Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability

108

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probablenumber-cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg ⅐ min/liter were needed to inactivate approximately 0.5 log 10 and 2.0 log 10 units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg ⅐ min/liter were required to achieve approximately 2.0 log 10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.

Section: Methodsmentioning

confidence: 99%

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Chauret

Radziminski

Lepuil

et al. 2001

108

“…However, dyeing is influenced by the degree of oocyst permeabilization and may not reflect parasite infectivity (3,21). Oocyst infectivity can be evaluated by monitoring in vitro parasite development in highly permissive cells (9,13,24).…”

mentioning

confidence: 99%

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

Delaunay

Gargala

Li³

et al. 2000

The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.Cryptosporidium parvum is presently identified as a common cause of diarrhea in immunocompetent individuals. In immunodeficient individuals, cryptosporidiosis may lead to lifethreatening chronic diarrhea, and, because of the incidence of AIDS, the disease poses a significant public health problem in developing countries where AIDS is endemic (8,14,17,22).Recent outbreaks of cryptosporidiosis support the concern about C. parvum oocyst contamination of treated and surface water (15). In Sydney, Australia, from July to September 1998, contamination of the drinking water supply involved over 3,000,000 residents (16).Water oocyst count is a commonly used parameter to evaluate the infectious risk. However, the significance of oocyst numbers is questionable, since storage duration and environmental conditions, such as pH, temperature, and/or the presence of oxidants, are likely to influence oocyst viability (5, 10, 18). Moreover, factors such as salinity, temperature, or storage duration may not decrease the infectivity enough to prevent infection in susceptible individuals (11,12).Oocyst viability is currently estimated by the quantitation of in vitro excystation rates or by incorporation of nucleic acid dyes (7). However, dyeing is influenced by the degree of oocyst permeabilization and may not reflect parasite infectivity (3,21). Oocyst infectivity can be evaluated by monitoring in vitro parasite development in highly permissive cells (9,13,24). In vivo, C. parvum infection is usually investigated using susceptible animal models such as immunocompromised or neonatal mice (19).The aim of this work was to assess C. parvum oocyst infectivity using a suckling mouse model (6). Flow cytometry, a rapid and simple alternative to microscopy, was used to detect viable oocysts and to docume...

“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Neumann¹,

Gyürék²,

Gammie³

et al. 2000

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...