Canadian Journal of Zoology

Intestinal parasites of wolves (Canis lupus L. 1758) in northern and western Canada

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2016-0017.R1

Manuscript Type: Article

Date Submitted by the Author: 08-Apr-2016

Complete List of Authors: Schurer, Janna; University of Saskatchewan, Veterinary Microbiology; University of Washington, Occupational and Environmental Health Sciences Pawlik, Michael; University of Saskatchewan Huber, Anna;Draft University of Saskatchewan Elkin, Brett; Government of the Northwest Territories, Environment and Natural Resources Cluff, Dean; Government of the Northwest Territories, Department of Environment and Natural Resources Pongracz, Jodie; Government of the Northwest Territories Gesy, Karen; University of Saskatchewan Wagner, Brent; University of Saskatchewan Dixon, Brent; Health Canada Merks, Harriet; Health Canada Bal, Mandeep; Guru Angad Dev Veterinary & Sciences University Jenkins, Emily; University of Saskatchewan

PARASITOLOGY < Discipline, MOLECULAR GENETICS < Discipline, Keyword: Zoonoses

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Intestinal parasites of wolves ( Canis lupus L., 1758) in northern and western Canada

Janna M. Schurer, Michael Pawlik, Anna Huber, Brett Elkin, H. Dean Cluff, Jodie Pongracz, Karen Gesy,

Brent Wagner, Brent Dixon, Harriet Merks, Mandeep S. Bal, Emily J. Jenkins

JMS, MP, AH, KG, BW, EJJ - Department of Veterinary Microbiology, University of Saskatchewan, Canada

BE, HDC, JP - Environment and Natural Resources, Government of the Northwest Territories, Canada

BD, HM - Health Canada, Canada

MSB – Guru Angad Dev Veterinary & Animal Sciences University, India

Corresponding Author: Janna M Schurer, 52Draft Campus Drive, Saskatoon, SK, S7N 5B4, email: [email protected] , telephone: 306-202-0827, fax: 306-966-7244

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Intestinal parasites of wolves ( Canis lupus ) in northern and western Canada

Janna M. Schurer, Michael Pawlik, Anna Huber, H. Brett Elkin, Dean Cluff, Jodie Pongracz, Karen Gesy,

Brent Wagner, Brent Dixon, Mandeep Bal, Emily J. Jenkins

Abstract:

Gray wolves ( Canis lupus L., 1758) are mobile opportunistic predators that can be infected by a wide range of parasites, with many acquired via predator-prey relationships. Historically, many of these parasites were identified only to genus or family, but genetic tools now enable identification of parasite fauna to species and beyond. We examined 191 intestines from wolves harvested for other purposes from regions in the Northwest Territories, British Columbia, Saskatchewan, and Manitoba. Adult helminths were collected from intestinal contents for morphological and molecular identification, and for a subset of wolves, fecal samples wereDraft also analyzed to detect helminth and protozoan (oo)cysts. Using both detection methods, we found that 83% of 191 intestines contained one or more parasite species, including cestodes (Taenia spp. , Echinococcus spp., and Diphyllobothrium sp.), ( Uncinaria stenocephala, Trichuris spp. , Physaloptera spp. , and ), a trematode ( Alaria sp.), and protozoa ( Sarcocystis spp. , Giardia spp. , and Cryptosporidium spp.).

Molecular characterization identified one species of Diphyllobothrium (D. latum ), three species of Taenia

(T. krabbei, T. hydatigena, and T. multiceps ), and two Giardia assemblages (B and C). These results demonstrate the diverse diet of wolves, and illustrate the possibility of parasite spillover among wildlife, domestic , and people.

Key Words:

Wolf; Canis lupus ; parasite; post-mortem; Canada; zoonotic

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Introduction:

Gray wolves ( Canis lupus L., 1758), including tundra and timber wolves, are large canids native to North

America and Eurasia that can disperse hundreds to thousands of kilometers in the wild (Merrill and

Mech 2000; Mech and Boitani 2004). Despite predator reduction programs that extirpated these canids

from many regions of Canada by the 1950s, the wolf population appears to have recovered, and is now

broadly distributed across the country except for the Maritime provinces and the island of

Newfoundland (Vila et al. 1999; Darimont and Paquet 2000; Mech and Boitani 2004). Gray wolves face

both natural and anthropogenic challenges to survival including prey availability, inter-species

competition, disease, habitat fragmentation, hunting/trapping, vehicle accidents, and changing

environmental and climatic conditions. As apex predators, they play a critical role in maintaining ecosystem health by limiting species in lowerDraft trophic levels, and contributing to species biodiversity throughout the food web (Darimont and Paquet 2000). The health status of gray wolves is also relevant

to the health of people, livestock and companion animals because pathogens move between these

groups in a complex web of direct and indirect pathways.

