Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.     

Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: A health risk for humans and animals

The objective of the present study was to determine the degree of contamination by Cryptosporidium spp. and Giardia duodenalis in a river basin in a livestock farming area in Galicia (NW, Spain). Water samples (50 l) were collected at 22 points in the main basin (including 5 recreational areas), and at the source and mouth of the 3 most important rivers and at the mouth of a smaller, secondary river. Faecal samples were collected from dairy cattle selected at random from 18 herds farmed in the area. A total of 139 neonatal calves, 480 heifers and 697 cows were sampled. The prevalence, intensity of infection and the risk associated with the spread of infection by both enteropathogens were determined. Water and faecal samples were collected in spring, summer, autumn and winter of 2007. The species and genotypes of these parasites present in the water samples were identified. In both water and faecal samples, more parasitic stages were collected in spring and summer than in autumn and winter. In spring, Cryptosporidium spp. oocysts were detected in 33 (9.4%) cows from 13 (72.2%) herds, and G. duodenalis cysts were detected in 56 (16.0%) cows from 15 farms (83.3%); the intensity of infection ranged from 5 to 7895 G. duodenalis cysts per gram of faeces. Infective stages of Cryptosporidium spp. and G. duodenalis were also detected in respectively 26 (89.6%) and 27 (93.1%) water samples, in spring. The mean concentrations of parasites ranged from 2 to 1200 Cryptosporidium spp. oocysts per litre and from 2 to 400 G. duodenalis cysts per litre. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The presence of both protozoans must be monitored in cattle, in sources of water used for recreational purposes and in artificial waterways used by farmers (water channels, animal drinking water and drainage systems).     

Detection and genotyping of Giardia duodenalis in wastewater: relation between assemblages and faecal contamination origin

Among the seven assemblages identified in Giardia duodenalis species, only assemblages A and B infect humans and numerous other mammals as well. On the other hand, assemblage E is considered to be host restricted to livestock. The aim of the present study was to compare the presence of G. duodenalis assemblages A, B and E in wastewater samples from two municipal treatment plants (n=24) and one slaughterhouse (n=12). Thus, PCR assays targeting the tpi gene were developed to detect specifically these three G. duodenalis assemblages. Assemblages A and B were detected in urban wastewater with a predominance of assemblage A, especially for one treatment plant. Concerning slaughterhouse wastewater, assemblage A was found in 58% of the samples, whereas assemblage B was not detected. Assemblage E was not detected in urban wastewater, but was found in 92% of the samples from slaughterhouse. Thus, combination of assemblages A and B seemed to indicate a human contamination origin, while combination of assemblages A and E appeared to correspond to a livestock contamination origin.     

Detection of enteric viruses, Giardia and Cryptosporidium in two different types of drinking water treatment facilities

In this study, two types of drinking water treatment facilities (two conventional drinking water treatment plants (DWTPs) and two compact units (Cus)) were compared referring to their production capacity. Water samples were collected from three main points: (a) different water treatment steps (b) washings of sand filters and (c) distribution system at different distances from the water treatment plants. Both viruses and protozoa were concentrated from each water sample by adsorption and accumulation on the same nitrocellulose membrane filters (0.45 microm pore size). Enteroviruses were detected by plaque infectivity assay in BGM cells and HAV, HEV and Norovirus were detected by RT-PCR. Giardia and Cryptosporidium were detected by conventional staining methods and PCR. The results revealed that enterovirus load at the intake ranged between 10-15 PFU/L for the two compact units and between 4.5 and 75 PFU/L for the two conventional DWTPs. The virus load in distribution system of the first type DWTPs at 1 km from the plant was the same as that of the intake. Viruses in the other type of treatment plants CUs at 1, 5 and 7 km, were much reduced. Investigation of raw water sediments of the two DWTPs showed enterovirus counts between 12 and 17.5 PFU/L. Virus count was reduced in sand of filters after washing. Giardia cysts were equally detected by microscopy and PCR in only intake samples of EL-Hawamdia CU (33.3%) and Meet Fares DWTP (50%). Cryptosporidium oocysts were equally detected by microscopy and PCR in intake samples of Abo EL-Nomros CU (100%), EL-Hawamdia CU (66.7%) and Fowa DWTP (50%). At Meet Fares DWTP three positive intake samples for Cryptosporidium were detected by PCR, compared with only two positive samples by microscopy. Giardia cysts and Cryptosporidium oocysts were detected in raw water sediment and sand of filters before washing. Only one sample from Meet Fares DWTP sand of filters after washing was positive for both Giardia and Cryptosporidium. It can be concluded that the poor microbial quality of the water may be due to improper operational skills and management of the various water treatment plants (especially at the two high capacity treatment plants).     

