Assessment of Giardia and Cryptosporidium spp. as a microbial source tracking tool for surface water: application in a mixed-use watershed.

Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed Natalie Prystajecky,a,b Peter M. Huck,c Hans Schreier,d Judith L. Isaac-Rentona,b

Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.

G

iardia lamblia and members of the Cryptosporidium genus are among the most common enteric parasites of humans. Affecting both human and animal populations, these organisms have a wide host range, infecting most mammalian as well as some avian and reptilian species (1). Human-infective G. lamblia strains are also zoonotic, while both zoonotic and host-adapted subtypes (including human-specific subtypes) of Cryptosporidium spp. exist (2). Human infections by Giardia and Cryptosporidium are transmitted via the fecal-oral route, either as a result of person-toperson transmission or through secondary transmission by contaminated food or water. Water is the most common transmission vehicle reported. These pathogens represent a significant challenge to public health and drinking water purveyors due to their persistence and survival in water, their resistance to some drinking water treatment methods, and their low infectious doses (3, 4). The gold standard laboratory method for the detection of Giardia and Cryptosporidium in water is U.S. Environmental Protection Agency (EPA) Method 1623 (5). This method includes commercially available immunofluorescence assays and immunomagnetic separation (IMS) kits, which use pan-specific antibodies against members of the Giardia and Cryptosporidium genera. Thus, Method 1623 may detect species and subtypes of these organisms that are not infectious to humans. As such, to understand human health risks associated with these organisms, supplementation of the routine detection method with genotyping is needed. There are eight G. lamblia assemblages (syn., subtypes or genotypes), of which two subtypes (assemblages A and B) are re-

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sponsible for the majority of human infections (7). Both of these assemblages are zoonotic; the remaining assemblages (assemblages C to H) are primarily host adapted (associated with specific animals) (7–9). It should be noted, however, that host-adapted assemblages (assemblages C, D, E, and F) have been detected in humans (10). There are 26 species of Cryptosporidium currently described in the literature, with a wide range of animal hosts (6, 11–13). Only C. parvum has a wide host range that includes humans and other mammalian species, such as livestock and mammals, with the remaining species appearing closely adapted to their host species (1). For example, C. hominis is infectious only to humans and C. andersoni primarily infects bovines (1, 13). Infections with “human-noninfectious” Cryptosporidium species are possible but occur infrequently and mostly in immunocompromised hosts (6). Knowledge of parasite host range may be exploited for microbial source tracking (MST) activities to investigate and remediate contamination events. Residents of British Columbia (BC) suffer from a greater incidence of gastrointestinal illness and have experienced a greater

Received 9 July 2013 Accepted 8 January 2014 Published ahead of print 24 January 2014 Editor: C. R. Lovell Address correspondence to Natalie Prystajecky, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02037-13

Applied and Environmental Microbiology

p. 2328 –2336

April 2014 Volume 80 Number 8

Downloaded from http://aem.asm.org/ on December 1, 2014 by UCSF Library & CKM

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canadaa; British Columbia Centre for Disease Control Public Health Microbiology & Reference Laboratory, Vancouver, British Columbia, Canadab; NSERC Chair in Drinking Water Treatment, Department of Civil and Environmental Engineering, University of Waterloo, Waterloo, Ontario, Canadac; Soil Science, Faculty of Land & Food Systems, University of British Columbia, Vancouver, British Columbia, Canadad

Parasites for Microbial Source Tracking in Water

Hopington Aquifer A, Aquifer B, and Aquifer C, respectively. The map was generated using an open-source orthophoto (taken in 1995) and ArcView GIS.

