Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.     

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.     

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications to United States Environmental Protection Agency methods 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% +/- 11.8%, while the mean cyst recovery was 57.1% +/- 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% +/- 16.3% for oocysts and 49.4% +/- 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% +/- 13.8%, while the mean cyst recovery percentages was 41.2% +/- 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% +/- 11.1% and 61.3% +/- 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

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.     

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

AIMS: To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Alava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters. METHODS AND RESULTS: A total of 284 samples of drinking and recreational water supplies were analysed. Cryptosporidium oocysts were found in 63.5% of river samples, 33.3% of reservoirs samples, 15.4% and 22.6% of raw water samples from conventional and small water treatment facilities (respectively), 30.8% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. Giardia cysts were found in 92.3% of river samples, 55.5% of reservoirs samples, 26.9% and 45.2% of raw water samples from conventional and small water treatment facilities (respectively), 19.2% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. The presence of Cryptosporidium and Giardia had significant Pearson's correlation coefficients (P < 0.01) with the turbidity levels of the samples, and a number of significant associations were also found with the count levels for total coliforms and Escherichia coli. The samples were positive for Cryptosporidium significantly (P < 0.05) more frequently during the autumn season than during the spring and winter seasons. No significant differences were found in the seasonal pattern of Giardia. A moderate association (r = 0.52) was found between rainfall and the presence of Cryptosporidium oocysts. CONCLUSIONS: Cryptosporidium and Giardia are consistently found at elevated concentrations in surface waters for human consumption from the province of Alava, northern Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Water treatments based on rapid filtration process and/or chlorination only are often unsatisfactory to provide safe drinking water, a situation that represents an important public health problem for the affected population because of the risk of waterborne outbreaks.     

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.     

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.     

Comparison of methods for the concentration of Cryptosporidium oocysts and Giardia cysts from raw waters

This study compared the recovery of Cryptosporidium oocysts and Giardia cysts ((oo)cysts) from raw waters using 4 different concentration-elution methods: flatbed membranes, FiltaMax foam, Envirochek HV capsules, and Hemoflow ultrafilters. The recovery efficiency of the combined immunomagnetic separation and staining steps was also determined. Analysis of variance of arcsine-transformed data demonstrated that recovery of Cryptosporidium oocysts by 2 of the methods was statistically equivalent (flatbed filtration 26.7% and Hemoflow 28.3%), with FiltaMax and Envirochek HV recoveries significantly lower (18.9% and 18.4%). Recovery of Giardia cysts was significantly higher using flatbed membrane filtration (42.2%) compared with the other 3 methods (Envirochek HV 29.3%, FiltaMax 29.0%, and Hemoflow 20.9%). All methods were generally acceptable and are suitable for laboratory use; 2 of the methods are also suitable for field use (FiltaMax and Envirochek HV). In conclusion, with recoveries generally being statistically equivalent or similar, practical considerations become important in determining which filters to use for particular circumstances. The results indicate that while low-turbidity or "finished" waters can be processed with consistently high recovery efficiencies, recoveries from raw water samples differ significantly with variations in raw water quality. The use of an internal control with each raw water sample is therefore highly recommended.     

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water.

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.     

Monitoring of waterborne pathogens in surface waters in amsterdam, the Netherlands, and the potential health risk associated with exposure to cryptosporidium and giardia in these waters

The water in the canals and some recreational lakes in Amsterdam is microbiologically contaminated through the discharge of raw sewage from houseboats, sewage effluent, and dog and bird feces. Exposure to these waters may have negative health effects. During two successive 1-year study periods, the water quality in two canals (2003 to 2004) and five recreational lakes (2004 to 2005) in Amsterdam was tested with regard to the presence of fecal indicators and waterborne pathogens. According to Bathing Water Directive 2006/7/EC, based on Escherichia coli and intestinal enterococcus counts, water quality in the canals was poor but was classified as excellent in the recreational lakes. Campylobacter, Salmonella, Cryptosporidium, and Giardia were detected in the canals, as was rotavirus, norovirus, and enterovirus RNA. Low numbers of Cryptosporidium oocysts and Giardia cysts were detected in the recreational lakes, despite compliance with European bathing water legislation. The estimated risk of infection with Cryptosporidium and Giardia per exposure event ranged from 0.0002 to 0.007% and 0.04 to 0.2%, respectively, for occupational divers professionally exposed to canal water. The estimated risk of infection at exposure to incidental peak concentrations of Cryptosporidium and Giardia may be up to 0.01% and 1%, respectively, for people who accidentally swallow larger volumes of the canal water than the divers. Low levels of viable waterborne pathogens, such as Cryptosporidium and Giardia, pose a possible health risk from occupational, accidental, and recreational exposure to surface waters in Amsterdam.     

Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds.

