Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

UV inactivation of Cryptosporidium hominis as measured in cell culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.     

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.     

Characterization of Cryptosporidium parvum by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers.     

Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples.

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.     

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.     

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

Characterization of Cryptosporidium parvum by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers.     

Patterns of Cryptosporidium oocyst shedding by eastern grey kangaroos inhabiting an Australian watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.     

Contamination of river water by Cryptosporidium parvum oocysts in western Japan

In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries.     

UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.     

The effect of sewage sludge treatment processes on oocysts of Cryptosporidium parvum

T.N. WHITMORE AND L.J. ROBERTSON. 1995. The effect of common sewage sludge treatment processes on oocysts of the coccidian protozoan Cryptosporidium was evaluated in laboratory simulations. The ability of primary sewage sedimentation to remove Cryptosporidium oocysts was found to be poor. Thermophilic (55°C) aerobic digestion and sludge pasteurization at the same temperature were found to be effective treatments to inactivate Cryptosporidium oocysts. Approximately 10% of the oocyst population were found to be viable after 18 d exposure to mesophilic (35°C) anaerobically digesting sludge. The viability of Cryptosporidium oocysts decreased within the range 20–40% in sludge-treated soil mesocosms over 30 d. The survival results obtained, however, indicated that oocysts would survive well beyond this period.     

Comparison of sensitivity of immunofluorescent microscopy to that of a combination of immunofluorescent microscopy and immunomagnetic separation for detection of Cryptosporidium parvum oocysts in adult bovine feces.

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.     

Identification of Cryptosporidium oocysts in river water.

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.     

Taking the eye strain out of environmental cryptosporidium analysis

The use of flow cytometry to detect Cryptosporidium oocysts in water was investigated using a Skatron Argos 100–5 instrument. Raw and treated drinking water samples seeded with oocysts and treated sewage samples known to contain oocysts were analysed by flow cytometry and by fluorescent microscopy. Oocysts could be rapidly detected in the treated sewage in raw and treated drinking water when the latter were seeded with levels of 1000 oocysts/1 or higher. Flow cytometry proved to be easier and quicker than fluorescent microscopy but an improvement in sensitivity is necessary before flow cytometery can be used for routine monitoring of drinking water supplies.