Comparison of Animal Infectivity and Nucleic Acid Staining for Assessment of Cryptosporidium parvum Viability in Water

Abstract

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...

Monitoring of Waterborne Pathogens in Surface Waters in Amsterdam, The Netherlands, and the Potential Health Risk Associated with Exposure to Cryptosporidium and Giardia in These Waters

“…It has been demonstrated that the results of viability assays do not always correlate with the outcome of in vivo (43) and in vitro infectivity assays (16). Moreover, parasite genotypes could not be confirmed and a fraction of the (oo)cysts may belong to species other than those infectious to humans.…”

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

“…Results from several investigations have demonstrated that both microscopic assays correlate with results from the standard mouse infectivity and in vitro excystation assays (10,26). However, the interpretation of viability from both assays must be undertaken cautiously, since the tests are known to overestimate viability (7,10,28,50). Therefore, the extent to which rRNA probes are useful for oocyst viability studies depends on the decay rate of SSU rRNA in the environment, which likely varies depending on different environmental conditions (28).…”

Flow Cytometric Detection of Leishmania Parasites in Human Monocyte-Derived Macrophages: Application to Antileishmanial-Drug Testing

“…These techniques have largely been used in the fields of medical science and cell biology (10). Recently, flow cytometry has been introduced in the medical field of parasitology for the study of human cells infected with protozoan parasites (11,18,19). Many reliable techniques are now extensively used to detect parasites of the genus Plasmodium in human red blood cells (22,24), and various fluorescent probes or monoclonal antibodies have been developed for use in the exploration of interactions between parasites and host cells (21,24).…”