number of waterborne outbreaks of disease than residents in other parts of Canada (14). There have been 29 documented waterborne outbreaks of disease since 1980; over half were caused by Giardia (13 outbreaks) or Cryptosporidium (3 outbreaks). Lack of adequate treatment of drinking water was the main reason for the majority of outbreaks (14, 15). While the incidence of giardiasis and cryptosporidiosis in BC has declined over the past decade, the incidence of these infections still exceeds rates in the rest of Canada (16). In BC in 2008, the rate of cryptosporidiosis was 2.6 cases per 100,000 population and the rate of giardiasis was 14.2 cases per 100,000 (17). These rates exceed the U.S. EPA’s tolerable risk of one waterborne infection per 10,000 persons per year, although it should be noted that multiple exposure pathways (i.e., food-borne and person-to-person) in addition to waterborne transmission contribute to reportable communicable disease (RCD) counts. As such, it is important to understand the sources and relative risks to human health of these parasites in residents of BC via waterborne transmission. This study was conducted as a substudy in a larger source-totap investigation of water quality in a BC mixed urban-rural community. This work focused on evaluating the benefit of adding genotyping as a risk assessment to traditional parasite surveillance


in water supplies. Parasite prevalence in select raw surface water was determined and compared to traditional chemical and bacteriological water quality indices. Giardia and Cryptosporidium isolates detected were sequenced to identify subtype prevalence and variability. These data were then interpreted in the context of source tracking potential. MATERIALS AND METHODS Sampling site description. Water samples were collected in the Township of Langley, an urban-rural community located 40 km east of Vancouver, BC, Canada. The Township’s residents use a range of drinking water sources, including private wells, treated municipal groundwater, and a mixture of treated municipal groundwater and treated surface water supplied by Metro Vancouver. Samples were collected from the Hopington Aquifer (Arcadia Municipal Well) and surface water samples from Coghlan Creek and Salmon River (Fig. 1). Coghlan Creek is a tributary of Salmon River, located above the Hopington Aquifer. The aquifer is largely influenced by surface water in the summer months (18). Both groundwater and surface water supplies in our study area are impacted by persistent high nitrate values, suggestive of ongoing fecal and/or agricultural contamination, and are therefore a concern of local authorities (18, 19). Giardia and Cryptosporidium sampling. Samples were collected biweekly over a 2-year study period (September to November [25 months])

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FIG 1 Sampling sites within the Salmon River watershed used in this study for genotyping of Giardia and Cryptosporidium isolates. A, B, and C refer to

Prystajecky et al.

TABLE 1 Summary of chemical and microbiological indicatorsa collected in Coghlan Creek, Township of Langley, British Columbia

Parameter Median SD Minimum Maximum % of samples above detection limit a

Total Fecal Giardia NO3-N Conductivity Turbidity coliforms coliforms Cryptosporidium (no./100 Cl⫺ PO43⫺ NH4-N (NTU) (CFU/100 ml) (CFU/100 ml) (no./100 liters) liters) (mg/liter) (mg/liter) (mg/liter) (mg/liter) (S) 11.83 3.32 2.61 31.45 NA

