Beaver Fever: Whole-Genome Characterization of Waterborne Outbreak and Sporadic Isolates To Study the Zoonotic Transmission of Giardiasis (2024)

Upinder Singh, Editor

Upinder Singh, Stanford University;

Giardia causes the diarrheal disease known as giardiasis; transmission through contaminated surface water is common. The protozoan parasite’s genetic diversity has major implications for human health and epidemiology. To determine the extent of transmission from wildlife through surface water, we performed whole-genome sequencing (WGS) to characterize 89 Giardia duodenalis isolates from both outbreak and sporadic infections: 29 isolates from raw surface water, 38 from humans, and 22 from veterinary sources. Using single nucleotide variants (SNVs), combined with epidemiological data, relationships contributing to zoonotic transmission were described. Two assemblages, A and B, were identified in surface water, human, and veterinary isolates. Mixes of zoonotic assemblages A and B were seen in all the community waterborne outbreaks in British Columbia (BC), Canada, studied. Assemblage A was further subdivided into assemblages A1 and A2 based on the genetic variation observed. The A1 assemblage was highly clonal; isolates of surface water, human, and veterinary origins from Canada, United States, and New Zealand clustered together with minor variation, consistent with this being a panglobal zoonotic lineage. In contrast, assemblage B isolates were variable and consisted of several clonal lineages relating to waterborne outbreaks and geographic locations. Most human infection isolates in waterborne outbreaks clustered with isolates from surface water and beavers implicated to be outbreak sources by public health. In-depth outbreak analysis demonstrated that beavers can act as amplification hosts for human infections and can act as sources of surface water contamination. It is also known that other wild and domesticated animals, as well as humans, can be sources of waterborne giardiasis. This study demonstrates the utility of WGS in furthering our understanding of Giardia transmission dynamics at the water-human-animal interface.

IMPORTANCEGiardia duodenalis causes large numbers of gastrointestinal illness in humans. Its transmission through the contaminated surface water/wildlife intersect is significant, and the water-dwelling rodents beavers have been implicated as one important reservoir. To trace human infections to their source, we used genome techniques to characterize genetic relationships among 89 Giardia isolates from surface water, humans, and animals. Our study showed the presence of two previously described genetic assemblages, A and B, with mixed infections detected from isolates collected during outbreaks. Study findings also showed that while assemblage A could be divided into A1 and A2, A1 showed little genetic variation among animal and human hosts in isolates collected from across the globe. Assemblage B, the most common type found in the study surface water samples, was shown to be highly variable. Our study demonstrates that the beaver is a possible source of human infections from contaminated surface water, while acknowledging that theirs is only one role in the complex cycle of zoonotic spread. Mixes of parasite groups have been detected in waterborne outbreaks. More information on Giardia diversity and its evolution using genomics will further the understanding of the epidemiology of spread of this disease-causing protozoan.

TABLES1

All isolates included in the investigation with epidemiological data, assemblage, and various statistical data from the raw next-generation sequencing (NGS) reads and assembled genomes. Highlighted samples have mixed assemblages. Download TABLES1, XLS file, 0.1 MB.

TABLE1

Prevalence of Giardia assemblages in isolates grouped by source of origin

TABLE2

Giardia isolates from waterborne outbreaks in Canada summarized by assemblage

TABLES2

Genomic similarity among all samples (assemblages A and B) using MUMmer. Download TABLES2, XLS file, 0.1 MB.

FIGS1

Correlation between pairwise genetic distance (SNVs) and geographical distance in Giardia duodenalis assemblage B isolates. Download FIGS1, TIF file, 0.1 MB.

TABLE3

Fifteen Giardia isolates from surface drinking water at Mission Creek, British Columbia, Canada, studied over time and summarized by assemblage

FIGS2

Neighbor net analysis based on genome-wide SNVs showing the genetic relationships of Giardia duodenalis assemblage A (a) and assemblage B (b). Isolates are colored according to their source (water [blue], human [red], and veterinary [green]). Download FIGS2, PPT file, 0.2 MB.

Zoonotic transmission.