Removal of Giardia and Cryptosporidium in drinking water treatment: a pilot-scale study

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern for public health. The aim of this study is to examine both parasites in the water samples taken from three pilot-scale plant processes located in southern Taiwan, to upgrade the current facilities. Three processes include: conventional process without prechlorination (Process 1), conventional process plus ozonation and pellet softening (Process 2), and integrated membrane process (MF plus NF) followed conventional process (Process 3). The detection methods of both parasites are modified from USEPA Methods 1622 and 1623. Results indicated that coagulation, sedimentation and filtration removed the most percentage of both protozoan parasites. The pre-ozonation step can destruct both parasites, especially for Giardia cysts. The microfiltration systems can intercept Giardia cysts and Cryptosporidium oocysts completely. A significant correlation between water turbidity and Cryptosporidium oocysts was found in this study. The similar results were also found between three kinds of particles (phi=3-5,5-8 and 8-10 microm) and Cryptosporidium oocysts.     

Settling velocity of Cryptosporidium parvum and Giardia lamblia

Understanding transport behavior of Cryptosporidium parvum oocysts and Giardia lamblia cysts (together referred to as (oo)cysts) in overland flow is important for beneficial uses of receiving water bodies. Like sediment, (oo)cysts are subjected to deposition once they are present in overland flow or low flow environments like reservoirs, wetlands and sedimentation basins. The objectives of this paper are to present the theory and experiment to determine the free settling velocity (v(s)) of (oo)cysts and to compare experimental settling velocities to estimates using Stokes' law. A settling experiment was designed to quantify the v(s) of (oo)cysts in an aqueous column. C. parvum oocysts used were spherical with average diameter (+/-1SD) of 6.6+/-1.1 microm. G. lamblia cysts were oval shaped (average eccentricity = 1.48+/-0.19) with average size of 11.8 +/-1.3 microm. Average densities were 1009 kg m(-3) for C. parvum oocysts and 1013 kg m(-3) for G. lamblia cysts. Observed experimental settling velocities are 0.27 microm s(-1) and 0.67 microm s(-1) for C. parvum and G. lamblia, respectively. Estimated average settling velocities using Stokes' law were 0.36 microm s(-1) for C. parvum and 0.84 microm s(-1) for G. lamblia. R-squared values of the observations from the settling experiments with the Stokes' law estimation are 0.87 and 0.88 for G. lamblia and C. parvum, respectively. Our results suggest that Stokes' law can be used to estimate settling velocities of (oo)cysts. Qualitatively, the low settling velocities indicate that (oo)cysts will very slowly settle out of suspension.     

Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters

Recreational beach water samples collected on weekends and weekdays during 11 consecutive summer weeks were tested for potentially viable Cryptosporidium parvum oocysts and Giardia lamblia cysts using the multiplexed fluorescence in situ hybridization (FISH) method. The levels of oocysts and cysts on weekends were significantly higher than on the weekdays (P<0.01). Concentrations of oocysts in weekend samples (n=27) ranged from 2 to 42 oocysts/L (mean: 13.7 oocysts/L), and cyst concentration ranged from 0 to 33 cysts/L (mean: 9.1 cysts/L). For the samples collected on weekdays (n=33), the highest oocyst concentration was 7 oocysts/L (mean: 1.5 oocysts/L), and the highest cyst concentration was 4 cysts/L (mean: 0.6 cysts/L). The values of water turbidity were significantly higher on weekends than on weekdays, and were correlated with the number of bathers and concentration of C. parvum oocysts and G. lamblia cysts (P<0.04). The study demonstrated positive relationships between number of bathers and levels of waterborne C. parvum oocysts and G. lamblia cysts in recreational beach water. It is essential to test recreational waters for Cryptosporidium and Giardia when numbers of bathers are greatest, or limit the number of bathers in a recreational beach area.     