Gray wolves generally depend upon a primary ungulate prey species in their diet, but feed

opportunistically on a variety of secondary prey species, according to season and availability (Kuyt 1972;

Mech and Boitani 2004). Outside of denning season, tundra wolves generally follow migratory prey,

while timber wolves generally protect a territory year-round, hunting suitable prey within a defined area

(Musiani et al. 2007). Timber wolves residing in Riding Mountain National Park (RMNP), in southern

Manitoba (MB), predate elk ( Cervus elaphus canadensis (Erxleben, 1777)) most frequently, followed by

moose ( Alces alces L., 1958), white-tailed deer ( Odocoileus virginianus (Zimmermann, 1780)), and

snowshoe hare ( Lepus americanus (Erxleben, 1777); Sallows 2007). In other areas of Canada, wolves

primarily prey on Sitka black-tailed deer ( O. hemionus sitkensis (Merriam, 1898); Darimont and Paquet

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2000), caribou ( Rangifer tarandus L., 1958; Musiani et al. 2007), mule deer ( O. hemionus (Rafinesque,

1817)), muskoxen ( Ovibos moschatus (Zimmermann, 1780); Gray 1983), or moose (Peterson and Page

1988). Secondary prey include bison ( Bison bison L. 1958), beaver ( Castor canadensis (Kuhl, 1820)) and various , mustelids, ursids, lagomorphs, and spawning fish (Darimont and Paquet 2000; Darimont and Reimchen 2002), especially when ungulate populations are scarce (Darimont and Reimchen 2002;

Garrott et al. 2007). Many of these prey species act as intermediate or paratenic hosts for parasites that utilize wolves as definitive hosts. In addition to parasites acquired from prey through indirect life cycles, wolves are exposed to numerous parasites that have a direct fecal-oral life cycle. The result of this diverse diet and environmental exposure is that tundra and timber wolves are exposed to a broad range of parasites, including at least 28 species, 27 cestode species, 16 trematode species, one acanthocephalan, as well as protozoans (CraigDraft and Craig 2005). Common parasites of Canadian wolves include taeniid spp. cestodes, nematodes ( Toxascaris leonina and Uncinaria stenocephala), the intestinal fluke Alaria , and coccidian protozoa such as Sarcocystis spp. (Choquette et al. 1973; Craig and Craig

2005; Stronen et al. 2011; Bryan et al. 2012).

The advent of molecular tools has resulted in many parasites being taxonomically re-classified and/or identified to levels beyond that possible by morphological examination (e.g., subspecies, genotypes, subgenotypes, and assemblages). With a few recent exceptions (Bryan et al. 2012; Schurer et al. 2014 b), surveys of wolves in Canada have reported parasites only to genus or species level, depending on whether samples were collected by necropsy or fecal examination. The present study surveyed wolves from 8 regions in 3 provinces (British Columbia [BC], Saskatchewan [SK], Manitoba [MB]) and the

Northwest Territories (NT), each with distinct differences in wolf dietary preferences, prey availability, and proximity to human settlements. We report gastrointestinal helminth and protozoa distribution and prevalence in these areas, including Taenia species, Echinococcus species and genotypes , and Giardia

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duodenalis assemblages. These baselines are increasingly relevant for ecologists, veterinarians, and

those concerned with public health aspects of these parasites, as rapid environmental change in

northwestern Canada may be redrawing the boundaries of hosts and parasites alike.

Materials and Methods:

Gross examination

Hunters and trappers obtained 191 wolves from the Northwest Territories ( N=143), British Columbia ( N

=28), Saskatchewan ( N =17), and Manitoba ( N =3), Canada for purposes other than research, between

2010 and 2014 (Figure 1). Intestines were stored at -20˚C until shipped to the University of

Saskatchewan, where they were frozen at -80˚C for at least 5 days prior to examination in order to inactivate eggs of Echinococcus , which areDraft potentially infective for people (Eckert et al. 2001). We harvested helminths from the intestines using the scraping, filtration, and counting technique (SFCT)

(Gesy et al. 2013), and stored them in 70% ethanol until they could be identified to genus by

morphological examination. The intensity of Echinococcus cestodes was estimated as per (Gesy et al.

2013). Where possible, we obtained feces from the rectum, colon, and/or distal ileum, and stored them

at -20˚C prior to conducting fecal counts (FEC). Parasite ova and/or cysts were identified and

quantified for 4 grams (wet weight) of feces using a modified Stoll double centrifugation fecal flotation

(Nielsen et al. 2010). Giardia and Cryptosporidium (oo)cysts were quantified for 1 gram of feces using a

sucrose gradient flotation and commercial immunofluorescence assay (Waterborne Inc., New Orleans,

LA; Olson et al. 1997).