Isolates from assemblages A1, A2, and B from human and domestic veterinary sources have previously been examined using WGS, including assemblage A1 and A2 from Sweden and Norway (5, 23,–26), assemblage B from Alaska, United States, and Norway (5, 27), and one assemblage E isolate from a piglet in the Czech Republic (28). However, the current study is the first to sequence the whole genome of G.duodenalis isolates retrieved from geographically and epidemiologically linked surface drinking water, human, and animal (including wildlife) sources. It is also the first study to use WGS to analyze surface water source isolates from one site collected over time. Waterborne giardiasis outbreaks occurred in widely separated geographic locations in this large Canadian province; some were clearly epidemiologically linked to untreated surface water contaminated by beavers. This study also examined water, human, and beaver isolates from these outbreaks, with a view to the one health approach.

In a past study, 12 G.duodenalis isolates from beaver fecal samples collected from six different riverbank sites in southern Alberta, Canada, were found to belong to assemblage B based on sequences of 18S rDNA (29). Beavers from Massachusetts and Maryland in the United States were also confirmed to carry assemblage B genotypes based on multilocus sequences (22, 30). The current study provides clear genomic evidence supporting the role of beavers epidemiologically identified as the cause of two small community waterborne outbreaks (10). One beaver (VANC/90/UBC/52) removed from near the Arrow Creek (cyst-contaminated drinking water) drinking water intake during the Creston outbreak exhibited the same assemblage A1 genotype as two human isolates retrieved during the outbreak (Fig.2 and ​and4).4). Similarly, another beaver isolate (VANC/90/UBC/55) trapped in a Goat River beaver lodge, which was not associated directly with the outbreak, had A2/B infections with a majority of the same assemblage B (also with minority assemblage A2 genotype shared by another Creston outbreak clinical isolate [VANC/90/UBC/42]) (Fig.3 and ​and4).4). In addition, isolates from two other beavers from the Goat River beaver lodge (as noted, not directly associated with the Creston outbreak but located in a river about 10km from Creston, near Kitchener, BC) clustered with the three water system isolates retrieved from the contaminated water source (Arrow Creek) causing the Creston outbreak and with five human isolates retrieved during the outbreak investigation (Fig.4). These isolates all shared >98% identity in SNVs and genomic content. We note that Arrow Creek and Goat River are connected some distance downstream of the drinking water intake. We also note that, since juvenile beavers are able to migrate for long distances in the watershed when pressured, it is possible that the isolate from the lone beaver living under the snow and ice at the drinking water intake in Arrow Creek originated from the nearby Goat River beaver colony (10). Similarly, in the Revelstoke outbreak, the beaver infected by an assemblage B genotype and residing in the culvert at the community drinking water intake, and considered by public health to be the cause of the outbreak, was identical at the whole-genome SNV level of that of the three outbreak-associated human isolates (Fig.3). This is consistent with the results based on PCR-restriction fragment length polymorphism (RFLP) of tpi in these Revelstoke isolates (31). In both Creston and Revelstoke waterborne outbreaks, the drinking water source was upstream of the town and in remote areas without a human residence or the presence of domestic animals (18, 31). The epidemiological conclusions from the two giardiasis outbreaks are consistent with current SNV findings suggesting that beaver-to-human spread is important and that beavers were the source of human infections in these events. We do not, however, rule out the possibility of human influence on infections of beavers (human-animal-human dynamic) overall.

Our findings are consistent with the proposed role of beavers as an amplification or reservoir host (14, 32) for waterborne spread of zoonotic G.duodenalis species (Fig.5). Increased numbers of cysts from amplification hosts that reside in surface water supplies with ongoing contamination of water downstream has been shown to relate to beaver breeding activities in the late spring and early fall activities preparing for winter (33, 34). Thus, nonimmune beavers exposed to cysts from humans or other animal hosts contaminating the surface water in which they live, when infected with a zoonotic species, could produce ongoing contamination of the water source (Fig.5). As G.duodenalis trophozoites proliferate in the intestinal tracts of new beaver hosts, more cysts are excreted into their water, perpetuating contamination particularly during spring and early fall activities, hence their role in giardiasis transmission, acting as an infection amplification host for zoonotic species (14, 32, 34, 35). This role is seen more clearly when beaver infections are spread to humans (zoonotic species) in outbreak events, particularly when these outbreaks are associated with watersheds with limited domestic animal or human activities, such as two of the BC events studied.