0.02 0.02 0.00 0.08 NA

0.08 0.12 0.00 0.53 NA

3.85 1.23 0.42 5.96 NA

16.46 43.34 0.60 231.00 NA

16.46 43.34 0.60 231.00 NA

3,831 4,236 90 23,000 100

327 636 1 3,600 100

333 2,554 0 20,600 56

107 469 0 3,800 91

and tested by following Method 1623 (5). Water samples were collected by filtration using an open-disc foam filter (IDEXX Filta-Max; Westbrook, ME) with a pump at a flow rate of 0.2 to 4.5 liters/min. Up to 550 liters of treated water and up to 50 liters of raw water were filtered, depending on the turbidity of the source water. Samples were transported in coolers on ice, stored at 4°C, and processed within 96 h. Sampling of the Arcadia municipal groundwater well was discontinued after a year due to no detection of parasites. Sample processing and immunomagnetic separation. All samples were processed using an automatic wash station according to the instructions of the manufacturer (IDEXX, Westbrook, ME). The resulting concentrates were further processed by immunomagnetic separation (IMS) (Dynabeads-GC Combo kit; Dynal Biotech, Brown Deer, WI). IMS purification followed the manufacturer’s procedure with the exception that the suggested acid dissociation was replaced by heat dissociation (20). Giardia cysts and Cryptosporidium oocysts were enumerated by immunofluorescence assay (IFA). When the pellet size exceeded 0.5 ml, two IMS tubes were set up. Immunofluorescence assay and microscopic examination. The purified sample was applied to a Dynal Spot-On slide and dried overnight at 4°C. The slides were then fixed using methanol and stained with a fluorescein isothiocyanate (FITC)-conjugated antibody (Easy-Stain; Biotechnology Frontiers, North Ryde, Australia) and DAPI (4=6-diamidino-2phenylindole). Presumptive positive cysts or oocysts identified by IFA were confirmed with DAPI staining and differential interference contrast (DIC) microscopy. Ongoing precision and recovery (OPR) procedures were conducted annually on each sampling site to ensure adequate method performance per the U.S. EPA-recommended procedures (5). Slide-scraping procedure. To remove cysts and oocysts from IFA slides for nucleic acid extraction, coverslips were removed and slides were washed to remove the DABCO (1,4-diazabicyclo[2.2.2]octane)-glycerol, dried at room temperature, and stored at ⫺20°C. Slides were processed as described by Ruecker et al. (21). A series of spiking experiments with laboratory-grade water, spiking slides with 0 to 30 cysts and oocysts (counted by flow cytometry) per slide and evaluating recoveries using PCR, were carried out to determine the limits of detection (LOD) and recoveries for slide scraping and PCR. Genomic DNA extraction. Genomic DNA was extracted by freezethaw with proteinase K digestion, using six freeze-thaw cycles that alternated between a 2-min freeze in liquid nitrogen and a 5-min thaw at 65°C. Proteinase K (Qiagen) (400 ␮g) was added, and samples were incubated overnight at 56°C. The resulting lysates were purified using a QiaAMP DNA Micro kit (Qiagen) according to the manufacturer’s instructions, with the addition of carrier RNA. When two heat dissociation steps were used, each slide resulting from each heat dissociation step was treated as a separate sample. A spiked positive control containing 100 cysts and 100 oocysts in 1 ml of laboratory-grade water and a negative control were also included in the genomic extraction step. Nested PCR. Nested-PCR protocols were used for the amplification of Giardia lamblia DNA and Cryptosporidium DNA. For genotyping of G. lamblia, the 18S rRNA gene was amplified by nested PCR (22, 23), which generated a 292-bp secondary product. The PCR was carried out as pre-

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viously published, with the exceptions of using HotStarTaq MasterMix (Qiagen, Mississauga, Ontario [ON], Canada) and the addition of 5% dimethyl sulfoxide (DMSO) and 10 mg/ml bovine serum albumin (BSA). To genotype Cryptosporidium-positive samples, a nested PCR targeting an ⬃82-bp segment of the 18S rRNA gene was used as previously described (24). The PCR was carried out as previously published, with the exception of using Platinum Taq polymerase (Invitrogen). Sequencing. PCR-positive samples were purified using a QiaQuick PCR purification kit according to the manufacturer’s instructions (Qiagen) and quantified using a spectrophotometer (Ultrospec 3100 Pro; BioChrom, Ltd., Cambridge, United Kingdom). Samples were sequenced using a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Life Technologies, Grand Island, NY) on an ABI 3100 automated sequencer (Applied Biosystems, Life Technologies, Grand Island, NY). Samples were sequenced in forward and reverse directions. Data analysis. Sequence data were assembled and analyzed using SeqMan and EditSeq software packages (DNAStar Inc., Madison, WI). Sequences were identified using the nucleotide-nucleotide BLAST program on the NCBI website (blastn algorithm). Published sequences for the 18S rRNA gene for G. lamblia and Cryptosporidium spp. were obtained from GenBank and used for phylogenetic analyses. Sequences were trimmed and aligned to published sequences using ClustalW. Phylogenetic trees were generated using a neighbor-joining tree and a Jukes-Cantor genetic distance model, resampled 1,000 times (Geneious Pro, Auckland, New Zealand). Nodes with values of less than 70 were excluded. The Microsoft Excel Statistical Package was used for all statistical analyses. Chemical and microbiological characterization. Water chemistry data were collected for the following parameters: nitrates (NO3-N), ammonia (NH3-N), phosphate, and chloride. Turbidity and conductivity were determined for each sample using a dual-function probe, calibrated before each use. Total and fecal coliform testing of all samples was conducted by membrane filtration. Climate data were obtained from the Environment Canada’s National Climate Data and Information Archive, using data from the nearest weather station (Abbotsford A station, climate identification no. [ID] 1100030). Statistical analyses (Pearson correlation and linear regressions) were performed in Excel to determine the relationships among chemical and microbiological parameters. Nucleotide sequence accession numbers. All sequences have been deposited in the NCBI GenBank database with accession numbers KF994564 to KF994610.