Wolf sampling sites in the Northwest Territories

Wolf samples were collected in four NT ecozones (Figure 1; Ecosystem Classification Group 2007;

Ecosystem Classification Group 2008; Ecosystem Classification Group 2010; Ecosystem Classification

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Group 2012). In the north, wolves sampled were from the Tundra Plains and Tundra Cordillera ecozones where summers are very short and cold, and winters are extremely cold and long (mean annual temperatures (MAT) -9 ˚C to -11˚C; Ecosystem Classification Group 2010). Continuous permafrost exists throughout these nearly treeless landscapes that are dominated by dwarf shrub tundra on uplands

(Ecosystem Classification Group 2010).

Samples came from wolves in the northern, mid, and southern areas of the large Taiga Plain ecozone.

The Taiga Plain ecozone ranges from very short cool summers and very cold winters in the north to warm moist summers and very cold and snowy winters in the south (MAT -8˚C to -13˚C in the north to -

1˚C to -4.5˚C in the south; Ecosystem Classification Group 2007). Permafrost is continuous in the north to discontinuous in the south and peatlandsDraft are extensive throughout. The highly variable climate results in forests that range from very open stunted forests of black ( Picea mariana (Mill.) B.S.P.) and white spruce ( P. glauca (Moench) Voss) with understory that is lichen in the north to closed canopy mixed-wood forest of aspen ( Populus L.) and white spruce with jack pine ( Pinus banksiana Lamb.) on drier sites in the south (Ecosystem Classification Group 2007).

There were also samples from the Taiga Shield ecozone, which has a climate of very short cool summers, very cold winters (mean annual temp -4˚C to -9˚C) towards treeline to warm moist summers, and very cold and snowy winters (mean annual temperature -3˚C to -4˚C) further southwest (Ecosystem

Classification Group 2008). Permafrost ranges along the gradient from widespread and continuous to discontinuous. Near treeline, trees only occur in small stands along lakeshores, lower slopes, eskers and gullies. At treeline, there are very open and usually stunted black and white spruce woodlands that progress to a closed-canopied mixed wood forests of aspen, white spruce and jack pine in the southwest

(Ecosystem Classification Group 2008).

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Molecular methods - cestodes

We extracted DNA from cestodes for PCR analysis using two methods. For Taenia and Diphyllobothrium ,

we macerated approximately 0.1 gram of tissue from 1-2 specimens per using needle tip scissors,

and extracted DNA using the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). For Echinococcus ,

we randomly selected three intact cestodes from each infected wolf, placed each in separate petri

dishes containing 500 μL lysis buffer for 10 mins, and extracted DNA as per Catalano et al. (2012).

Amplification of Taenia and Echinococcus DNA focused on regions of the nicotinamide adenosine

dinucleotide dehydrogenase subunit 1 (NAD1) and cytochrome c oxidase subunit 1 (CO1) mitochondrial

genes, with lengths of 471 and 446 base pairs, respectively (Bowles et al. 1992; Bowles and McManus 1993). A separate 437 bp region of the CO1Draft mitochondrial gene was used to amplify Diphyllobothrium - specific DNA (Wicht et al. 2010). These PCR products were resolved by electrophoresis (110V, 25 min)

on a 1.5% agarose gel stained with RedSafe nucleic acid (ChemBio Ltd, Herfordshire, UK), and viewed

under UV light. Samples that produced positive bands were purified (QIAquick PCR Purification Kit,

Qiagen Inc., Valencia, CA), sequenced in both directions using the same primers as the original

amplifications (Macrogen Inc., Seoul, Korea), and then assembled, edited and aligned using Staden

v4.10. Consensus sequences were identified by comparison to previously published sequences in

GenBank (National Center for Biotechnology Information) by BLAST search (blastn).

Molecular methods – Giardia

Molecular methods to identify G. duodenalis assemblages were performed on a subset of positive

samples from NT and SK ( N=35), using a PCR assay targeting a 292 bp region of the 16S-rRNA gene.

Specifically, DNA was extracted from cysts concentrated in a sucrose suspension using the DNeasy Blood

and Tissue Kit (Qiagen Inc., Mississauga, ON), using a protocol modified as follows: 100 μL of the

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suspended cysts were transferred to 1.5 mL microcentrifuge tubes containing 300 μL lysis buffer. Tubes

were subjected to five freeze/thaw cycles followed by overnight incubation with 20 μL of proteinase K

(20 mg/ml) at 56°C. Manufacturer’s instructions were then followed to purify the DNA. Nucleic acid

was eluted with 100 μL of elution buffer. Positive control, G. duodenalis cysts (Waterborne, Inc., New

Orleans, LA), and negative control, RNase/DNase-free water (Life Technologies, Carlsbad, CA), were also

extracted in parallel. Nested-PCR was performed to amplify a fragment of the Giardia 16S rRNA gene as

described in Appelbee et al. (2003). DNA sequencing of the PCR products was performed using a 3130xl

Genetic Analyzer (Applied Biosystems, Foster City, CA). PCR products were purified and bi-directional

sequencing was performed using the same primers as the original amplifications. DNA sequences were

assembled, edited and aligned using SeqScape v2.5 (ABI). Resulting consensus sequences were then aligned with representative sequence dataDraft from G. duodenalis assemblages, and trimmed to identical lengths of 189 bp for 16S rRNA, using Bioedit v7.1.3.0. Consensus sequences were compared to

reference sequences in GenBank using NCBI standard nucleotide BLAST (blastn).