We also note that three other beavers (one with mixed assemblage isolates) from a beaver lodge near Aldergrove, a southeastern BC town, were infected with identical A1 genotypes. Since assemblage A1 is lacking in genetic variability, less can be inferred about their genetic relatedness. In a similar fashion to the Goat River beaver lodge group, since the beavers live together in the same contaminated water source, highly genetically related isolates from beaver lodge members is not unexpected. It is possible that, similar to human-to-human spread (1, 2) reported in settings such as child day care centers, animal-to-animal spread within the beaver lodge also occurs (19) (Fig.5). Another possibility is that, since one beaver in the Goat River beaver group showed a divergent isolate compared to the assemblage B isolates of others, the Aldergrove beaver lodge had different types of infections, but this was not detected either because of the clonality of assemblage A1 (Fig.4 and ​and5)5) or because of the bias inherent to culture-based isolate retrieval (36).

Although no beaver hosts were directly identified as the source of the outbreaks in Barriere and Kitimat, both watersheds providing community drinking water were known to have beavers, along with other wildlife, living in them. We note that all BC outbreak communities studied used surface water sources from watersheds with very little human influence. It is well-known, however, that many types of wildlife such as deer, wolves, voles, moose, elk, coyote, beaver, and as noted above, many kinds of domesticated animals, including dogs, cats, sheep, cattle, as well as humans may cause spread (14, 18, 37,–40). Although not acting as amplification hosts, domestic animals in close contact with humans may also play an important role in human infection and environmental contamination. One study showed that G.duodenalis cysts from fecal samples from humans and dogs in a remote community in India typed at multiple genetic loci, and similar assemblages (A and B) were observed in humans and dogs in the same household (41). One of our study dog samples (Vancouver Island, Nanaimo, BC) was found to be assemblage B and clustered with a water isolate from interior BC (Mission Creek) as well as a beaver isolate (not associated with an outbreak) from northern BC. The second dog sample from Calgary, Alberta, Canada, was found to have an A1 genotype and clustered with other human, sheep, and water isolates collected from sources across Canada, United States, and New Zealand. Thus, animal-to-human transmission is clearly possible. A systematic review of giardiasis in Brazil also suggested that dogs and cats could be disseminators of Giardia, but at the same time, the results indicated that humans were a major source of transmission (40).

The SNV-based clustering of an assemblage B human isolate (collected in 1987 from an ill patient residing in Kelowna), with 10 assemblage B water isolates (collected between 1990 and 1994 from Mission Creek, 15km east of Kelowna [Fig.1]) could again be considered in two ways with regard to the original source of watershed contamination. First, humans infected with zoonotic species contaminate the environment, and then wildlife or domestic animals amplify and continue to circulate the parasite in that watershed (14). In this example, the resident of the nearby town (Kelowna) could have contaminated the Mission Creek watershed (a rural area with minimal human habitation) with ongoing watershed contamination observed (over a 4-year study period). Thus, a zoonotic Giardia assemblage B was maintained, circulating among animals living in this watershed and resulting in ongoing surface water contamination. Alternatively, it was also possible that this assemblage B genotype originated from and was maintained by animal hosts in this watershed over time with the patient from Kelowna, BC (near Mission Creek) becoming infected by exposure to Mission Creek surface water. We also note that the several different assemblages and SNV genotypes within assemblage B, all from one surface water body studied over years (Table3), are consistent with multiple sources of parasites contaminating this river (42, 43).

Cattle are another source of zoonotic spread of G.duodenalis through surface water (44). It was observed that higher concentrations of Giardia cysts were detected in Mission Creek during peak calving time (45). The prevalence of giardiasis in BC cattle has been reported as ranging from 16.7% to 70% in six locations from four watersheds (46, 47). It is also known that although G.duodenalis assemblage A and assemblage B have both been detected in cattle, nonzoonotic assemblage E is the predominant assemblage in most studies, including those of western Canada (1, 2, 48). No assemblage E isolates, however, were detected in our study (42).

