3830  |  Molecular Ecology. 2020;29:3830–3840.wileyonlinelibrary.com/journal/mec Received: 7 May 2020  |  Revised: 30 July 2020  |  Accepted: 6 August 2020 DOI: 10.1111/mec.15602 O R I G I N A L A R T I C L E Large-scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility Maria Immaculata Arifin1 | Antanas Staskevicius2 | Su Yeon Shim1 | Yuan-Hung Huang1 | Heather Fenton3 | Philip D. McLoughlin4 | Gordon Mitchell2 | Catherine I Cullingham5  | Sabine Gilch1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd 1Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada 2National and OIE Reference Laboratory for Scrapie and CWD, Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, ON, Canada 3Ross University School of Veterinary Medicine, Basseterre, St. Kitts 4Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada 5Department of Biology, Carleton University, Ottawa, ON, Canada Correspondence Sabine Gilch, Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada. Email: sgilch@ucalgary.ca Catherine I Cullingham, Department of Biology, Carleton University, Ottawa, ON, Canada. Email: catherinecullingham@cunet.carleton. ca Funding information Alberta Prion Research Institute; Canada Research Chairs; Genome Canada; Genome Alberta; Natural Sciences and Engineering Research Council of Canada Abstract Polymorphisms within the prion protein gene (Prnp) are an intrinsic factor that can modulate chronic wasting disease (CWD) pathogenesis in cervids. Although wild European reindeer (Rangifer tarandus tarandus) were infected with CWD, as yet there have been no reports of the disease in North American caribou (R. tarandus spp.). Previous Prnp genotyping studies on approximately 200 caribou revealed single nu- cleotide polymorphisms (SNPs) at codons 2 (V/M), 129 (G/S), 138 (S/N), 146 (N/n) and 169 (V/M). The impact of these polymorphisms on CWD transmission is mostly unknown, except for codon 138. Reindeer carrying at least one allele encoding for asparagine (138NN or 138SN) are less susceptible to clinical CWD upon infection by natural routes, with the majority of prions limited to extraneural tissues. We se- quenced the Prnp coding region of two caribou subspecies (n = 986) from British Columbia, Saskatchewan, Yukon, Nunavut and the Northwest Territories, to iden- tify SNPs and their frequencies. Genotype frequencies at codon 138 differed sig- nificantly between barren-ground (R. t. groenlandicus) and woodland (R. t. caribou) caribou when we excluded the Chinchaga herd (p < .05). We also found new vari- ants at codons 153 (Y/F) and 242 (P/L). Our findings show that the 138N allele is rare among caribou in areas with higher risk of contact with CWD-infected species. As both subspecies are classified as Threatened and play significant roles in North American Indigenous culture, history, food security and the economy, determining frequencies of Prnp genotypes associated with susceptibility to CWD is important for future wildlife management measures. K E Y W O R D S caribou, caribou conservation, chronic wasting disease, genotyping, prion protein www.wileyonlinelibrary.com/journal/mec mailto: https://orcid.org/0000-0002-6715-0674 mailto: https://orcid.org/0000-0001-5923-3464 http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:sgilch@ucalgary.ca mailto:catherinecullingham@cunet.carleton.ca mailto:catherinecullingham@cunet.carleton.ca http://crossmark.crossref.org/dialog/?doi=10.1111%2Fmec.15602&domain=pdf&date_stamp=2020-09-10      |  3831ARIFIN et Al. 1  | INTRODUC TION Chronic wasting disease (CWD) was first discovered in 1967 in cap- tive mule deer in Colorado (Williams & Young, 1980). Since then, the disease has spread to wild and farmed cervids in 26 US states and three Canadian provinces (https://www.usgs.gov/media/ image s/distr ibuti on-chron ic-wasti ng-disea se-north -ameri ca-0; Hannaoui, Schatzl, & Gilch, 2017). Recently, CWD has also been reported in wild cervids in Europe (Benestad, Mitchell, Simmons, Ytrehus, & Vikøren, 2016). CWD is a prion disease, also known as a transmis- sible spongiform encephalopathy (TSE), of cervid species. Prion diseases are caused by conversion of the endogenous cellular prion protein (PrPC) into its infectious isoform, PrPSc, which is the major component of prions (Prusiner, 1998). This conformational transition is catalysed by PrPSc coming into contact with PrPC (Prusiner, 1998), and PrPC is most abundantly