Statistical analysis and mapping

Data were entered into a spreadsheet and analysed using SPSS (version 19; IMB Corporation, Armonk,

New York, USA). Age was categorized as juvenile (<12 mos) or adult (≥12 mos), and wolves were

considered to be infected if parasite adults or ova were observed by FEC or SFCT. A two-sided Fisher’s

Exact test at the 5% level was conducted to determine if gender and age were statistically associated

with parasite infection, and if parasite prevalence differed between geographic regions. Freeman-

Halton’s extension was used when contingency tables were greater than 2x2. Four fecal samples with

extremely high numbers of Sarcocystis sarcocysts were not quantified, and were omitted from the

overall intensity calculation.

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Precise geographic coordinates for wolf-kill locations were only reported for a subset of samples. To

illustrate sampling regions for the remaining samples, we mapped the closest city or town to the

reported wolf location. Sampling sites were mapped using ArcGIS (version 10; Esri, Redlands, California,

USA).

Results:

Of the 191 wolves (83 female, 106 male, two unreported), 15 were juvenile, 102 were adult, and 74 had

unreported age. We observed at least one parasite species in 63% of 191 intestines (Table 1), which was

not statistically different ( P=0.82) from the 64% infection prevalence (not including protozoa) observed

in a subset of fecal samples ( N =151). Combining both methods to include protozoans showed that 83% (95% CI: 78-83%) of wolves were infected withDraft at least one gastrointestinal parasite, and that species richness in infected animals ranged from one to seven species (Figure 2).

Analysis of gut contents revealed six helminth genera (Diphyllobothrium spp., Taenia spp. , Echinococcus

spp. , Toxascaris leonina, Uncinaria stenocephala , and Alaria sp.), with cestodes observed most

frequently, followed by nematodes and flukes. Overall, 71 of 191 (37%) wolves were infected by

Echinococcus, with a median intensity of 2,258 ± 24,397 cestodes per infected wolf (range: 15-149,600).

DNA was successfully amplified from at least one adult cestode in 54 of 71 (76%) infected wolves.

Among BC wolves, 25 of 28 (89%) were infected with Echinococcus , including E. canadensis G8 (6 of 28;

21%), E. canadensis G10 (13 of 28; 46%), and E. multilocularis (3 of 28; 11%). Echinococcus results

obtained by gut necropsy from all SK/MB and 73 NT wolves are reported elsewhere (Schurer et al.

2014 b). Of the remaining NT wolves, nine of 70 were infected with Echinococcus (13%), including E.

canadensis G8 (1 of 70; 1%) and E. canadensis G10 (9 of 70; 13%). The minimum prevalence for all

wolves, including those reported elsewhere, was as follows: 46 of 191 (24%) E. canadensis G10 , 15 of

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191 (8%) E. multilocularis , and 12 of 191 (6%) E. canadensis G8 (15). Infection prevalence was

significantly different between NT, BC and SK/MB (P-value < 0.001). Mixed taeniid infections were

present in BC, NT, SK and MB, and were characterized as E. canadensis-E. multilocularis (6%) , E.

canadensis G8 -G10 (5%), T. multiceps -T. hydatigena (1%), and Echinococcus-Taenia (20%). Taenia spp.

cestodes were present in 36% of wolves and were identified, in order of descending frequency, as T.

krabbei , T. hydatigena , and T. multiceps . Of the five wolves (3% overall) infected with Diphyllobothrium,

four harboured D. latum . Mixed infections of D. latum with T. krabbei were also seen ( N =2). The

similarity of aligned PCR sequences to those previously catalogued in GenBank was generally high: T. krabbei (99-100%), T. hydatigena (98-100%), T. multiceps (96-97%), D. latum (99-100%), E. canadensis

G8 (98-99%), E. canadensis G10 (97-100%), E. multilocularis (98-100%). One Echinococcus isolate targeted by the CO1 had lower similarity –Draft with 93% shared identity to E. canadensis G10 - and was not successfully sequenced with ND1 primers. Giardia sp., Taenia spp., Echinococcus spp., and D. latum

sequences were submitted to GenBank and were assigned accession numbers as follows: KX058165-

KX058193, xxxxxxxx-xxxxxxxx.