Mixed infections and tools for further study.

Our data demonstrated the occurrence of mixed assemblages during waterborne outbreaks. Direct evidence for mixed infections in beavers (two beaver infections, one from an Aldergrove pond beaver lodge and one from a Goat River beaver lodge) are reported for the first time. Considering the Creston outbreak with only a single possible source of the deep winter waterborne outbreak, this study also provides indirect evidence of a mixed infection/coinfection occurring in the single (beaver source) host. While this observation has not been made previously in any other giardiasis outbreak (42, 49), infections with mixed assemblages have been reported in both humans and animals (1, 2, 50). The overall indirect evidence for this includes the following: this was an abrupt onset, well-defined outbreak; some isolates from humans infected during the outbreak exhibited assemblages different than those of beavers but with the same assemblages as those from the contaminated drinking water, and mixes were well documented. Thus, given the short-lived and explosive outbreak with a single source of contamination, we suspect that the beaver in Arrow Creek was infected with assemblages A1, A2, and B. We suggest that mixed assemblages in host and environmental samples could be more common than currently thought. It is probable that mixes are underestimated even in the current study, since genetic evidence may have been lost during isolate retrieval and culture.

We note that genotyping by conventional PCR and sequencing of a single locus may produce preferential amplification of one assemblage over another assemblage, also resulting in an underestimate of mixes (2, 49). Whole-genome SNV phylogeny may offer an advantage, in this regard, over other genotyping methods, with a better ability to detect mixed assemblages and having better ability to generate higher-resolution isolate characterization. At the same time, it is noted that the research method used in the current study has its own biases (primarily related to trophozoite propagation as noted above). For example, the lack of assemblages C, D, and E detected in animal samples and the relatively low proportion of assemblage A2 in the study collection (51) may be based on the phenotypic ability of the isolates to adapt to in vitro growth.

Reference genome selection may also bias SNV analysis particularly when query genomes are of variable evolutionary distances from the reference genome used and when the reference genome is not completely sequenced (52) (for example, the reference genome in assemblage B was incomplete). Since our data set represents the largest collection of Giardia genomic sequences thus far, instead of using a single published reference genome for SNV analysis, we used a reference-free SNV analysis algorithm and constructed assemblages A and B specific pan-genomes as the reference. This allowed direct comparison of isolates without using a reference genome as an intermediate. Such an approach has the same sensitivity as the mapping-based approach (53, 54), while including pan-genome lineage-specific regions not present in the published reference genome.

Genomic data confirmed that assemblages A1 and B of G.duodenalis are widespread in water, human, and veterinary source samples in British Columbia, Canada. The predominance of assemblage A1 in sporadic human infections in the current study is consistent with other studies, although assemblages A2 and B were also observed to cause some human infections (1, 55). No other assemblages were detected in this large number of water, veterinary, and human isolates studied. No unique, adapted assemblage was detected in beavers; assemblage B has been noted to be prevalent in previous reports (1), while A1 and A2 have been rarely reported in beavers (15, 56).

The genetic divergence between assemblages A and B is congruent with the theory that assemblages A and B are in fact two distinct biological species (1, 5, 27). The lack of evidence for interassemblage gene flow and recombination suggests that these two assemblages can be reproductively isolated (57). The majority of assemblage A1 isolates have been reported to be highly clonal, consistent with our finding; no substructuring and genetic variability from isolates obtained from human, beaver, sheep, or dog sources was observed. This suggests that assemblage A1 is a highly adapted, panglobal, zoonotic genotype (1). The genetic variability in the samples from surface water (VANC/90/UBC/62) and a cat (ATCC 50163) may reflect specific local adaptations. Assemblage A2 has been reported from humans and domesticated animals, such as cattle, horses, dogs, and cats (1, 49). Assemblage A2 isolates are genetically and biologically more variable than A1 isolates. It was impossible, however, to assign our A2 isolates to a meaningful subcluster because of the small number of isolates available. Our findings also revealed assemblage A2 to be rather distant from A1 based on percent SNV variation. Ankarklev et al. (25) estimated the genomic divergence between strain WB and two assemblage A2 isolates to be 1% based on 100,000 SNVs; however, the current study demonstrated greater genetic divergence (ca. 15 to 20%) between assemblages A1 and A2. The divergence between assemblages A1 and A2 may be underestimated in the former study, as the two A2 samples shared only 57,680 single nucleotide polymorphisms (SNPs), while each differed from the reference genome WB by 93,900 and 100,273 SNPs, respectively (25). Differences in the sources of the samples, sequencing technology, variant calling strategies, and coverage are also possible factors contributing to disparity in heterogeneity estimates (52).