expressed in the central nervous sys- tem (CNS) (Linden et al., 2008). The CNS is a major site of PrPSc conversion and accumulation, leading to neurodegeneration and ultimately death (Prusiner, 1998). In naturally occurring CWD, pri- ons are acquired through peripheral routes, such as oral uptake from environmental reservoirs (Gilch et al., 2011). Prions are highly re- sistant to many major forms of inactivation (Giles, Woerman, Berry, & Prusiner, 2017). It is exceptionally challenging and not possible to contain and eradicate CWD to date, as prions are shed in ex- crement and bodily fluids of infected free-ranging cervids (Cheng, Hannaoui, et al., 2017; Haley et al., 2011; Henderson et al., 2015; Safar et al., 2008). This results in contamination of the environ- ment, particularly soil, with prions that remain infectious for years (Bartelt-Hunt & Bartz, 2013; Kuznetsova, McKenzie, Cullingham, & Aiken, 2020; Pritzkow et al., 2018; Somerville et al., 2019). So far, wild and farmed cervid species naturally infected with CWD include white-tailed deer (Odocoileus virginianus), mule deer (O. hemionus), elk (Cervus canadensis), red deer (C. elaphus) and moose (Alces alces sp.) in North America (Baeten, Powers, Jewell, Spraker, & Miller, 2007; Hannaoui, Amidian, et al., 2017; Kurt & Sigurdson, 2016; Pirisinu et al., 2018; Williams & Young, 1980, 1982, 1993), as well as rein- deer (Rangifer tarandus tarandus), red deer (C. elaphus) and moose (A. a. alces) in Europe (Benestad et al., 2016; Pirisinu et al., 2018; Vikøren et al., 2019). There are currently four caribou subspecies residing in North America: the Peary (R. t. pearyi), Grant's (R. t. granti), barren-ground (R. t. groenlandicus), and woodland (R. t. caribou) caribou (National Museum of Canada, 1962). Peary caribou populations are located in the Canadian Arctic, on islands north of the Northwest Territories (NT) and Nunavut (Mallory & Boyce, 2019). Barren-ground caribou herds migrate throughout vast areas of NT, Nunavut and Yukon, with historical ranges in Northern Alberta and Saskatchewan (COSEWIC, 2016). Grant's caribou, consisting mainly of the Porcupine herd, are found in Yukon and Alaska, and are also often classified as barren-ground caribou (Festa-Bianchet, Ray, Boutin, Côté, & Gunn, 2011). Woodland caribou are divided into two major eco- types comprising boreal and mountain populations (Festa-Bianchet et al., 2011). Boreal woodland caribou populations span from British Columbia (BC) to Québec, while mountain woodland caribou mainly reside in BC, Yukon and NT (Festa-Bianchet et al., 2011). Many caribou populations in Canada are listed as either Threatened or Endangered (COSEWIC, 2016; Thomas & Gray, 2002). Recent ob- servations of cervid populations in Saskatchewan show an overlap in habitat between white-tailed deer, including CWD-infected deer, and boreal caribou herds (Hebblewhite & Fortin, 2017; Hervieux et al., 2013; McLoughlin et al., 2019; Saskatchewan Ministry of Environment, 2013). Because CWD is becoming increasingly prev- alent in free-ranging cervids in Saskatchewan (CWD map of posi- tive tests, 2020; Kahn, Dubé, Bates, & Balachandran, 2004), this brings into light the possibility of CWD invading the R. tarandus populations in this province. All cervid species tested so far have been shown to be susceptible to CWD infection, albeit some only in experimental settings (Balachandran et al., 2010; Hamir et al., 2011; Mitchell et al., 2012; Moore et al., 2016; Nalls et al., 2013; Sigurdson et al., 1999). Furthermore, wild reindeer in Europe acquired CWD (Benestad et al., 2016). While as yet wild caribou in Canada are re- portedly free from CWD, transmission is likely to happen naturally under current conditions. The prion protein gene (Prnp) sequence is largely identical among cervid species, with only a small number of single nucleotide poly- morphisms (SNPs) present in the gene pool (Robinson, Samuel, O’Rourke, & Johnson, 2012). Nevertheless, Prnp polymorphisms can modulate susceptibility to CWD (Angers et al., 2014; Green et al., 2008; Hannaoui, Amidian, et al., 2017; Hannaoui, Schatzl, et al., 2017; Johnson et al., 2011). Previous studies reported a Prnp polymorphism resulting in a substitution from serine to asparag- ine at codon 138 (S138N) unique to fallow deer (Dama dama) and reindeer/caribou (Hamir et al., 2011; Mitchell et al., 2012; Moore et al., 2016; Rhyan