Fecal analysis and intestinal examination both indicated that taeniids ( Taenia and Echinococcus ) and T. leonina were the dominant gastrointestinal helminths of wolves in the sample regions. Slightly less prevalent were Giardia sp. , Cryptosporidium spp. , Alaria sp., and Sarcocystis spp. (range: 18-25%). Least common, and with limited distributions were Diphyllobothrium sp. , U. stenocephala, Physaloptera spp. , and Trichuris spp. (range 1-8%) . Molecular analysis of Giardia demonstrated that most infections were zoonotic Assemblage B only (33 of 35 samples in NT and SK), but that two wolves (one each in NT and

SK) harboured mixed infections of Assemblage B and canid-specific Assemblage C.

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Overall infection status (by either method) was not significantly correlated to age ( P=0.16) or gender

(P=0.56). Age class was not a risk factor for infection by Echinococcus spp. (P=0.77), Taenia spp.

(P=0.23), T. leonina (P=0.55), Sarcocystis spp. (P=0.15), Giardia spp. (P=1.0) or Cryptosporidium spp.

(P=0.22). Gender was also not associated with infection by individual parasites [ Echinococcus (P=0.36),

Taenia (P=0.65), Toxascaris (P=0.74), Diphyllobothrium (P=0.16), Alaria (P=0.24), Sarcocystis (P=0.15),

Giardia (P=0.26) or Cryptosporidium (P=0.63)]. Parasite genera observed by fecal flotation but not

detectable by intestinal examination included Trichuris, Physaloptera, Sarcocystis, Giardia, and

Cryptosporidium .

At the province/territory level (Tables 1-2), we observed statistically significant geographic differences in parasite prevalence for Echinococcus spp.,Draft Taenia spp., and Sarcocystis spp. In NT, significant differences in parasite prevalence occurred among ecozones for D. latum, T. multiceps , and Alaria sp.

(Table 3). While E. canadensis was distributed across all ecozones of the NT and was present in all

provinces, E. multilocularis was detected only in the Taiga Plains/Taiga Cordillera ecozone in NT and Fort

St. John in BC. Taenia krabbei was also widely distributed, whereas T. hydatigena was absent from two

NT ecozones, and T. multiceps was found only in Tundra Plains. No significant differences in T. leonina

prevalence were observed at the province/territory or ecozone level.

Discussion:

Our study reports baseline endoparasite prevalence estimates for wolves in several regions of western

Canada, using molecular techniques to identify species and genotypes, when required. Such

information will become increasingly important for those using One Health approaches to investigate

pathogen transmission dynamics among animals, people and the environment. Our study demonstrated

statistically similar helminth prevalence using fecal flotation and gastrointestinal necropsy (64% and

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63%, respectively). In situations where canid necropsy is unacceptable (e.g., zoological specimens, pets, free ranging wildlife of conservation concern), fecal flotation is a non-invasive technique with high sensitivity for detecting certain endoparasite species such as T. leonina (Liccioli et al. 2012). Using sensitive techniques (such as sucrose gradient concentration and immunofluorescence assays) and in the hands of experienced users, it has the added benefit of detecting protozoa not visible to the naked eye on necropsy or commonly observed using basic fecal flotation techniques. In comparison to necropsy, fecal analysis is less time intensive, less expensive, and carries less risk of exposure to biological hazards if eggs and cysts contained in stool samples are inactivated at -80˚C prior to processing. However, there are several advantages to necropsy such as the relative ease in detecting and recovering taeniid cestodes (identified morphologically, if specimens are fresh and of good quality, or by molecular methods, which are especiallyDraft useful when sample quality and handling are not optimal for parasite morphology) and distinguishing among mixed infections of parasite species shedding morphologically indistinguishable eggs. Taeniid cestodes ( Taenia and Echinococcus spp.) cannot be differentiated on the basis of egg morphology, eggs may not float consistently in routine fecal tests, and extraction of DNA from eggs can be challenging. As well, some helminths shed eggs sporadically or produce eggs that crack during freezing (such as hookworm eggs of Ancylostoma and Uncinaria spp.), and so infection intensity for these species is more accurately estimated by intestinal necropsy (Liccioli et al. 2012; Schurer et al. 2014 a).

This study confirms the sympatric distribution of E. canadensis G8 and G10 genotypes in western

Canada, and broadens the known distribution of wolves as definitive hosts for E. multilocularis to

include BC (Wang et al. 1989; Rausch 1995; Craig and Craig 2005; Thompson et al. 2006; Schurer et al.

