable. The presence of dead parasites in finished water is of little concern for disease transmission. Animals have been used as surrogates for determining the infectious potential of C. parvum oocysts (13). However, the animal infectivity method is tedious, difficult, and expensive and is not readily amenable to normal laboratory analysis in the water industry. Several methods have been used to estimate the viability of parasites, including in vitro excystation (1,6,7,25,26), infection of cell lines (12,24,27), parasite morphology by light microscopy, the uptake or exclusion of fluorogenic dyes (4,5,8,9,23), and animal infectivity (2-5, 15, 18, 19, 21, 22, 29, 30). Other assays that allow determination of viability of C. parvum oocysts include immunomagnetic capture PCR (28) and fluorescence in situ hybridization (FISH) techniques (11,32,33). Of all of these methods, only animal infectivity provides direct information about the ability of the parasite to cause disease.In previous studies, we examined the potential use of fluorescent nucleic acid binding dyes as indicators of C. parvum oocyst viability under different experimental conditions. We reported that the staining of C. parvum oocysts with the nucleic acid binding dyes SYTO-9 and SYTO-59 correlated with the viability of these organisms, with heat-killed oocyst preparations used as a positive control (4, 5). In the present study, we demonstrate that fluorescence intensity of SYTO-9-and SYTO-59-stained C. parvum oocysts directly correlates with animal infectivity.
Source of C. parvum oocysts.The strain of C. parvum used in this study was originally isolated by Harley Moon (National Animal Disease Center, Ames, Iowa) and is referred to as the Iowa strain. C. parvum oocysts were isolated from the fece...