While it has been reported that assemblage B undergoes frequent recombination events, the assemblage B isolates of surface water, human, and beaver origins in the Creston and Revelstoke outbreaks, as well as the assemblage B surface water isolates in the region of Mission Creek, clustered together with high genetic similarity. This can be attributed to asexual reproduction and clonal population expansion (35, 58). Genetic heterogeneity may be driven by pressures from the host immune systems (59). On the other hand, greater genetic variation (longer branch length) was detected between isolates in the Barriere and Kitimat outbreaks based on WGS SNVs. This may indicate different selective pressures within or among hosts or a rapid microevolution between different subtypes of assemblage B (leading to the genetic differences detected).

Assemblage B, the predominant assemblage from surface water samples collected in different regions across BC, also varied genetically; thus, geographical or environmental barriers could be driving such genetic divergence. It is possible that different groups or lineages of this parasite diverged or evolved in geographic isolation due to the lack of recombination or gene flow among localized parasite population types (58). It is important to continue to investigate genomic variation in isolates from the different geographical regions.

Detecting mixed Giardia duodenalis infections is important to public health epidemiology because it may have significant clinical implication in the treatment of patients. Our study showed that multiple Giardia genotypes can occur within an outbreak. We also showed that different genotypes may be found in the same surface water source studied over time. Recent in vitro studies indicated that this parasite appears to have the capacity to modulate host immune responses and, pertinent to the unique observations from community outbreak events, that mixed infections of different assemblages or genotypes can cause enhanced intestinal cell damage (59, 60). The importance of mixes, which are probably underestimated, warrants further study. While we note that population-level “virulence” as expressed by outbreak events, is clearly related to lack of herd immunity (61), further study of intra-assemblage genetic variation and interassemblage interactions along with an exploration of the as yet unexplained species-level, polyparasite host interaction (possibly producing increased virulence), is needed (61). Better methods for optimal identification of mixed infections, in single hosts and in outbreak events, are also needed.

Human health is closely linked to the health of animals and our environment. The growing understanding of the complex ecology of G.duodenalis continues to reinforce the fundamental need for multiple layers of protection of surface water supplies used for drinking water (including watershed protection and management as well as adequate treatment). This reinforces the need for adequate treatment for small communities. The small rural BC communities impacted by the historical outbreaks described have gone on to make drinking water protection and treatment improvements. The unique niche of aquatic mammals, such as beavers in the amplification of G.duodenalis, also requires recognition for its part in the zoonotic cycle of transmission.

Isolate information.

A total of 89 isolates were included in whole-genome sequencing (WGS) and single nucleotide variant analyses (see TableS1for a complete list of isolates and their sources). All isolates used in this study are part of an archive of Giardia isolates housed at the British Columbia Centre for Disease Control Public Health Laboratory (BCCDC PHL). They were collected from 1985 to 1996 with appropriate human and animal ethics protocols from the University of British Columbia. Additional ethics approval was not sought for this study, as these isolates were archival. Most isolates in this study have previously been characterized using pulsed-field gel electrophoresis, PCR-based fingerprinting, and/or Sanger sequencing methods (10, 18, 42, 43).

