The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidiumparvum oocysts

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0·998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum -specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.     

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.     

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water

AIM: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. METHODS AND RESULTS: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. CONCLUSIONS: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

The effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum on beef surfaces

AIMS: The aim of this research was to examine the effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum oocysts attached to a beef surface. METHODS AND RESULTS: This study examined the effects of heat treatment (60 or 75 degrees C) on the viability of C. parvum oocysts inoculated onto the surface of beef muscle estimated by vital dye assay. The infectivity of the oocysts was assessed against monolayers of HCT-8 cells. At 60 degrees C viability of the oocysts decreased from 100% at T0 to 64.2% at T60. At 75 degrees C the viability of the oocysts decreased from 100% at T0 to 53.7% at T15 and finally to 11.2% at T60. Oocysts were rendered noninfective against monolayers of HCT-8 cells following treatments of 60 degrees C/45 s and 75 degrees C/20 s. CONCLUSION: The washing of carcasses with hot water and standard thermal treatments is sufficient to kill C. parvum on beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that relatively mild heat, currently used to decontaminate and heat treat beef carcasses and to cook meat products, is capable of inactivating C. parvum.     

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.     

Impact of zooplankton grazing on the excystation, viability, and infectivity of the protozoan pathogens Cryptosporidium parvum and Giardia lamblia

Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 x 10(4) per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 x 10(4) cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20 degrees C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

The effects of time and temperature on flow cytometry enumerated live Cryptosporidium parvum oocysts

AIMS: Recoveries of spiked standard suspensions are used to evaluate method performance. For many applications, gamma-irradiated Cryptosporidium oocysts are appropriate. In contrast, methods that determine viability, such as Cryptosporidium cell culture, require the use of live oocysts. Oocyst standards are usually prepared at a flow cytometry laboratory for use at another laboratory, and thus the samples are shipped. The goal of this study was to evaluate the shipping and storage stability of flow cytometry enumerated live oocysts over time at three temperatures: 4 degrees C, room temperature and 37 degrees C. METHODS AND RESULTS: Replicate samples containing 100 live C. parvum oocysts were prepared by flow cytometry and stored at 4 degrees C, room temperature and 37 degrees C. These samples were counted at various time points. Significant oocyst losses were observed after storage for 1 day at 37 degrees C, 7 days at room temperature and 21 days at 4 degrees C. CONCLUSIONS: Live C. parvum oocysts internal standards should be used within 10 days of preparation, and stored and shipped at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: When evaluating method performance with live oocysts, both the storage temperature and time are critical factors for obtaining reliable and accurate results.     

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.     

Survival of Cryptosporidium species in environments relevant to foods and beverages

AIMS: To provide data on the survival of Cryptosporidium oocysts in a range of conditions relevant to foods and beverages. METHODS AND RESULTS: Cryptosporidium parvum and C. hominis oocysts were stored in buffered media at different pH values and with various acids. In addition, neutral solutions with high salt (4.5% w/v), glycerol (20% v/v), sucrose (50% w/v) or ethanol (9 and 40% v/v) were used to determine their effects on survival. After storage periods of between 1 h and 14 days, viability was assessed using sporozoite ratio or infection of MRC-5 cell monolayers (not previously reported for culture of this organism). With all treatments, and with both assay techniques, viable oocysts were found at the end of the storage periods. However, treatments with one of the following additions: high salt, glycerol, sucrose or ethanol showed a negative and statistically significant effect on survival. Decline was noted after 1 day or even 1 h of treatment. CONCLUSIONS: MRC-5 cells are suitable for infection by C. parvum and C. hominis. Both tissue culture and sporozoite ratio gave broadly similar survival results and the greatest effects were seen with addition of components which reduced water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful additional information to the food industry when considering the risk posed by this organism.     

Development of a TaqMan quantitative PCR assay specific for Cryptosporidium parvum

A rapid detection method that is both quantitative and specific for the water-borne human parasite Cryptosporidium parvum is reported. Real-time polymerase chain reaction (PCR) combined with fluorescent TaqMan technology was used to develop this sensitive and accurate assay. The selected primer-probe set identified a 138-bp section specific to a C. parvum genomic DNA sequence. The method was optimized on a cloned section of the target DNA sequence, then evaluated on C. parvum oocyst dilutions. Quantification was accomplished by comparing the fluorescence signals obtained from test samples of C. parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. This real-time PCR assay allowed reliable quantification of C. parvum oocysts over six orders of magnitude with a baseline sensitivity of six oocysts in 2 h.     

Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.     

Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland

AIMS: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996-1999, and to compare conventional and molecular methods of detection. METHODS AND RESULTS: Four hundred and seventy-four waters were investigated by conventional methods, namely immuno-fluorescent antibody detection (IFA; 380) and immuno-magnetic separation-IFA (IMS-IFA; 94), of which 14/474 (3%) were positive. Two hundred and fourteen samples (214/474) were also investigated by PCR techniques, targeting both the 18S rRNA and TRAP-C2 genes, of which 11/214 (5.1%) were positive. These 11 samples were classified as genotype II following sequence analysis of the TRAP-C2 amplicon. CONCLUSIONS: This study demonstrated the low incidence of oocysts of C. parvum in water sources in Northern Ireland. SIGNIFICANCE AND IMPACT OF THE STUDY: Such molecular-based techniques offer a number of advantages over conventional detection methodologies, namely greater sensitivity and specificity as well as the ability to provide accurate genotyping data rapidly, which may be valuable in directing operational management in potential outbreak situations.     

Evaluation of Cryptosporidium parvum oocyst recovery efficiencies from various filtration cartridges by electrochemiluminescence assays

AIMS: To evaluate four types of filtration cartridges for their capacities, efficiency for capture and release of Cryptosporidium parvum oocysts for detection. METHODS AND RESULTS: Filtration cartridges included in this evaluation were IDEXX Filta-Max, Gelman Envirochek HV, Corning CrypTest, and Filterite Sigma+. Various dosages of C. parvum oocysts were spiked into water samples with a wide range of turbidity (10-50 NTU). Electrochemiluminescence assays were employed to enumerate viable or total number of C. parvum oocysts in these eluates. Among the cartridges tested, Filta-Max consistently showed higher oocyst recovery efficiency, especially with large volume, highly turbid water samples. CONCLUSIONS: Filta-Max filter is the best performer because of its higher oocyst recovery efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall sensitivities of various C. parvum oocyst detection assays in water samples can be improved if highly efficient oocyst recovery filtration cartridges such as Filta-Max are incorporated in sample preparation.