Monitoring of Cryptosporidium and Giardia river contamination in Paris area

This study evaluates the protozoan contamination of river waters, which are used for drinking water in Paris and its surrounding area (about 615,000 m(3) per day in total, including 300,000 m(3) for Paris area). Twenty litre samples of Seine and Marne Rivers were collected over 30 months and analyzed for Cryptosporidium oocysts and Giardia cysts detection according to standard national or international methods. Cryptosporidium oocysts and Giardia cysts were found, respectively, in 45.7% and 93.8% of a total of 162 river samples, with occasional high concentration peaks. A significant seasonal pattern was observed, with positive samples for Cryptosporidium more frequent in autumn than spring, summer and winter, and positive samples for Giardia less frequent in summer. Counts of enterococci and rainfalls were significantly associated with Giardia concentration but not Cryptosporidium. Other faecal bacteria were not correlated with monitored protozoan. Marne seems to contribute mainly to the parasitic contamination observed in Seine. Based on seasonal pattern and rainfall correlation, we hypothesize that the origin of contamination is agricultural practices and possible dysfunction of sewage treatment plants during periods of heavy rainfalls. High concentrations of protozoa found at the entry of drinking water plants justify the use of efficient water treatment methods. Treatment performances must be regularly monitored to ensure efficient disinfection according to the French regulations.     

Applying fluorescence based technology to the recovery and isolation of Cryptosporidium and Giardia from industrial wastewater streams

As increasing water shortages continue, water re-use is posing new challenges with treated wastewater becoming a significant source of non-potable water. Rapid detection strategies that target waterborne pathogens of concern to industry are gaining importance in the assessment of water quality. This study reports on the ability to recover spiked Cryptosporidium and Giardia from a variety of industrial wastewater streams of varied water quality. Incorporation of an internal quality control used commonly in finished water-enabled quantitative assessments of pathogen loads and we describe successful analysis of pre- and part-treated wastewater samples from four industrial sites. The method used combined calcium carbonate flocculation followed by flow cytometry and epifluorescence microscopy. Our focus will now aim at characterising the ambient parasites isolated from industrial wastewater with the objective of developing a suite of highly specific platform detection technologies targeted to industrial needs.     

The prevalence and characterisation of Cryptosporidium spp. in beef abattoir water supplies

The prevalence of Cryptosporidium spp. in 50 l samples of water used to wash beef carcasses at (a) an abattoir with a borehole water (BH) supply (n = 46) and (b) an abattoir with a river water (RW) supply (n = 48) was determined. In addition, a 100 l water sample and post-wash carcass samples (n = 24) were collected from the RW supply on a single day in July. Cryptosporidium spp. was detected in 0% and 26.1% of samples from the BH and RW supply abattoirs, respectively, with oocyst concentrations ranging from 0.02 to 8.6/l. Cryptosporidium spp. was not isolated from post-wash beef carcasses, while it was detected in water samples from that day at a concentration of 0.06 oocysts/l. The species of 3/5 isolates were identified as C. parvum, and the remaining were C. andersoni. This study has demonstrated that water used to wash beef carcasses can be contaminated with Cryptosporidium of human health importance and is a potential source of carcass contamination.     

Comparing the partitioning behavior of Giardia and Cryptosporidium with that of indicator organisms in stormwater runoff

Microbial association with particles can significantly affect the fate and transport characteristics of microbes in aquatic systems as particle-associated organisms will be less mobile in the environment than their free phase (i.e. unattached) counterparts. As such, similarities or dissimilarities in the partitioning behavior of indicator organisms and pathogens may have an impact on the suitability of a particular indicator to act as a surrogate for a pathogen. This research analyzed the partitioning behavior of two pathogens (Cryptosporidium, Giardia) and several common indicator organisms (fecal coliform, Escherichia coli, Enterococci, Clostridium perfringens spores, and coliphage) in natural waters under both dry and wet weather conditions. Samples were taken from several streams in two distinct sampling phases: (i) single grab samples; and (ii) intrastorm samples obtained throughout the duration of four storms. Partitioning behavior varied by microbial type, with 15-30% of bacterial indicators (fecal coliform, E. coli, and Enterococci) associated with settleable particles compared to 50% for C. perfringens spores. Both pathogens exhibited similar levels of particle association during dry weather (roughly 30%), with increased levels observed during wet weather events (Giardia to 60% and Cryptosporidium to 40%). The settling velocities of particle-associated microbes were also estimated, with those of the bacterial indicators (fecal coliform, E. coli, and Enterococci), as well as C. perfringens spores, being similar to that of the Giardia and Cryptosporidium, suggesting these organisms may exhibit similar transport behavior. With respect to intrastorm analysis, the highest microbial concentrations, in both particle-associated and free phase, occurred during the earlier stages of a storm. The total loadings of both indicators and pathogens were also estimated over the course of individual storms.     