RESULTS

Chemical and microbiological characterization. Summaries of all study parameters are provided in Table 1 (Coghlan Creek) and Table 2 (Salmon River). Total coliform and fecal coliforms were detected in 100% of samples collected at the Coghlan Creek and Salmon River sites. Total coliform and fecal coliform concentrations were higher in the Salmon River site, suggesting that as a tributary, Coghlan Creek is not the predominant source of fecal contamination of the Salmon River. Giardia and Cryptosporidium concentrations. Cryptospo-



n ⫽ 65. NTU, nephelometric turbidity units; NA, not applicable.


TABLE 2 Summary of chemical and microbiological indicatorsa collected in Salmon River, Township of Langley, British Columbia

Parameter Median SD Minimum Maximum % of samples above detection limit a

Total Fecal Giardia NO3-N Conductivity Turbidity coliforms coliforms Cryptosporidium (no./100 Cl⫺ PO43⫺ NH4-N (NTU) (CFU/100 ml) (CFU/100 ml) (no./100 liters) liters) (mg/liter) (mg/liter) (mg/liter) (mg/liter) (S) 16.04 3.72 6.15 25.00 NA

0.02 0.03 0.00 0.10 NA

0.09 0.12 0.00 0.56 NA

2.59 0.64 0.86 4.14 NA

162.17 41.15 60.00 238.80 NA

14.95 21.85 2.40 86.00 NA

6,295 12,496 0 85,000 100

737 1,806 0 9,600 100

11 20 0 126 74

32 104 0 730 77

ridium spp. were detected in 63% of surface water samples, at concentrations ranging from 0 to 20,600 oocysts per 100 liters of water (Fig. 2). Cryptosporidium oocysts were detected more frequently in the Salmon River site (73%, n ⫽ 49) than in the Coghlan Creek site (55%, n ⫽ 65). The average Cryptosporidium concentrations were 333 oocysts/100 liters in Coghlan Creek and 11 oocysts/100 liters in Salmon River. Giardia was detected more frequently than Cryptosporidium. Giardia cysts were detected in 86% of surface water samples, at concentrations ranging from 0 to 3,800 cysts per 100 liters of water

(Fig. 3). Giardia cysts were detected more frequently in the Coghlan Creek site (92%, n ⫽ 65) than in the Salmon River site (78%, n ⫽ 49). The average Giardia concentrations were 107 cysts/100 liters in Coghlan Creek and 32 cysts/100 liters in Salmon River. At the Coghlan Creek site, two separate incidents of highly elevated Giardia and Cryptosporidium concentrations were observed. On one day (in October), Cryptosporidium concentrations were 20,600 oocysts per 100 liters of water, and on another (in March), Giardia concentrations were 3,800 cysts per 100 liters of

FIG 2 Prevalence of Cryptosporidium detected at two sampling sites (Coghlan Creek and Salmon River) in the Township of Langley, British Columbia, using Method 1623. Sep, September; Dec, December; Mar, March; Jun, June; Oct, October; Jan, January; Apr, April; Aug, August; Nov, November.