2014 b). We demonstrate that G8 and G10 not only co-exist within geographic locales, but that co-

infection of wolves with multiple taeniid genera, species, and genotypes occurs. Mixed infections of

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Echinococcus species (6%), E. canadensis genotypes (5%), Taenia species (1%), and of Echinococcus and

Taenia (20%) were all observed; however, we did not conduct PCR on all Taenia , Diphyllobothrium , or

Echinococcus adults, and such estimates undervalue the true prevalence of mixed infection. This

supports laboratory based studies demonstrating that adaptive immunity against recurrent taeniid

infection is unlikely to occur in canids (D. Jenkins and Rickard 1985). Echinococcus was present in more

wolves than any other parasite, highlighting the importance of wolves in maintaining the sylvatic

predator-prey life cycle. Domestic dogs, particularly those with access to carcasses and not regularly

dewormed, can act as bridging hosts between wildlife and people, serving as a source of Echinococcus

infection for both. Dogs do not experience adverse health effects from infection by adult Echinococcus

cestodes; however, ingestion of eggs of E. multilocularis shed in feces of wild canids can result in infection with the larval form (alveolar echinococcDraftosis), which has a poor clinical prognosis (Peregrine et al. 2012; Skelding et al. 2014). We report an Echinococcus infection prevalence (37%) that is higher than

previous reports from MB, BC and NT (Choquette et al. 1973; Stronen et al. 2011; Bryan et al. 2012),

which might be explained by differences in sampling techniques or changing environmental, ecological

or climatic conditions. The high prevalence of E. canadensis relative to E. multilocularis likely

demonstrates the preference among wolves for ungulate prey relative to rodents, but may also be

explained by the smaller geographic distribution of E. multilocularis in North America, defined by two

focal regions (i.e., the northern tundra zone and the north central region), although this may be

changing with detection of the parasite in new regions such as BC (Eckert et al. 2001; Mech and Boitani

2004; Schurer et al. 2013).

Nine Taenia species are known to infect wolves in Canada, and of these we observed three: T. krabbei,

T. hydatigena, and T. multiceps (Craig and Craig 2005) . Taenia krabbei was most prevalent but occurred

only in BC and the NT, highlighting the importance of cervids such as moose, elk, and caribou as primary

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prey to those wolves (Jones and Pybus 2001; Craig and Craig 2005). In contrast, T. hydatigena, which is

transmitted by bovid, cervid or caprid intermediate hosts, was present in BC, SK, and 2 ecozones of the

NT, but at lower prevalence than T. krabbei . Caribou are possible intermediate hosts for Taenia

multiceps within the sampling regions. Suggestions that T. krabbei is most common in tundra wolves,

while T. hydatigena is most prevalent in timber wolves, conflict with dietary studies that suggest tundra wolves consume a greater variety of prey, including rodents, than timber wolves (Pimlott et al. 1969;

Kuyt 1972; Choquette et al. 1973; Craig and Craig 2005). The presence of E. multilocularis and T. hydatigena in both tundra and boreal regions suggests that wolves in both areas hunt opportunistically, utilizing ungulates as well as a wide variety of smaller prey. Piscivores are infected by Diphyllobothrium when they consume infected marine or freshwater fish (e.g., pike [ Esox lucius L., 1758], yellow perch [Perca flavescens (Mitchill, 1814)], and walleyeDraft [ Sander vitreus (Mitchill, 1818)]; Scholz et al. 2009). The presence of D. latum in NT was expected, as wolves may have direct and/or indirect access to freshwater and anadromous fish, but other surveys have also reported D. dendriticum (reviewed in E.

Jenkins et al. 2013). We did not detect Diphyllobothrium in BC, where D. nihonkaiense occurs in coastal wolves, even though three wolves in this survey were captured on Vancouver Island. This could be due to seasonal switching of primary prey (Darimont and Reimchen 2002), but is more likely explained by the fact that most BC samples were from non-coastal areas (Figure 1).

We observed four nematode species: T. leonina (most prevalent), U. stenocephala, Physaloptera spp. , and Trichuris spp. We did not observe Spirocerca, , Eucoleus aerophilus , Filaroides osleri or Ancylostoma caninum, likely because they are relatively rare in Canada and because our isolation method was not optimized to detect all nematode species (Choquette et al. 1973; Craig and

Craig 2005; Bryan et al. 2012). , a nematode common to young canids and transmitted through direct, peri-natal, and paratenic life cycles, was also not observed despite previous reports of

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infected wolves in BC and MB (Bryan et al. 2012; Stronen et al. 2011). This suggests that wolves are less

suitable hosts than domestic dogs, which have been observed with moderate levels of in

close proximity to the sampling areas (Salb et al. 2008; Himsworth et al. 2010; Schurer et al. 2012). It is

also possible that Toxascaris life stages are better adapted to survival in cold arctic climates than

Toxocara , for which a northern distributional limit exists (E. Jenkins et al. 2013) .

The finding of Giardia Assemblage C was expected, as this assemblage is canid-specific and was

previously reported in wild ( Canis latrans (Say, 1823)) in Alberta and SK (Thompson et al. 2009).