et al., 2011; Robinson et al., 2019). This poly- morphism has been associated with lower susceptibility to CWD in both species under experimental conditions, depending on the route of prion infection (Mitchell et al., 2012; Moore et al., 2016; Rhyan et al., 2011). Fallow deer, naturally homozygous for asparagine at codon 138 (138NN), did not develop clinical disease nor accumulate detectable amounts of PrPSc in their CNS or lymphatic organs, after 6 years of exposure to CWD-infected mule deer (Rhyan et al., 2011). Nevertheless, fallow deer were susceptible to intracerebral CWD in- fection, where PrPSc and spongiform degeneration was detected in the CNS after a prolonged incubation period of 51 months post-in- fection, whereas deer usually succumb to the disease between 12–34 months (Hamir et al., 2011; Kahn et al., 2004). In two other studies, no PrPSc was found in the CNS of reindeer homozygous or heterozygous for asparagine at codon 138 (138SN or 138NN) in- fected peripherally with prions, with the exception of one animal (Mitchell et al., 2012; Moore et al., 2016). A prolonged CWD incuba- tion period with the absence of typical clinical CWD symptoms was also seen in a 138SN reindeer infected orally (Mitchell et al., 2012). The S138N polymorphism is present in caribou herds in Alaska and Alberta, but not reported in wild reindeer in Norway so far (Cheng, Musiani, Cavedon, & Gilch, 2017; Güere et al., 2020; Happ, Huson, Beckmen, & Kennedy, 2007). Other Prnp polymorphisms in the 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://www.usgs.gov/media/images/distribution-chronic-wasting-disease-north-america-0 https://www.usgs.gov/media/images/distribution-chronic-wasting-disease-north-america-0 3832  |     ARIFIN et Al. Rangifer species include codons V2M, G129S, N146n (synonymous substitution), V169M, N176D, S225Y, and a 24 base pair deletion in the octapeptide repeat region (Cheng, et al., 2017; Robinson et al., 2012; Wik et al., 2012). Recent studies reported that the prevalence of the 225S allele and a combination of the 2M-129S- 169M variant was higher in CWD-positive wild reindeer from the Nordfjella region in Norway (Güere et al., 2020). Here, we report the prevalence and distribution of R. tarandus prion protein polymorphisms obtained from 986 caribou samples from BC, Yukon, NT, Nunavut and Saskatchewan. We identified two novel prion protein polymorphisms, one in barren-ground (Y153F) and one in woodland (P242L) caribou. The frequencies of previously reported polymorphisms found in this study were not significantly different across herds and subspecies. At the herd level, the 138N allele was exceptionally prevalent in the Chinchaga boreal wood- land caribou population in BC, in agreement with previous studies in Alberta (Cheng, Musiani, et al., 2017). When we excluded this woodland herd from the analyses, we found that codon 138 was significantly different between the two subspecies, with 138NN and 138SN genotypes being more frequent in barren-ground than woodland caribou. Notably, spatial differences were found among different herds, with the lowest prevalence of the 138N allele in woodland caribou of the mountain ecotype in BC, and the boreal woodland caribou herds in SK. 2  | MATERIAL S AND METHODS 2.1 | Samples We obtained a total of 986 frozen caribou blood samples or tissue from herds in BC (n = 397), NT/Nunavut (n = 462), Saskatchewan (n = 101) and Yukon (n = 26). These samples were collected between 2004–2018 and represent woodland and barren-ground caribou populations in western Canada (Figure 1). The NT/Nunavut samples consist of the barren-ground herds Bathurst (n = 87), Beverly and Ahiak (n = 107), Bluenose East (n = 90), Bluenose West (n = 50), Cape Bathurst (n = 27), Tuktoyaktuk Peninsula (n = 15) and woodland boreal herds Hay and Cameron (n = 54) and those from the North Slave region (n = 19). Yukon samples comprise of the Porcupine F I G U R E 1   Left panel shows the distribution of caribou samples used in this study with colours representing the herds, and symbol shapes representing the subspecies/ecotype. Black symbols represent herds without individual coordinates. Top panel indicates significant pairwise genotypic comparisons among herds following p-value correction for multiple tests. Right panel is an interpolation of the 138N allele frequency across herds, individual coordinates were averaged for