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n ⫽ 49.

Prystajecky et al.

1623.

water. All organisms examined were confirmed by PCR and sequencing to be Cryptosporidium (October) or Giardia (March). These high concentrations of parasites may reflect a bolus of parasites captured during sampling, which could arise from animal fecal material mobilized into water or could be due to defecation by animals directly into the water supply or to septic system failures. Because only zoonotic strains were detected at these time points, possible sources of the parasite could not be elucidated. Neither Giardia nor Cryptosporidium organisms were detected in any of the Arcadia Municipal Well samples; thus, collection and testing was discontinued after 1 year. Comparison between parameters. There was no correlation between chemical or microbial parameters and Cryptosporidium and Giardia concentrations (Table 3). A statistically significant relationship was observed between parasite concentration and turbidity at both sites. Turbidity was a better predictor of the Cryptosporidium concentration in Coghlan Creek, while turbidity was a good predictor of Giardia occurrence at both sites. In the Salmon River site, Giardia and Cryptosporidium concentrations were strongly correlated (r ⫽ 0.86, P ⬍ 0.05), suggesting common or similar sources of the organisms. However, Cryptosporidium and Giardia concentrations were weakly correlated in the Coghlan

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TABLE 3 Pearson correlation comparing chemical and bacteriological parameters to Cryptosporidium and Giardia concentrations at two study sites in the Township of Langleya Correlation (r) with parasite concn Coghlan Creek

Salmon River

Parameter

Cryptosporidium

Giardia

Cryptosporidium

Giardia

Total coliforms Fecal coliforms Cl P NO3-N NH4-N Conductivity Turbidity Giardia Cryptosporidium 24-h rainfall

⫺0.10 0.02 ⫺0.05 ⫺0.05 ⫺0.05 ⫺0.05 ⫺0.05 0.76* 0.00 NA 0.02

⫺0.27 ⫺0.27 ⫺0.27 ⫺0.27 ⫺0.27 0.00 0.03 0.81* NA 0.00 0.05

0.02 0.13 ⫺0.03 0.16 0.00 ⫺0.07 ⫺0.27 0.56* 0.86* NA 0.03

⫺0.15 0.00 ⫺0.04 ⫺0.04 ⫺0.05 0.08 ⫺0.26 0.56* NA 0.86* 0.04

a r values greater than 0.7 were considered representative of a strong relationship. Asterisks (*) denote a statistically significant relationship, as determined by linear regression (P ⬍ 0.05).



FIG 3 Prevalence of Giardia detected at two sampling sites (Coghlan Creek and Salmon River) in the Township of Langley, British Columbia, using Method


TABLE 4 Summary of the rate of positive PCRs (combined across sites) at various numbers of (oo)cysts per slide for Giardia and Cryptosporidiuma % of slides positive No. of (oo)cysts per slide