Two adjacent British Columbia, Canada, watersheds with similar topographical features were studied. Both the Black Mountain Irrigation District (BMID) and the Vernon Irrigation District (VID) serve rural agricultural communities which are active in cattle ranching. The present study was carried out in five phases, during which a total of 249 surface water samples were tested in the study watersheds. The aims of these phases were to determine levels of parasite contamination in raw water samples collected from the intakes as well as from other sites in each watershed and to investigate cattle in the watersheds as potential sources of parasite contamination of surface drinking water supplies. Giardia cysts were not detected in the raw water samples collected from lake sources at the headwaters of both watersheds but were found in 100% (70 or 70) of water samples collected at the BMID intake and 97% (68 of 70) of water samples collected at the VID intake. Significantly higher levels (P < 0.05) of Giardia cysts were found at the BMID intake (phase 1, 7 to 2,215 cysts per 100 liters; phase 3, 4.6 to 1,880 cysts per 100 liters) when compared with that of the VID intake (2 to 114 cysts per 100 liters). The BMID watershed has a more complex system of surface water sources than the VID watershed. Cattle have access to creeks in the BMID watershed, whereas access is restricted in the VID watershed. Collection of raw water samples from a creek upstream and downstream of a cattle ranch in the BMID watershed showed that the downstream location had significantly higher (P < 0.05) levels (0.6 to 42.9 cysts per 100 liters and 1.4 to 300.0 oocysts per 100 liters) of both Giardia cysts and Cryptosporidium oocysts than those of the upstream location (0.5 to 34.4 cysts per 100 liters and 0.5 to 34.4 oocysts per 100 liters). Peak concentrations of both parasites coincided with calving activity. Fecal samples, collected from cattle in both watersheds, showed 10% (3 of 30) in the BMID and 50% (5 of 10) in the VID watersheds to be Giardia positive. No Cryptosporidium-positive fecal samples were found. Giardia cysts isolated from the BMID watershed were repeatedly infective to gerbils in contrast to those from the VID watershed. The 10 BMID drinking water Giardia isolates retrieved into culture and biotyped showed zymodeme and karyotype heterogeneity. The differences in patterns of parasite contamination and cattle management practices contribute to the unique watershed characteristics observed between two areas which are topographically similar and geographically adjacent.     

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.     

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.     

Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples

AIMS: An internal positive control for Cryptosporidium and Giardia monitoring was evaluated for use in routine water monitoring quality control. The control, known as ColorSeed C&G (BTF Pty Ltd, Sydney, Australia), is a suspension containing exactly 100 Cryptosporidium oocysts and 100 Giardia cysts that have been modified by attachment of Texas Red to the cell wall, allowing them to be differentiated from unmodified oocysts and cysts. The control enables recovery efficiencies to be determined for every water sample analysed. METHODS AND RESULTS: A total of 494 water samples were seeded with ColorSeed C&G and with unlabelled Cryptosporidium and Giardia and then analysed. Additionally, the robustness of the ColorSeed labelling was challenged with various chemical treatments. Recoveries were significantly lower for the ColorSeed Texas Red labelled Cryptosporidium and Giardia than recoveries of unlabelled Cryptosporidium and Giardia. However, the differences in recoveries were small. On average ColorSeed Cryptosporidium recoveries were 3.3% lower than unlabelled Cryptosporidium, and ColorSeed Giardia recoveries were 4% lower than unlabelled Giardia. CONCLUSIONS: ColorSeed C&G is suitable for use as an internal positive control for routine monitoring of both treated and raw water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The small differences in recoveries are unlikely to limit the usefulness of ColorSeed C&G as an internal positive control. The ColorSeed labelling was found to be robust after different treatments.     

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Fran?aise de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.     

Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species

Cryptosporidium spp. and Giardia spp. are protozoan parasites that are often associated with severe diarrheal disease in a variety of mammals. Although these parasites have been extensively studied in terrestrial ecosystems, little is known about either parasite in the marine environment. Therefore, the objective of this study was to determine the prevalence of both Cryptosporidium spp. and Giardia spp. in 5 marine mammal species. Fecal samples were collected from 39 bowhead whales (Balaena mysticetus), 49 North Atlantic right whales (Eubalaena glacialis), 31 ringed seals (Phoca hispida), 22 bearded seals (Erignathus barbatus), and 18 beluga whales (Delphinapterus leucas) between 1998 and 2003. Using an immunofluorescent assay, parasites were detected in the feces of bowhead whales, right whales, and ringed seals, while neither parasite was detected in samples from bearded seals or beluga whales. Overall, prevalences were highest in ringed seals (Cryptosporidium spp., 22.6%; Giardia spp., 64.5%) and right whales (Cryptosporidium spp., 24.5%; Giardia spp., 71.4%) and lowest in bowhead whales (Cryptosporidium spp., 5.1%; Giardia spp., 33.3%). To our knowledge, this is the first report of Cryptosporidium spp. and Giardia spp. in either whale species and of Cryptosporidium spp. in the ringed seal.