Within western Canada, Assemblage B has been reported in BC wolves (Bryan et al. 2012); however,

between the two zoonotic assemblages (A and B), Assemblage A has been reported more often, with hosts including dogs (Salb et al. 2008; HimsworthDraft et al. 2010; Schurer et al. 2012), wolves (Bryan et al. 2012), coyotes (Thompson et al. 2009), muskoxen (Kutz et al. 2008) and cattle ( Bos taurus L., 1758;

Appelbee et al. 2003). The widespread geographic distribution and host range of zoonotic Giardia

assemblages suggests that pathogen sharing among people, wildlife and domestic animals occurs even

in remote areas; however, the direction of pathogen transmission remains unclear.

One limitation to our opportunistic sampling method was that geographic areas were not equally

represented. For MB in particular, we do not report a complete picture of parasite richness; those

parasites that were present at low prevalence may have remained unobserved. This study maximized

parasite detection by conducting fecal analysis and adult helminth parasite recovery from intestinal

necropsy; however, intensity estimates based on fecal flotation for some helminths (e.g., Uncinaria ),

may have appeared artificially low due to morphological changes in eggs that prevent detection or

identification after freezing (Schurer et al. 2014 a). Other endoparasites (ascarids, taeniids and

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Sarcocystis ) produce ova/cysts that tolerate extreme temperature conditions well, and infection can be accurately assessed by FEC (Schurer et al. 2014 a).

This study highlights the utility of combining fecal analysis and necropsy to estimate overall gastrointestinal , and the need for new, non-invasive techniques to detect parasites common to wolves. We observed a broad geographic distribution of certain zoonotic parasites ( Echinococcus,

Cryptosporidium, Giardia ), as well as localized differences in parasite diversity and prevalence. While prevalence of these helminth zoonoses appears to be decreasing in people across the Canadian North, there remain a disproportionate number of cases in northern Canadians relative to the rest of Canada, and this may exacerbate existing health disparities (Jenkins et al. 2013). There is increasing concern that these protozoans are already a significant Draftcontributor to human enteric illness in the Canadian North and that outbreaks may increase in frequency and magnitude in a rapidly changing northern climate.

For these reasons, there remains a need to determine baselines for parasite diversity in free-ranging wildlife, and to further characterize the occurrence and direction of pathogen transmission among wildlife, domestic animals, and people in northern environments, in order to better assess and manage current and emerging risks to animal and public health.

Acknowledgements:

We gratefully acknowledge Helen Schwantje, Karl Cox, Todd Shury, Chelsea Himsworth, Christine

Menno, Marsha Branigan, and the hunters and trappers who provided animal tissues. We also thank

Rajnish Sharma for technical support. Student training at the Zoonotic Parasite Research Unit was generously funded by the Public Health and the Agricultural Rural Ecosystem (PHARE), the Western

College of Veterinary Medicine Interprovincial Undergraduate Summer Student Fellowship, and the

Integrated Training Program in Infectious Diseases, Food Safety, and Public Policy (ITraP). Laboratory

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funding was provided by the Natural Sciences and Engineering Research Council of Canada and the

Canadian Foundation for Innovation Leaders Opportunity Fund.

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Table 1: Parasite prevalence, not including Echinococcus , in wolves (Canis lupus L., 1758) as observed by

intestinal necropsy and compared by province/territory.

Prevalence N (%) 95% CI P-value 1

Province/Territory 2 NT BC SK/MB Total

Sample size 143 28 20 191

Toxascaris 25 (17) 6 (21) 3 (15) 34 (18) 13 -23 0.81

Diphyllobothrium 3 5 (3) 0 (0) 0 (0) 5 (3) 1-5 0.77

D. latum 4 4 (3) 0 (0) 0 (0) 4 (2) 0-4

Unidentified 1 (1) 0 (0) 0 (0) 1 (1) 0-1

Taenia 3 38 (27) 23 (28) 8 (40) 69 (36) 29 -43 <0.001 T. krabbei 4 26 (18) Draft16 (57) 0 (0) 42 (22) 16-28 T. hydatigena 4 6 (4) 6 (21) 1 (5) 13 (7) 4-11

T. multiceps 4 3 (2) 0 (0) 0 (0) 3 (2) 0-4

Unidentified 4 (3) 6 (21) 7 (35) 17 (9) N/A

Alaria 8 (6) 0 (0) 3 (15) 11 (6) 3-9 0.06

Uncinaria 1 (1) 0 (0) 0 (0) 1 (1) 0-1 1.0

Parasite Prevalence 5 75 (52) 28 (100) 17 (85) 120 (63) 56 -70 <0.001

1Fisher’s Exact Test; 2NT-Northwest Territories, BC-British Columbia, SK-Saskatchewan, MB-Manitoba;

3Based on morphological identification; 4Based on molecular identification; 5Percent wolves infected

with one or more adult helminth species observed during necropsy

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Table 2: Parasite prevalence in wolves (Canis lupus L., 1758) as observed by fecal flotation and compared by province/territory.

