Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of > 20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and > 80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

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.     

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.     

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)     

Giardia and Cryptosporidium in mammalian wildlife--current status and future needs

Environmental pollution with human and domestic-animal fecal material is recognized as a potential pathogen pathway for wildlife infections with zooanthropomorphic protozoan parasites such as Giardia and Cryptosporidium. In this article, we review current knowledge about the diversity of free-living and captive terrestrial and marine mammalian wildlife species infected with Giardia and Cryptosporidium. The combination of prevalence studies with modern molecular-genotyping techniques is providing valuable insights into the host specificity and possible transmission routes of these two important parasites.     

Giardia and Cryptosporidium removal from waste-water by a duckweed (Lemna gibba L.) covered pond

AIMS: To determine the ability of duckweed ponds used to treat domestic waste-water to remove Giardia and Cryptosporidium. METHODS AND RESULTS: The influent and effluent of a pond covered with duckweed with a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%, respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. There was a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts by the pond (P < 0.001). Influent turbidity and parasite removal were also significantly correlated (Cryptosporidium and turbidity, P=0.05; Giardia and turbidity, P=0.01). CONCLUSIONS: The larger organisms (parasites) probably settled to the bottom of the pond, while removal of smaller bacteria and coliphages in the pond was not as effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Duckweed ponds may play an important role in wetland systems for reduction of Giardia and Cryptosporidium.     

Cryptosporidium,Giardia, and Cyclospora in ancient Peruvians

Twenty-two coprolites of human origin, collected from excavations along the north-central coast of Peru, were examined using fluorescent microscopy for the presence of fecal parasites, with emphasis on Cryptosporidium sp., Giardia sp., and Cyclospora sp. Three samples were positive. One coprolite dated between ca. 2,375 and 1,525 BC contained Giardia sp. cysts. This coprolite corresponded to the Peruvian preceramic period. Another positive coprolite ca. AD 770-830 corresponded to Epoch 3 of the Middle Horizon and contained Cryptosporidium sp. oocysts. The third positive coprolite (corresponding to the Middle Horizon. ca. AD 500-900) contained Giardia sp. cysts. This report demonstrates that Giardia sp. and Cryptosporidium sp. were present in Peruvian coastal populations for at least 4,300 and 1,100 BP.     

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.     

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).     

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.     

Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.     

Comparative analysis of Cryptosporidium, Giardia and indicator bacteria during sewage sludge hygienization in various composting processes

AIMS: To evaluate the suitability of Clostridium perfringens, Escherichia coli and enterococci as indicator organisms for Cryptosporidium and Giardia in treated sludge. METHODS AND RESULTS: Occurrence of Cryptosporidium oocysts and Giardia cysts, detected and enumerated by direct immunofluorescence microscopy, were compared with counts of indicator bacteria during six different sewage sludge hygienization processes, including closed reactor and open windrow composting, and sludge sanitation by quicklime or peat addition. No statistical correlation existed between the counts of indicator bacteria, Cl. perfringens, E. coli, and enterococci and occurrence of Cryptosporidium or Giardia. In sludge end-products, Giardia cysts were detected more frequently than Cryptosporidium oocysts. SIGNIFICANCE OF THE STUDY: Direct analysis is the best method to confirm the presence of (oo)cysts in sludge.     

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.     

Lack of detectable shedding of Cryptosporidium parvum oocysts by periparturient dairy cattle

We examined whether periparturient dairy cattle shed Cryptosporidium parvum oocysts within 12 hr of calving on 3 commercial dairy farms endemic for calfhood cryptosporidiosis. Using a diagnostic method that can detect as few as 1 oocyst per gram of feces, we found no evidence of C. parvum oocysts in 86 fecal samples collected within 12 hr of calving from 43 dairy cows.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world.     

Cryptosporidium sp. and Giardia sp. infections in mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda

For conservation purposes and because of growing ecotourism, some mountain gorilla (Gorilla gorilla beringei) populations have been habituated to humans. Fecal specimens (n = 100) of nonhabituated and human-habituated gorillas (5 populations; 6 age classes) were tested for Cryptosporidium sp. oocysts and Giardia sp. cysts by conventional staining and immunofluorescent antibody (IFA). Cryptosporidium sp. infections (prevalence 11%) were not restricted to very young gorillas but were observed in 3-yr-old to >12-yr-old gorillas; most of the infections (73%) occurred in human-habituated gorillas. The prevalence of Giardia sp. infections was 2%; 1 nonhabituated gorilla was concomitantly infected. Oocysts of Cryptosporidium sp. in the gorilla stools were morphologically, morphometrically, and immunologically undistinguishable from a bovine isolate of Cryptosporidium parvum oocysts. Mean concentration of Cryptosporidium sp. oocysts and Giardia sp. cysts in gorilla stools was 9.39x10(4)/g, and 2.49x10(4)/g, respectively. There was no apparent relationship between oocyst concentration and gorilla age, sex, or habituation status. Most Cryptosporidium sp. infections found in gorillas with closest proximity to people may be a result of the habituation process and ecotourism. This study constitutes the first report of Cryptosporidium sp. infections in the family Pongidae, in the free-ranging great apes, and in the species of gorilla.     

Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain

AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.