Giardiab

Cryptosporidiumc

1 2–10 11–50 51⫹

45 58 82 83

59 56 100 100

Creek site, suggesting a difference in the source or microbial transport of cysts versus oocysts between the tributary and main river. There was no correlation between 24-h-antecedent rainfall and parasite occurrence at any of the sites. Giardia and Cryptosporidium molecular analyses. Spiking experiments using cells that were enumerated by flow cytometry were performed to determine the limits of detection (LOD) using laboratory-grade water. Results showed that nested PCR would detect Giardia and Cryptosporidium from as few as two cysts and one oocyst per slide, respectively. Slides with less than five organisms per slide demonstrated variable results, particularly for Giardia (results not shown). Since each PCR used 2 to 5 ␮l of the 50-␮l nucleic acid preparation, the LOD per PCR in spiked laboratorygrade water samples were 0.12 to 0.2 cysts and 0.04 to 0.1 oocysts. Detection levels per slide (multiple slides were examined per sample, depending on the number of IMS tubes set up) were greater and more variable in environmental specimens than in laboratory spikes, likely due to the presence of PCR inhibitors, degradation of nucleic acid due to environmental exposures, and the age of the (oo)cyst(s) (Table 4). By nested PCR, 77% of microscopy-positive samples for Giardia were also positive by at least one nested PCR (PCR analyses were conducted in duplicate), indicating that some positive samples would have been negative if only a single PCR had been conducted. Performing the PCR in triplicate on a subset of samples increased PCR sensitivity from 77% to 80%, but, because of the limited improvement and increased costs, performing PCR in triplicate for Giardia was discontinued. Retrospective analysis comparing the microscopy record (fluorescence intensity, DAPI staining of nuclei, and DIC characteristics) to the PCR results did not demonstrate any consistent relationship for a sample(s) that was positive by microscopy but negative by PCR. However, it was noted that cysts observed in PCR-negative but microscopically positive samples often contained fewer than four nuclei as revealed by DAPI staining. Sequencing was performed to determine the assemblage designation of the Giardia cysts detected. The majority of samples were from zoonotic assemblages (assemblages A and B). Assemblage B was more predominant (56%) than assemblage A (35%). Despite livestock operations in the area, assemblage E was detected infrequently (2%). Giardia sequence data were compared to published sequences (GenBank) and to sequences from a BCspecific library of G. lamblia clinical, environmental, and animal isolates. Interestingly, some historical isolates had 100% sequence similarity, despite over 15 years between collection dates. This


DISCUSSION

The Township of Langley is a mixed urban-rural community in southwestern British Columbia that is representative of many communities across Canada. Residents of the Township are increasingly concerned by diminished water quality associated with changes in land use practices in the region. Five of the major aquifers within the Township are unconfined and show a rapid recharge rate (19). The aquifer-stream interactions of Coghlan Creek and Salmon River show that both streams contributed significantly to aquifer levels in the summer months; therefore, contamination of surface water directly impacts groundwater quality during that time of the year. Due to the range of land use in the area, this study site was determined to be ideal for investigation of the suitability of parasite genomic sequencing as a tool to determine public health risk and for microbial source tracking. Results of this study showed that parasite concentrations in this watershed were elevated year round compared to other urban-rural watersheds. A study of a mixed agricultural watershed in Southern Ontario, Canada, found Cryptosporidium and Giardia in 44% and 31% of samples tested, respectively (25). In contrast, Cryptosporidium and Giardia were detected more frequently in the current study, in 63% and 86% of samples, respectively. A study of a mixed-use watershed in Italy found Cryptosporidium and Giardia concentrations as high as 1,050 oocysts/100 liters and 1,280 cysts/100 liters, respectively (26). In comparison, the maximum concentrations in the current study were as high as 20,600 oocysts/ 100 liters and 3,800 cysts/100 liters for Cryptosporidium and Giardia, respectively. Results confirm that traditional bacterial surrogates of water microbial quality are poor indicators of Giardia and Cryptosporidium occurrence in surface water. While parasites are of fecal origin, their survival in water differs significantly from that of fecal

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a Note that at least two (and up to four) microscopy slides were prepared for each sample; only parasite-positive slides were examined by PCR. b At least one of two PCRs targeting Giardia was positive. c At least one of three PCRs targeting Cryptosporidium was positive.