As the investigation of the related samples from the outbreak in Creston, British Columbia (BC), Canada, were important to this study, further details of this outbreak are available (10, 23). Since isolates from the Mission Creek surface water supply were the largest number of isolates (from one single site), further information is briefly described (Table1 and Table S1). Raw water samples were collected over 4years, during the same overall 10-year isolate collection period as the others in this library. Water samples were collected to determine the frequency of parasite contamination and to assess the effects of settling ponds (reservoirs) on Giardia cyst concentration (45) (Fig.1). Ten isolates were retrieved from raw water samples collected at the drinking water intake from Mission Creek; five additional isolates were collected from the reservoir/settling ponds and also from further down the water distribution system, after the reservoir (acting as settling ponds). Water samples were collected from all seasons over a 4-year period (Table3). At that time, the town used settling ponds and a long contact chlorination contact time for treatment (it now fully treats water using membrane filtration and chemical disinfection). They reported no excess cases of giardiasis during the study period. Three other geographically separate surface water sites associated with outbreaks (Barriere, Kitimat, and Revelstoke) (Fig.1) were also tested for cysts. An additional four water source isolates not associated with outbreaks were also retrieved from geographically separate water sites. No excess cases of giardiasis were reported from the four towns using these four surface drinking water supplies. Two water source isolates associated with a waterborne outbreak in another Canadian province (Newfoundland) occurring during the collection period (1991) were also included (62). Animal source isolates comprised 17 isolates from BC beavers, 2 from dogs (BC, Alberta), 2 from sheep (United States [ATCC 50170] and Canada), and 1 from a cat (United States [ATCC 50163]). These isolates were retrieved from the BCCDC PHL archive (including those provided by external research laboratories and reference collections) and are described in the studies above. We included strain WB (ATCC 50803) (assemblage A1) as the reference isolate; this isolate has been characterized as being acquired by a traveler to Afghanistan (63).

Isolate retrieval.

Giardia isolates were retrieved previously by purifying cysts from surface water samples (23) and human and beaver fecal samples containing cysts (64) using both in vitro (65) and in vivo excystation methods. While previous work showed that both methods are effective (36), almost all study isolates were retrieved using the in vivo gerbil model, given the low numbers of cysts. All animal studies were conducted in the Faculty of Medicine animal facilities at the University of British Columbia (UBC), and animals were handled according to the guidelines and in full compliance with the UBC Animal Care and Use Program (ACUP). Giardia trophozoites were cultured in TYI-33 medium and cryopreserved in 10% dimethyl sulfoxide (DMSO) at −80°C (65). For the present molecular characterization study, cryopreserved trophozoites were retrieved, and DNA was obtained from the 89 study isolates as noted below.

DNA extraction, library preparation, and whole-genome sequencing.

Genomic DNA was extracted from parasite trophozoites using a QIAamp DNA minikit (Qiagen, Mississauga, ON, Canada) with modifications. Briefly, trophozoites were centrifuged, washed with phosphate-buffered saline (PBS), and incubated at 58°C with proteinase K and buffer ATL for digestion. Extracted DNA was quantified, and its quality was assessed as described in reference 23. Paired-end (PE) DNA libraries were constructed with Nextera XT DNA kit (Illumina, San Diego, CA, USA) according to the protocols with the following modification. To remove small inserts, DNA libraries were purified twice using Ampure XP beads (Beckman-Coulter) at a 0.5× ratio. After DNA was cleaned up, traces were verified using High Sensitivity DNA chips in a 2100 BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA). Samples were then normalized, denatured, and pooled following the manufacturer’s instructions (Illumina, San Diego, CA, USA). The pooled library consisted of 6 to 10 samples (per run) (each with a unique DNA index from the Illumina Nextera Index kit) prepared as recommended by Illumina (MiSeq v2 reagent preparation guide) and loaded onto a cartridge (V2 chemistry) with 250-bp paired-end output. Additionally, 1% of PhiX, an adapter-ligated single-stranded DNA (ssDNA) virus, was spiked in the pooled libraries as a control in every sequencing run.

Whole-genome de novo assembly.

The quality of the reads was assessed by Fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and the adapter sequences and the sequences of poor quality were trimmed by Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads were assembled using assembler SPAdes v.3.1.1 (66) with default parameters, and the resulting contigs were filtered to be >500bp and have a coverage of >4. We assessed the assemblage type of each isolate by mapping reads from each isolate to the reference genomes of G.duodenalis (assemblages A, B, and E) that were retrieved from the GiardiaDB (http://giardiadb.org) (67). To remove any potential contaminant contigs (non-Giardia reads), contigs were mapped to the NCBI nt database using BLASTn megablast (-best_hit_overhang 0.25 -best_hit_score_edge 0.05). If 80% of hits within two times the minimum E value mapped to G.duodenalis genomes, the contigs were kept. Additionally, any contigs that did not fit this criterion were mapped to a combined reference of assemblage A (WB) and assemblage B (GS_B) using BLASTn megablast with the same parameters, and those for which >500bp matched the reference were added to the final contig set.