centroids. Herds are labelled using the following abbreviations: AT, Atlin; B-A, Beverly/Ahiak; BE, Bluenose east; BH, Bathurst; BV, Beverly; BW, Bluenose west; CA, Calendar; CB, Cape Bathurst; CH, Chinchaga; F-G, Frog/Gataga; FI, Finlay; GR, Graham; H-C, Hay/Cameron; HS, Hart south; I-I, Itcha-Ilgachuz; MX, Maxhamish; NS, North Slave; PC, Porcupine; SK, SK1; S-S, Snake-Sahtaneh; TK, Tuktoyaktuk 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense      |  3833ARIFIN et Al. herd (R. t. granti) (n = 19), which will be included as barren-ground from here onwards, and woodland caribou of the mountain ecotype including Horseranch (n = 2), Little Rancheria (n = 2) and Carcross (n = 2) herds. BC woodland caribou herds included in this study were Calendar (n = 35), Chinchaga (n = 59), Parker (n = 3), Prophet (n = 9), Maxhamish (n = 37), Snake-Sahtaneh (n = 70), and Fort Nelson (n = 4) of the boreal ecotype and Muskwa (n = 6), Frog (n = 6), Itcha- Ilgachuz (n = 59), Wolverine (n = 10), Atlin (n = 26), Finlay (n = 8), Gataga (n = 8), Graham (n = 11), Pink Mountain (n = 17), and Hart South (n = 29) of the mountain ecotype. Out of the 986 samples, 14 animals did not have herd information. 2.2 | DNA extraction, polymerase chain reaction (PCR) and sequencing We extracted DNA from frozen clotted blood using the MagMAX-96 DNA Multi-Sample Kit (ThermoFisher Scientific, cat #4413022) in a 96-well format following the manufacturer's protocol. The Prnp open reading frame in exon 3 was amplified using a protocol from Cheng, Musiani, et al. (2017). PCR reaction conditions were as fol- lows: 10 µl of genomic DNA (equivalent to 100 ng of genomic DNA), 2 µl of 10 µM forward primer (5′- CCT AGT TCT CTT TGT GGC CAT GTG -3′ or 5′- GGG CAT ATG ATG CTG ACA CCC TCT TT -3′), 2 µl of 10 µM reverse primer (5′- TGA GGA AAG AGA TGA GGA GGA TCA C 3′ or 5′- GAG AAA AAT GAG GAA AGA GAT GAG GAG G -3′), 10 µl of 10x Pfu buffer (Agilent), 1 µl of 10 mM dNTP (Invitrogen), 0.5 µl of Pfu polymerase (Agilent) and nuclease-free water in a total reaction volume of 50 µl. Conditions for the PCR were 4 min of initial denaturation at 94°C, followed by 39 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. The runs were performed in 96-well plates using the GeneAmp PCR System 9700 (Applied Biosystems) or the T100 (Bio-Rad). No-template controls were randomly included in every run to ensure no cross-contami- nation of samples. Amplified samples were run on a 2% agarose gel at 95V for 1 hr. Bands were visualized under UV light, excised using a clean blade, and amplified DNA was retrieved and purified using a commercial gel extraction kit (Qiagen, cat #28706). Sequencing of products were performed at Eton Bioscience (San Diego) with the first primer set used in the PCR reaction above. 2.3 | TOPO cloning Gel-extracted PCR products from samples with novel SNPs de- tected in the chromatograms were cloned using the TOPO Zero Blunt cloning kit (ThermoFisher Scientific, cat #450245) and trans- formed into TOP10 cells (ThermoFisher Scientific, cat #C404010) according to the manufacturer's protocol. Ten colonies from each sample were screened for the Prnp insert using colony PCR with Taq DNA Polymerase (GenScript, cat #E00043) and the first primer set mentioned above. Plasmid DNA was extracted from positive colonies using a commercial kit (Omega Bio-tek, cat #D6945-2) and sent for sequencing at the University of Calgary core sequencing facility or Eton Bioscience (San Diego) using the common M13F and M13R primers present in the vector sequence. 2.4 | SNPs and statistical analyses To find SNPs, both known and novel, caribou Prnp sequences were analysed using the sangeranalyseR package (Aneichyk et al., 2018; https://github.com/robla nf/sange ranal yseR) in R Studio and Geneious v.10.2.6 (Kearse et al., 2012; http://www.genei ous.com). Statistical significance of genotype and allele frequencies between herds and subspecies were determined using using an exact G-test, chi-squared or Fisher's exact test in Genepop and/or the Genetics package (https://CRAN.R-proje ct.org/packa ge=genetics) in R Studio. p-values were adjusted using the fdr correction with the p. adjust function in R Studio. Prnp genotypes for each SNP were also analysed for deviations from Hardy-Weinberg Equilibrium, and for linkage disequilibrium using the Genetics package. To visualize the spatial variation in the frequency of 138N we generated a heat- map using the interpolation tool within ArcGIS Pro (ESRI Software). Centroid locations were estimated for herds without geographic co- ordinates, and we did not include herds with fewer than six individu- als. Frog and Gataga herds were merged as they had small sample sizes, were spatially adjacent, and their genotype frequencies did not differ. 