Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples

Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to 272, average 1,503+/-475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water; however, the method requires further development to improve sensitivity and account for background signals.     

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples.

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inefficient. We recently described an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS-PCR assay to detect all cryptosporidial oocysts was developed, and both IMS-PCR assays were optimized on river water samples. A comparative study of the two IMS-PCR assays and the classical detection method based on an immunofluorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS-PCR took the form of IFA-negative/IMS-PCR-positive results, and was caused mainly by the greater sensitivity of IMS-PCR as compared with IFA. Of the 50 water samples, only five tested positive for C. parvum using IMS-PCR, and could constitute a threat to human health. These results show that both IMS-PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts.     

Survival of Cryptosporidium parvum oocysts under various environmental pressures.

The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures.     

Survival of Cryptosporidium parvum oocysts under various environmental pressures.

The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures.     

A rapid method for producing highly purified Cryptosporidium parvum oocysts

Most procedures that have been described for purifying Cryptosporidium parvum oocysts are designed to either identify the parasites in clinical specimens or isolate oocysts from a small volume of feces from infected animals. The present study describes a rapid method for purifying high numbers of C. parvum oocysts from feces of infected calves that contains minimal contaminating fecal material and bacteria. The isolation method is based on differential flotation of C. parvum oocysts in NaCl, followed by ether extraction to solubilize lipids in calf feces. This procedure regularly yields > 10(9) purified C. parvum oocysts within 1-2 days of feces collection.     

Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection

Abstract In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)), and infeictivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr ≤ 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.     

Evaluation of immunofluorescence techniques for detection of Cryptosporidium oocysts and Giardia cysts from environmental samples

Cryptosporidium and Giardia species are enteric protozoa which cause waterborne disease. The detection of these organisms in water relies on the detection of the oocyst and cyst forms or stages. Monoclonal and polyclonal antibodies were compared for their abilities to react with Giardia cysts and Cryptosporidium oocysts after storage in water, 3.7% formaldehyde, and 2.5% potassium dichromate, upon exposure to bleach, and in environmental samples. Three monoclonal antibodies to Cryptosporidium parvum were evaluated. Each test resulted in an equivalent detection of the oocysts after storage, after exposure to bleach, and in environmental samples. Oocyst levels declined slightly after 20 to 22 weeks of storage in water, and oocyst fluorescence and morphology were dull and atypical. Oocyst counts decreased after exposure to 2,500 mg of sodium hypochlorite per liter, and fluorescence and phase-contrast counts were similar. Sediment due to algae and clays found in environmental samples interfered with the detection of oocysts on membrane filters. Two monoclonal antibodies and a polyclonal antibody directed against Giardia lamblia cysts were evaluated. From the same seeded preparations, significantly greater counts were obtained with the polyclonal antibody. Of the two monoclonal antibodies, one resulted in significantly lower cyst counts. In preliminary studies, the differences between antibodies were not apparent when used on the environmental wastewater samples. After 20 to 22 weeks in water, cyst levels declined significantly by 67%. Cysts were not detected with monoclonal antibodies after exposure to approximately 5,000 mg of sodium hypochlorite per liter.     

Rotifers ingest oocysts of Cryptosporidium parvum

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.     

Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Evaluation of immunofluorescence techniques for detection of Cryptosporidium oocysts and Giardia cysts from environmental samples.

Cryptosporidium and Giardia species are enteric protozoa which cause waterborne disease. The detection of these organisms in water relies on the detection of the oocyst and cyst forms or stages. Monoclonal and polyclonal antibodies were compared for their abilities to react with Giardia cysts and Cryptosporidium oocysts after storage in water, 3.7% formaldehyde, and 2.5% potassium dichromate, upon exposure to bleach, and in environmental samples. Three monoclonal antibodies to Cryptosporidium parvum were evaluated. Each test resulted in an equivalent detection of the oocysts after storage, after exposure to bleach, and in environmental samples. Oocyst levels declined slightly after 20 to 22 weeks of storage in water, and oocyst fluorescence and morphology were dull and atypical. Oocyst counts decreased after exposure to 2,500 mg of sodium hypochlorite per liter, and fluorescence and phase-contrast counts were similar. Sediment due to algae and clays found in environmental samples interfered with the detection of oocysts on membrane filters. Two monoclonal antibodies and a polyclonal antibody directed against Giardia lamblia cysts were evaluated. From the same seeded preparations, significantly greater counts were obtained with the polyclonal antibody. Of the two monoclonal antibodies, one resulted in significantly lower cyst counts. In preliminary studies, the differences between antibodies were not apparent when used on the environmental wastewater samples. After 20 to 22 weeks in water, cyst levels declined significantly by 67%. Cysts were not detected with monoclonal antibodies after exposure to approximately 5,000 mg of sodium hypochlorite per liter.     

Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts

Cryptosporidium parvum is an enteric protozoan parasite of medical and veterinary importance. Dissemination of environmentally resistant oocysts in surface water plays an important role in the epidemiology of cryptospridiosis. Although the polymerase chain reaction (PCR) is a well-established technique and is widely used for detecting microorganisms, it is not routinely applied for monitoring waterborne C. parvum. In order to facilitate the application of PCR to the detection of waterborne C. parvum oocysts, a comparison of published PCR protocols was undertaken and different sample-preparation methods tested. The sensitivity of a one-step PCR method, consisting of 40 temperature cycles, was 10 purified oocysts or fewer than 100 oocysts spiked in raw lake water. The detection limit of two primer pairs, one targeting the ribosomal small subunit and another specific for a C. parvum sequence of unknown function, was approximately ten-fold lower than achieved with a primer pair targeting an oocyst shell protein gene. Three cycles of freezing/thawing were sufficient to expose oocyst DNA and resulted in higher sensitivity than proteinase K digestion, sonication or electroporation. Inhibition of PCR by surface water from different local sources was entirely associated with the soluble fraction of lake water. Membrane filtration was evaluated in bench-scale experiments as a means of removing lake water inhibitors and improving the detection limit of PCR. Using gel and membrane filtration, the molecular size of inhibitory solutes from lake water was estimated to less than 27 kDa. Received: 14 November 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts

Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l^?1) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4′,6‐diamidino‐2‐phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0·005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from ～80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH₃ l^?1 and 50 mg NH₃ l^?1 for 4 days reduced their viability from between ～80% to 41·5% and 14·8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga‐based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga‐based wastewater‐treatment systems in the northwest of Spain.     

Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples

Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze-thaw cycles. The second method involved filtration, addition of EnviroAmp lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (> 10(5)/g) of C. parvum oocysts, this correlation was 100%.     

Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.     

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.