may be indicative of the limited geographical range of the isolates with published sequences or may represent genetic parasite variants specific to this region. A small number of isolates did not group with other sequences; these may represent unique genetic isolates, mixed genotypes, or interfering genetic material (amplicons were not cloned for sequencing). It should be noted that it is difficult to fully assess variant diversity at a single locus. For Cryptosporidium isolates, 60% of samples positive by microscopy were also positive by at least one nested PCR (PCR analyses were conducted in triplicate). This lower positivity rate likely reflects lower concentrations of oocysts on IFA slides; 48% of the slides contained only a single oocyst. Therefore, any losses during sample processing, along with nonhomogenous distributions of nucleic acids in extracts, impacted PCR results. Sequencing of the 18S rRNA gene for Cryptosporidium demonstrated that C. andersoni was detected most frequently (24% of sequences), followed by C. baileyi (15%) and the Cryptosporidium skunk genotype (12%). These species/genotypes likely pose little threat to human health; neither the skunk genotype nor C. baileyi has been reported in humans, and there is only one report of C. andersoni human infection. C. andersoni is associated with bovine species, and C. baileyi is associated with fowl. However, C. parvum (12%) and closely related genotypes (9%) were detected, along with C. hominis (9%), which all represent a potential threat to human health. While C. parvum can occur in both humans and animals, C. hominis is found only in human sources and therefore is indicative of human fecal contamination of water.

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predominance of livestock in our study area, cattle-specific G. lamblia (assemblage E) isolates were infrequently detected. This suggests that, in the Township, livestock were infected with the zoonotic G. lamblia assemblages, rather with than the livestockadapted strain, or, alternatively, assemblage E is present in the livestock in the watershed but occurrence in the watershed is limited due to proper manure management. The predominance of Giardia assemblage B in this watershed is consistent with the findings in an unpublished library of historical isolates collected in British Columbia since the 1980s. This suggests that assemblage B may dominate the ecological landscape in British Columbia. Assemblage B has a wide host range and is capable of infecting most mammalian species. It should be noted that assemblage A has been found more frequently than assemblage B in livestock. It was observed that not all samples positive by microscopy could be amplified by nested PCR. PCR performance could be impacted by poor quality or low concentrations of nucleic acid. The inability to amplify all (oo)cyst(s) detected by microscopy could arise from methodological challenges associated with slide scraping. Ruecker et al. (21) demonstrated better PCR yields than in this study for Cryptosporidium (they did not assess the method for Giardia); however, that study had a limited number of samples over a narrower range of oocyst concentrations than were detected in the current study. Furthermore, matrix difference may impact the slide-scraping recovery. The recovery experiments described in this paper used laboratory-spiked water rather than matrix water, which may have overestimated the LOD for the slide-scraping method. It is possible that lower PCR recoveries arise from the presence of materials inhibitory to PCR such as humic acids and sediments, which can coconcentrate during the filtration of large volumes of water (25, 32). Furthermore, cross-reactivity of IFA antibodies with algal species is possible, which could lead to an overestimation of the IFA results (40). Lastly, it was noted that often, under microscopy, the oo(cysts) lacked a full complement of visible nuclei by DAPI staining, which may suggest that nucleic acid may have been missing due to lack of structural integrity or age of the (oo)cyst. The loss of nucleic acid could yield reduced PCR success. It should be noted that the primary applications of parasite enumeration and genotyping (source water characterization, quantitative microbial risk assessment, and MST) are generally risk focused and that the characterization of nonrecent (aged) or noninfective (damaged) parasite contamination is not as meaningful. However, this has not been demonstrated empirically. As it is likely that multiple sources of parasites exist within a catchment, it is likely there are multiple species, genotypes, and assemblages within a water body. To generate meaningful and accurate MST outputs for waterborne parasites, it is important to be able to detect parasites at an LOD of a single (oo)cyst; otherwise, only the dominant species/genotypes/assemblages will be identified through sequencing. This may be accomplished through altering PCR conditions or newer molecular techniques such as digital PCR and single-cell sequencing. In the absence of an LOD of a single (oo)cyst, which may be difficult to achieve in environmental samples, sequencing of multiple amplicon copies from a sample can disambiguate multiple species/genotypes/ assemblages within a single copy. Historically, this was achieved through sequencing of a few (or hundreds of) clones; however, this can now be accomplished using next-generation sequencing (NGS) platforms such as the Illumina Miseq platform, where hun-