3  | RESULTS Seven Prnp polymorphisms and a deletion from nucleotide (nt) 249 to 272 have been reported in caribou/reindeer (R. tarandus spp.). The polymorphisms are V→M at codon 2, G→S at codon 129, S→N at codon 138, N→n (synonymous substitution) at codon 146, V→M at codon 169, N→D at codon 176 and S→Y at codon 225 and (41,48,49,51). The 24 base pair (bp) deletion, the 176 N→D and the 225 S→Y polymorphisms have only been reported in European reindeer. All sequences from our North American caribou samples were homozygous for serine at codon 225 and none had the 24 bp deletion. The polymorphism at codon 2 is located in the signal pep- tide sequence of PrPC, which is cotranslationally cleaved off, thus we excluded this codon from our analyses. Out of 986 samples, 708 successful reads were obtained for Prnp codon 129 (71.81%), 726 for codon 138 (73.63%), 697 for codon 146 (70.69%), 724 for codon 169 (73.43%) and 708 for codon 225 (71.81%). Only individuals with genotype data at codons 129, 138, 146 and/or 169 and herd data, and only herds with greater than five individuals, were included in the herd level analyses (n = 756). Statistical tests show that all alleles in the Prnp codons analysed here were in Hardy-Weinberg equilib- rium (chi-squared and Fisher's exact tests), suggesting that none of these Prnp polymorphisms were under selection pressure. Pairwise linkage disequilibrium analyses also showed us that the majority of 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://github.com/roblanf/sangeranalyseR http://www.geneious.com https://CRAN.R-project.org/package=genetics 3834  |     ARIFIN et Al. SNP haplotype pairs, with the exception of codon 146, were geneti- cally linked (D′ = 0.86–0.99; p < .05). This is expected as these SNPs are located physically close to each other within the Prnp open read- ing frame (ORF) on exon 3. We found two barren-ground caribou that were heterozygous for N and D at codon 176 (Figure S1). European reindeer are known to carry the D variant at codon 176, but this is the first report of its presence in caribou in North America. Furthermore, we found two novel Prnp SNPs that have not been previously reported in any species; a Y→F substitution at codon 153 in three NT barren-ground caribou (all heterozygous Y/F) and a P→L substitution at codon 242 in 12 BC woodland caribou (nine heterozygous P/L and three ho- mozygous L/L). We performed TOPO cloning on all samples with possible SNPs to exclude any sequencing artifacts. We assessed the genotypic differentiation between all caribou herds (n = 21 herds, with individuals >5 per herd, Frog-Gataga com- bined) at codons 129, 138, 146 and 169 in Genepop. Only codon 138 had significantly different pairwise comparisons following p- value correction; the majority of the significant comparisons were between Chinchaga and the other herds (Figure 1 and Table 1). We also performed Grubbs’ test for outliers, and based on the N allele frequency at codon 138, the Chinchaga herd was determined an outlier amongst all caribou herds (Z = 2.27, p < .05). Mean allele fre- quency of 138N was 0.32 across all herds, while the frequency in the Chinchaga herd was 0.64. There was no significant difference between the barren-ground and woodland caribou subspecies at all codons (Table S1), but when we omitted the Chinchaga herd, the genotype frequencies at codon 138 between barren-ground and woodland populations differed significantly (p < .01) (Figure 2). In ad- dition, codon 138 also significantly differed between barren-ground and woodland caribou of the mountain ecotype (p < .05) (Figure 2). These differences are highlighted in the heatmap for the 138N al- lele (Figure 1). We also show that individual animals can carry more than one polymorphic allele (Table 2). We report that the Prnp codon associated with reduced CWD susceptibility is found in higher fre- quencies in the Chinchaga woodland population, and higher in mi- gratory barren-ground than woodland caribou populations when this herd is excluded from analyses. 