Even after removing contaminating reads, five samples had a total contig length greater than 14Mb. This is significantly larger than the expected genome size of 10 to 11Mb, suggesting further contamination from within Giardia species. Reads of these five samples were each mapped to a combined reference of assemblages A and B (WB, DH_A, and GS_B) and using BLASTn megablast, with the same parameters. In mixed samples, reads which hit only the minority assemblages (3.5 to 28.7% of total reads) were removed. SPAdes assembly and subsequent filtering were then performed on these filtered reads as described above. This resulted in two of the five samples having a combined contig length of <12,000,000 bp; however, three samples were still above this length and were excluded from analysis, as we could not separately the contamination from the major assemblage. Final contigs from each sample, along with the references WB and GS_B, were used to construct two assemblage specific pan-genomes using Panseq (68) with default parameters. The assembled contigs of each sample were arranged and compared using MUMmer (69).

Whole-genome reference-based assembly and variant detection.

Reads were trimmed using cutadapt v1.8 (70) to remove Illumina adapters and reads with quality of <20 or length of <200. Assemblage assignment was then performed by mapping trimmed reads to a combined reference consisting of assemblage A (WB) and assemblage B (GS_B) using bowtie2 v2.1.0 (71) (with parameters --phred33 --local --dovetail --maxins 850) and determining the assemblage based on what the majority (70 to 99%) of reads mapped to. Trimmed reads were then remapped to the single majority assemblage using bowtie2 (--local).

Four samples had >5% of reads mapped to the minor assemblage after the initial bowtie2 alignment, so these were included in the analysis for both assemblages. For analysis in their minor assemblage, reads were mapped to a combined reference of WB, DH_A, and GS_B, using BLASTn megablast, with the same parameters as described above, and reads which hit only the majority assemblages were removed. The remaining reads were then included in the discoSNP analysis as below.

Single nucleotide variant (SNV) calling was performed by entering trimmed reads into discoSNP v2.1.2 (54) with parameters -c 4 –b 1 –k 31 –P 3, and a separate run for each assemblage. SNVs generated using discoSNP are output in “bubbles,” where each bubble consists of two distinct sequences (or branches) of k + 2 nodes, with the start and the end nodes in common. Each bubble was aligned to the assemblage-specific pan-genome using BLASTn and filtered so that only bubbles that hit a single unique position in the pan-genome were considered for further analysis. Additionally, bubbles were removed if any sample had a combined coverage over both branches of the bubble of <4 or any sample had <75% coverage in the majority branch. Filtered discoSNP output was then converted into a FASTA file using the SNV base from the branch with the highest coverage from each bubble. Phylogenetic trees were generated using FastTree v2.1.8 (default parameters) (72) and drawn using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic relationships among isolates were also inferred using SplitsTree, and a Φ-test was conducted to estimate the level of recombination (73). The correlation between genetic and geographic distances was established using MEGA (74) and GenAlEx (75).

Accession number(s).

Genome assemblies and raw reads are available at NCBI under BioProject no. PRJNA280606.

Funding support of the Canadian Institutes for Health Research (CIHR) in retrieving the isolate library is acknowledged, along with the technical support of the province’s public health and local officials and members of the Environmental Microbiology Program at the British Columbia Public Health Laboratory (BC PHL).

In particular, we thank Anna Li, Renata Zanchettin, Kirby Cronin, Justin Dirk, Sara Tan, M.-K. Lee, Brian Auk, and C. P. Fung for their ongoing dedication and technical assistance. We thank Merle Olsen, P. Wallis, G. Faubert, and P. Boreham for providing Giardia cultures, Jaroslav Klápště and Sunny Mak for providing the maps, and Jason McDiarmid for providing information on the improved Creston water supply.

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