Detection of Cryptosporidium Oocysts in Water: Effect of the Number of Samples and Analytic Replicates on Test Results

Due to the small number of Cryptosporidium oocysts in water, the number of samples taken and the analyses performed can affect the results of detection. In this study, 42 water samples were collected from one watershed during 20 storm events over 1 year, including duplicate or quadruplicate samples from 16 storm events. Ten samples from four events had three to eight subsamples. They were processed by EPA method 1623, and Cryptosporidium oocysts present were detected by immunofluorescent microscopy or PCR. Altogether, 24 of 39 samples (47 of 67 samples and subsamples) analyzed by microscopy were positive for Cryptosporidium. In contrast, 36 of 42 samples (62 of 76 samples and subsamples) were positive by PCR, including 10 microscopy-negative samples (13 microscopy-negative samples and subsamples). Six of the 24 microscopy-positive samples were negative by PCR, and all samples had one or less oocyst in a 0.5-ml packed pellet volume calculated. Discordant results were obtained by microscopy and PCR from six and three of the storm events, respectively, with multiple samples. Discordant microscopy or PCR results were also obtained among subsamples. Most of the 14 Cryptosporidium genotypes were found over a brief period. Cryptosporidium-positive samples had a mean of 1.9 genotypes per sample, with 39 of the 62 positive samples/subsamples having more than one genotype. Samples/subsamples with more than one genotype had an overall PCR-positive rate of 73%, compared to 34% for those with one genotype. The PCR amplification rate of samples was affected by the volume of DNA used in PCR.     

Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.     

Distribution of Cryptosporidium Genotypes in Storm Event Water Samples from Three Watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.     

Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples.     

Cryptosporidium genotypes in wildlife from a new york watershed

To identify the animal sources for Cryptosporidium contamination, we genotyped Cryptosporidium spp. in wildlife from the watershed of the New York City drinking water supply, using a small-subunit rRNA gene-based PCR-restriction fragment length polymorphism analysis and DNA sequencing. A total of 541 specimens from 38 species of wildlife were analyzed. One hundred and eleven (20.5%) of the wildlife specimens were PCR positive. Altogether, 21 Cryptosporidium genotypes were found in wildlife samples, 11 of which were previously found in storm runoff in the watershed, and six of these 11 were from storm water genotypes of unknown animal origin. Four new genotypes were found, and the animal hosts for four storm water genotypes were expanded. With the exception of the cervine genotype, most genotypes were found in a limited number of animal species and have no major public health significance.     

Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters

Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.     

Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.     

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.     

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.     

Genotypes of Cryptosporidium from Sydney water catchment areas

AIMS: Currently cryptosporidiosis represents the major public health concern of water utilities in developed nations and increasingly, new species and genotypes of Cryptosporidium are being identified in which the infectivity for humans is not clear. The complicated epidemiology of Cryptosporidium and the fact that the majority of species and genotypes of Cryptosporidium cannot be distinguished morphologically makes the assessment of public health risk difficult if oocysts are detected in the raw water supplies. The aim of this study was to use molecular tools to identify sources of Cryptosporidium from the Warragamba catchment area of Sydney, Australia. METHODS AND RESULTS: Both faecal and water samples from the catchment area were collected and screened using immunomagnetic separation (IMS) and immunofluorescence microscopy. Samples that contained Cryptosporidium oocysts were genotyped using sequence and phylogenetic analysis of the 18S rDNA, and the heat-shock (HSP-70) gene. Analysis identified five Cryptosporidium species/genotypes including C. parvum (cattle genotype), C. suis, pig genotype II, the cervid genotype and a novel goat genotype. CONCLUSIONS: Monitoring and characterization of the sources of oocyst contamination in watersheds will aid in the development and implementation of the most appropriate watershed management policies to protect the public from the risks of waterborne Cryptosporidium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that quantification by IMS analysis can be combined with the specificity of genotyping to provide an extremely valuable tool for assessing the human health risks from land use activities in drinking water catchments.     

Cryptosporidium proventriculi sp. n. (Apicomplexa: Cryptosporidiidae) in Psittaciformes birds

Cryptosporidiosis is a common parasitic infection in birds that is caused by more than 25 Cryptosporidium species and genotypes. Many of the genotypes that cause avian cryptosporidiosis are poorly characterized. The genetic and biological characteristics of avian genotype III are described here and these data support the establishment of a new species, Cryptosporidium proventriculi. Faecal samples from the orders Passeriformes and Psittaciformes were screened for the presence of Cryptosporidium by microscopy and sequencing, and infections were detected in 10 of 98 Passeriformes and in 27 of 402 Psittaciformes. Cryptosporidium baileyi was detected in both orders. Cryptosporidium galli and avian genotype I were found in Passeriformes, and C. avium and C. proventriculi were found in Psittaciformes. Cryptosporidium proventriculi was infectious for cockatiels under experimental conditions, with a prepatent period of six days post-infection (DPI), but not for budgerigars, chickens or SCID mice. Experimentally infected cockatiels shed oocysts more than 30 DPI, with an infection intensity ranging from 4,000 to 60,000 oocysts per gram (OPG). Naturally infected cockatiels shed oocysts with an infection intensity ranging from 2,000 to 30,000 OPG. Cryptosporidium proventriculi infects the proventriculus and ventriculus, and oocysts measure 7.4 × 5.8 μm. None of the birds infected C. proventriculi developed clinical signs.     

Occurrence, source, and human infection potential of cryptosporidium and Giardia spp. in source and tap water in shanghai, china

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative.     

Simultaneous detection of Cryptosporidium parvum and Giardia in sewage sludge by IC-PCR

AIMS: The aim of this study was to develop a method based on immunomagnetic capture and polymerase chain reaction (IC-PCR assay) for detection of Cryptosporidium parvum and Giardia intestinalis in sewage sludge. METHODS AND RESULTS: The detection limit of the IC-PCR assay for both organisms was 625 oocysts and cysts ml(-1). By hybridization of PCR products the sensitivity could be increased to 125 oocysts and cysts ml(-1). Forty-four sludge samples from 12 wastewater treatment plants were examined. The samples positive for Giardia (9 out of 44) were from eight wastewater plants and the C. parvum genotype 2 samples (3 out of 44) originated from different sewage works. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: IC-PCR offers the possibility to distinguish between Cryptosporidium and Giardia genotypes. This assay can be used to monitor the presence of these organisms in a community and to determine contamination of sludge used as soil amendment.     

Prevalence and identity of Cryptosporidium spp. in pig slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

Genotypes of Cryptosporidium species infecting fur-bearing mammals differ from those of species infecting humans

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.     

Occurrence of Cryptosporidium and Giardia in wild ducks along the Rio Grande River valley in southern New Mexico.

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean +/- standard deviation, 47.53 +/- 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean +/- standard deviation, 436 +/- 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.     

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.