Detection of Cryptosporidium in miniaturised fluidic devices

Contamination of drinking water with the protozoan pathogen, Cryptosporidium, represents a serious risk to human health due to the low infectious dose and the resistance of this parasite to chlorine disinfection. Therefore, several countries have legislated for the frequent monitoring of drinking water for Cryptosporidium presence. Existing approved monitoring protocols are however time-consuming and do not provide essential information on the species, virulence or viability of detected oocysts. Rapid, more information-rich and automatable systems for Cryptosporidium detection are highly sought-after, and numerous miniaturised devices have been developed to address this need. This review article aims to summarise the state-of-the-art and compare the performance of these systems in terms of detection limit, ability to determine species, viability and performance in the presence of interferents. Finally, conclusions are drawn with regard to the most promising methods and directions of future research.     

Cryptosporidium and Giardia detection in water bodies of Galicia, Spain

The objective of this study was to determine the mean concentration (per litre) of Cryptosporidium oocysts and Giardia cysts in recreational river areas (n = 28), drinking water treatments plants (DWTPs; n = 52) and wastewater treatment plants (WWTPs; n = 50) in Galicia (NW Spain). Water samples from rivers and from the influent (50–100 l) and the treated effluent (100 l) of the water plants were filtered using Filta-Max filters (IDEXX Laboratories, Inc., Westbrook, ME, USA). A total of 232 samples were processed and the (oo)cysts were concentrated, clarified by IMS and then detected by IFAT. The viability was determined by applying fluorogenic vital dye (PI).In the recreational areas, infective forms of Cryptosporidium and Giardia were detected in 16 (57.1%; 1–60 oocysts per litre) and 17 (60.7%; 1–160 cysts per litre) samples, respectively. In the water flowing into the water treatment plants, oocysts were detected in 21 DWTPs (40.4%; 1–13 oocysts per litre) and cysts were observed in 22 DWTPs (42.3%; 1–7 cysts per litre). In the effluents from the treatment plants, Cryptosporidium oocysts and Giardia cysts were identified in 17 DWTPs (32.7%; 1–4 oocysts per litre) and in 19 DWTPs (36.5%; 1–5 cysts per litre), respectively. The highest concentrations of (oo)cysts were found in the WWTPs; specifically, oocysts were detected in 29 (58.0%; 1–80 oocysts per litre) and cysts in 49 (98.0%; 2–14.400 cysts per litre) WWTP effluents. Cryptosporidium and Giardia were detected in 32 (64.0%; 1–120 oocysts per litre) and 48 (96.0%; 2–6.000 cysts per litre) WWTP effluents, respectively. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0%. In all samples analysed. Moreover, it was found that the effluents from coastal WWTPs were discharged directly into the sea, while inland WWTPs were discharged directly into rivers. The concentrations of both enteropathogens detected in effluents from WWTPs therefore represent a significant risk to human and animal health.These results demonstrate the wide distribution of Cryptosporidium and Giardia in the environment, the ineffectiveness of treatments in DWTPs and WWTPs in reducing/inactivating both protozoa and the need to monitor the presence, viability and infectivity of Cryptosporidium and Giardia in water bodies. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination.     

Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms (Stephanodiscus hantzschii) in slow sand filters

The decimal elimination capacity (DEC) of slow sand filtration (SSF) for Cryptosporidium parvum was assessed to enable quantitative microbial risk analysis of a drinking water production plant. A mature pilot plant filter of 2.56m(2) was loaded with C. parvum oocysts and two other persistent organisms as potential surrogates; spores of Clostridium perfringens (SCP) and the small-sized (4-7microm) centric diatom (SSCD) Stephanodiscus hantzschii. Highly persistent micro-organisms that are retained in slow sand filters are expected to accumulate and eventually break through the filter bed. To investigate this phenomenon, a dosing period of 100 days was applied with an extended filtrate monitoring period of 150 days using large-volume sampling. Based on the breakthrough curves the DEC of the filter bed for oocysts was high and calculated to be 4.7log. During the extended filtrate monitoring period the spatial distribution of the retained organisms in the filter bed was determined. These data showed little risk of accumulation of oocysts in mature filters most likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was 3.6 and 1.8log, respectively. On basis of differences in transport behaviour, but mainly because of the high persistence compared to the persistence of oocysts, it was concluded that both spores of sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for oocyst removal by slow sand filters. Further research is necessary to elucidate the role of predation in Cryptosporidium removal and the fate of consumed oocysts.     