Prevalence N (%) 95% CI P-value 1 Median

Intensity 2

(min-max)

Province/Territory 3 NT BC SK/MB Total

Sample size 118 13 20 151

Toxascaris leonina 30 (25) 3 (23) 1 (5) 34 (23) 16 -30 0.13 70 (3 -2425)

Diphyllobothrium 13 (11) 0 (0) 0 (0) 13 (9) 4-14 0.22 118 (5 -4010)

Taeniid 20 (17) 10 (77) 12 (60) 42 (28) 21 -35 <0.001 68 (3 -9593) Alaria 25 (21) 0 (0) Draft5 (25) 30 (20) 14 -26 0.12 10 (3 -1535) Uncinaria 1 (1) 0 (0) 0 (0) 1 (1) 0-3 1.0 N/A

stenocephala

Sarcocystis 21 (18) 0 (0) 8 (40) 29 (19) 13 -25 0.01 2067 (147 -

7600)

Physaloptera 2 (2) 0 (0) 0 (0) 2 (1) 0-3 0.99 N/A

Trichuris 2 (2) 0 (0) 0 (0) 2 (1) 0-3 0.99 N/A

Giardia 34 (29) 1 (8) 3 (15) 38 (25) 18 -32 0.15 800 (2 -18476)

Cryptosporidium 14 (19) 4 N/A 6 (30) 20 (13) 8-18 0.36 367 (67 -1467)

Parasite 87 (74) 13 (100) 17 (85) 117 (77) 70 -84 0.06

Prevalence 5

1Fisher’s Exact Test; 2Eggs or cysts per gram feces; 3NT-Northwest Territories, MB-Manitoba, BC-British

Columbia, SK-Saskatchewan; 4Subset of NT samples analyzed for Cryptosporidium (N=93); 5Wolves infected with at least one gastrointestinal parasite observed by fecal analysis

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Table 3: Comparison of parasite prevalence in Northwest Territory wolves (Canis lupus L., 1758; N=143)

by ecozone.

Ecozone Taiga Plains & Tundra Taiga Tundra P-value 1

Taiga Cordillera Plains Shield Cordillera

Fecal analysis or necropsy – N (%)

Sample Size 72 35 19 17

Toxascaris 19 (26) 11 (31) 8 (42) 5 (29) 0.6

Diphyllobothrium 11 (15) 0 (0) 3 (16) 1 (6) 0.04

Taenia 19 (26) 10 (29) 2 (11) 7 (41) 0.21

T. krabbei 11 (15) 7 (20) 2 (11) 6 (35) 0.23 T. hydatigena 5 (7) Draft1 (3) 0 (0) 0 (0) 0.59 T. multiceps 0 (0) 3 (9) 0 (0) 0 (0) 0.04

Echinococcus 23 (32) 4 (11) 3 (16) 2 (12) 0.06

E. canadensis G8 5 (7) 0 (0) 0 (0) 0 (0) 0.31

E. canadensis G10 12 (23) 4 (11) 2 (11) 2 (12) 0.91

E. multilocularis 6 (8) 0 (0) 0 (0) 0 (0) 0.19

Alaria 18 (25) 0 (0) 10 (53) 0 (0) <0.01

Fecal Analysis only – N (%)

Sample Size 65 23 19 11

Sarcocystis 16 (25) 1 (4) 4 (21) 0 (0) 0.05

Giardia 20 (31) 2 (9) 7 (37) 5 (45) 0.06

Cryptosporidium 11 (20) 2 N/A 3 (16) N/A 0.62

Parasite Prevalence 3 60 (83) 22 (63) 17 (89) 13 (76) 0.047

1Fisher’s Exact Test; 2N=54; 3Wolves infected with at least one gastrointestinal parasite

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Figure 1: Wolf sampling sites (circle - individual wolf; square – group of wolves)

Figure 2: Parasite richness in wolves ( Canis lupus L., 1758; N=193) from the Northwest Territories, British

Columbia, Saskatchewan, and Manitoba as determined by intestinal necropsy and fecal analysis (Bars -

95% confidence intervals).

Draft

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NT Ecozones

Boreal Cordillera Northern Arctic Southern Arctic: Tundra Plains Sachs ^_Harbour Taiga Cordillera Tuktoyaktuk Taiga Plains ^_ ^_ Taiga Shield Inuvik ^_Ulukhaktok Tundra Cordillera Northwest northern limit of trees

Norman ^_ Wells Yukon Nunavut DraftTerritories

Fort ^_ Yellowknife Simpson ^_

Hay River ^_ British Fort Smith^_ o Fort 60 Smithers St.John

Alberta Key Prince Lake George Saskatchewan Manitoba Port Columbia Hardy Prince Albert Big River Pemberton National Tofino Park

Mill Bay Lamb Creek Riding Mountain National 0 125 250 500 Park km

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40

35

30

25

20

% wolves % 15

10

5

0 0 1 2 3 4 5 6 7 # parasite genera

Draft

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