coliforms, due to the structural integrity of the protozoan (oo)cyst wall and low metabolic state, allowing prolonged survival in water (27, 28). The best predictor of Giardia and Cryptosporidium occurrence in this study was turbidity, and yet the strengths of the relationships differed for the two water sources studied. A stronger relationship between turbidity and parasite occurrence was observed in Coghlan Creek, which has lower streamflow discharge than Salmon River. A link between drinking water turbidity and emergency room visits, hospitalizations, and calls to a health information telephone line has previously been shown (29, 30). However, many studies have failed to show consistently strong relationships between turbidity and parasite occurrence in raw water supplies (31, 32). In addition, a stronger relationship between Giardia and Cryptosporidium was observed for Salmon River than for Coghlan Creek, suggesting differences in pathogen transport patterns in the different streams. Parasite occurrence was also compared to 24-h-antecedent rainfall, since it has been reported that rainfall precedes waterborne outbreaks of disease (33, 34). In this study, there was no correlation between parasite detection and rainfall. The detection of elevated parasite counts in the summer, in the absence of rainfall, is not consistent with other studies (26, 35). This suggests that the parasites may be originating from grazing animals nearby, from in-stream wildlife such as muskrat, or from various waterfowl species. Microbial source tracking (MST) using pathogens, including Giardia and Cryptosporidium, has been cited as one of the possible methods in the MST toolbox (21, 36, 37). While still costly compared to bacterial MST methods, the costs of PCR and sequencing have decreased in recent years, and genotyping of Giardia and Cryptosporidium can be added to routine parasite testing when information on the source of parasite contamination is needed. However, the literature indicates that parasite genotyping is infrequently applied to MST. These organisms, like other MST markers such as Bacteroides and Preveotella spp., demonstrate different host ranges that may be exploited for MST. Sequencing revealed that the majority (98%) of the Giardia isolates belonged to zoonotic assemblages (assemblages A and B) whereas only a small number (12%) of Cryptosporidium isolates belonged to the most widely recognized zoonotic Cryptosporidium species, C. parvum. These results indicate that in the Township of Langley, giardiasis risk from contaminated water is higher than the risk of cryptosporidiosis because not only are Giardia species detected more frequently than Cryptosporidium but a higher proportion are infective to humans. The most commonly detected Cryptosporidium species were C. andersoni (23%), which is most likely to originate from cattle, and C. baileyi (14%) from avian sources. The presence of C. hominis, infectious only to humans and representing 6% of the isolates in this study, indicates that contamination of surface water in the Township of Langley by human waste is occurring. Thus, while the predominance of Giardia assemblages that are primarily zoonotic in this study limits the usefulness of Giardia sequencing as an MST tool in this area, the range of Cryptosporidium species detected makes Cryptosporidium sequencing a beneficial pathogen source tracking tool. Infection of cattle with only zoonotic assemblages has been reported in some geographical areas, such as New Zealand (38), whereas other studies in Western Canada found that more than 90% of cattle samples contained assemblage E (39). Despite the


ACKNOWLEDGMENTS We thank staff at the Enhanced Water Laboratory at the BC Health Microbiology and Reference Laboratory, Provincial Health Services Authority, including Joe Fung, Selena Shay, Anna Li, Renata Zanchettin, and Mohamad Khan, for their contributions. This project was funded by Canadian Institutes of Health Research (CIHR) (SFW-66537), Canadian Water Network (CWN), Natural Science and Engineering Research Council of Canada (NSERC), and Partners of the NSERC Industrial Research Chair in Water Treatment at the University of Waterloo.

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