Quantitative risk assessment of Cryptosporidium in tap water in Ireland

Cryptosporidium species are protozoan parasites associated with gastro-intestinal illness. Following a number of high profile outbreaks worldwide, it has emerged as a parasite of major public health concern. A quantitative Monte Carlo simulation model was developed to evaluate the annual risk of infection from Cryptosporidium in tap water in Ireland. The assessment considers the potential initial contamination levels in raw water, oocyst removal and decontamination events following various process stages, including coagulation/flocculation, sedimentation, filtration and disinfection. A number of scenarios were analysed to represent potential risks from public water supplies, group water schemes and private wells. Where surface water is used additional physical and chemical water treatment is important in terms of reducing the risk to consumers. The simulated annual risk of illness for immunocompetent individuals was below 1 × 10^− 4 per year (as set by the US EPA) except under extreme contamination events. The risk for immunocompromised individuals was 2-3 orders of magnitude greater for the scenarios analysed. The model indicates a reduced risk of infection from tap water that has undergone microfiltration, as this treatment is more robust in the event of high contamination loads. The sensitivity analysis highlighted the importance of watershed protection and the importance of adequate coagulation/flocculation in conventional treatment. The frequency of failure of the treatment process is the most important parameter influencing human risk in conventional treatment. The model developed in this study may be useful for local authorities, government agencies and other stakeholders to evaluate the likely risk of infection given some basic input data on source water and treatment processes used.     

Removal of viable and inactivated Cryptosporidium by dual- and tri-media filtration

The limited efficacy of disinfectants, other than ultraviolet irradiation and ozonation, as a barrier against Cryptosporidium parvum in drinking water treatment has underscored the increased importance of oocyst removal by filtration. Currently, no reliable surrogates have been identified for C. parvum removal by filtration. As a result, evaluations of the Cryptosporidium removal by treatment operations have been performed using oocysts. It has typically been assumed that chemically inactivated oocysts are suitable surrogates for viable oocysts. Measurements of electrophoretic mobility, however, have shown that chemical inactivation changes the surface charge of Cryptosporidium oocysts. The present bench-scale research indicated that formalin-inactivated oocysts are reliable surrogates for viable oocysts during both stable filter operation and periods where filtration processes are challenged, such as coagulation failure. This finding is important because of the practical difficulties associated with using viable oocysts in filtration investigations. Poor coagulation conditions severely compromised removal of viable and inactivated oocysts by dual- and tri-media filters compared to stable operating conditions and filter ripening, emphasizing the importance of optimized chemical pre-treatment (coagulation) for the successful removal of oocysts during filtration. The treatment optimization experiments also indicated that tri-media filters offered only marginally higher oocyst removals than dual-media filters.     

Modeling Cryptosporidium parvum oocyst inactivation and bromate in a flow-through ozone contactor treating natural water

A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters was coupled with an empirical ozone decay model and a one-dimensional axial dispersion reactor (ADR) model to represent the performance of a lab-scale flow-through ozone bubble-diffuser contactor. Dissolved ozone concentration, bromate concentration (in flow-through experiments only), hydroxyl radical exposure and C. parvum oocyst survival were measured in batch and flow-through experiments performed with filtered Ohio River water. The model successfully represented ozone concentration and C. parvum oocyst survival ratio in the flow-through reactor using parameters independently determined from batch and semi-batch experiments. Discrepancies between model prediction and experimental data for hydroxyl radical concentration and bromate formation were attributed to unaccounted for reactions, particularly those involving natural organic matter, hydrogen peroxide and carbonate radicals. Model simulations including some of these reactions resulted in closer agreement between predictions and experimental observations for bromate formation.     

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.     

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.     

Molecular and phylogenetic approaches for assessing sources of Cryptosporidium contamination in water

The high sequence diversity and heterogeneity observed within species or genotypes of Cryptosporidium requires phylogenetic approaches for the identification of novel sequences obtained from the environment. A long-term study on Cryptosporidium in the agriculturally-intensive South Nation River watershed in Ontario, Canada was undertaken, in which 60 sequence types were detected. Of these sequence types 33 were considered novel with no identical matches in GenBank. Detailed phylogenetic analysis identified that most sequences belonged to 17 previously described species: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, muskrat I, muskrat II, deer mouse II, fox, vole, skunk, shrew, W12, W18, W19 and W25 genotypes. In addition, two new genotypes were identified, W27 and W28. C. andersoni and the muskrat II genotype were most frequently detected in the water samples. Species associated with livestock made up 39% of the total molecular detections, while wildlife associated species and genotypes accounted for 55% of the Cryptosporidium identified. The human pathogenic species C. hominis and C. parvum had an overall prevalence of 1.6% in the environment, indicating a small risk to humans from the Cryptosporidium present in the watershed. Phylogenetic analysis and knowledge of host-parasite relationships are fundamental in using Cryptosporidium as a source-tracking or human health risk assessment tool.