4  | DISCUSSION Currently, there have been no reports of CWD in wild caribou in North America. However, CWD has been identified in their wild relatives in Europe (reindeer) (Benestad et al., 2016). They are mem- bers of the same species, R. tarandus; hence, there is concern that CWD will expand, or has already transmitted, to caribou in the wild. The S138N Prnp polymorphism has been described in caribou and Herd Subspecies Ecotype Abbrev. Sample size Codon 138 N-freq S-freq Atlin Woodland Mountain AT 9 0.222 0.778 Bathurst Barren-ground NA BH 79 0.342 0.658 Beverly Barren-ground NA BV 18 0.417 0.583 Beverly/Ahiak Barren-ground NA B-A 77 0.435 0.565 Bluenose E Barren-ground NA BE 83 0.343 0.657 Bluenose W Barren-ground NA BW 46 0.348 0.652 Calendar Woodland Boreal CA 34 0.294 0.706 Cape Bathurst Barren-ground NA CB 26 0.346 0.654 Chinchaga Woodland Boreal CH 51 0.637a  0.363 Finlay Woodland Mountain FI 6 0.083 0.917 Frog/Gataga Woodland Mountain F-G 11 0.318 0.682 Graham Woodland Mountain GR 7 0.214 0.786 Hart South Woodland Mountain HS 17 0.206 0.794 Hay/Cameron Woodland Boreal H-C 45 0.256 0.744 Itcha-Ilgachuz Woodland Mountain I-I 30 0.200 0.800 Maxhamish Woodland Boreal MX 12 0.333 0.667 North Slave Woodland Boreal NS 14 0.321 0.679 Porcupine Barren-ground NA PC 19 0.316 0.684 SK woodland Woodland Boreal SK 82 0.250 0.750 Snake- Sahtaneh Woodland Boreal S-S 33 0.439 0.561 Tuktoyaktuk Barren-ground NA TK 15 0.400 0.600 aSignificantly different from most other herds. TA B L E 1   Herd allele frequencies at Prnp codon 138 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense      |  3835ARIFIN et Al. fallow deer (Cheng, Musiani, et al., 2017; Hamir et al., 2011; Happ et al., 2007; Wik et al., 2012) and the pseudogene of mule deer and white-tailed deer (Brayton, O’Rourke, Lyda, Miller, & Knowles, 2004; O’Rourke, 2004). Here, we report the prevalence of the S138N polymorphism in Prnp sequences of 726 caribou samples from two provinces (BC, Saskatchewan) and three territories (Yukon, NT, and Nunvaut) in western Canada. Our results give an overall picture of the 138N allele frequency being higher in the northern migratory barren-ground herds when compared to both boreal and moun- tain woodland caribou populations, with the exception of the bo- real woodland Chinchaga herd. We also report the presence of the N176D polymorphism in caribou for the first time as well as two novel Prnp polymorphisms, Y153F and P242L. As the genotype frequencies in the other three polymorphisms (G129S, N146n and V169M) do not differ significantly between subspecies and herds, we will not discuss them further. This S138N polymorphism is linked with strongly reduced sus- ceptibility to clinical CWD upon natural routes of prion infection (Hamir et al., 2011; Mitchell et al., 2012; Moore et al., 2016; Rhyan et al., 2011). In fallow deer, where both alleles at codon 138 encode asparagine (138NN), peripheral exposure to CWD-infected deer did not result in disease (Rhyan et al., 2011). However, they are sus- ceptible to intracerebral infection with white-tailed deer and elk CWD prions, with prolonged survival times ranging between 24 to 59 months post-infection (Hamir et al., 2011), indicating that 138N PrPC per se can be converted into PrPSc and does not confer abso- lute resistance to prion infection. In reindeer, the 138N allele has been associated with reduced susceptibility to natural CWD infec- tion routes in experimental settings. Only one animal had detectable prions in the obex, while in the rest prions were limited to periph- eral organs (Mitchell et al., 2012; Moore et al., 2016). All reindeer that tested positive for CWD in the Nordfjella region in Norway are of the 138SS genotype, and the 138N allele has not been reported in that population of reindeer (Güere et al., 2020). In addition, the substitution from serine to asparagine at position 138 impacted in vitro prion conversion in a previous study (Raymond et al., 2000). Together, these data suggest this may be an important site for dis- ease modulation. Structurally, serine is a small polar (hydrophilic) noncharged residue. An exchange to asparagine, which is a larger polar noncharged residue, can alter packing of side chains as serine may fit into pockets of bigger residues. While the newly identified polymorphisms have no known expo- sure to CWD, we provide some insight into their potential role in dis- ease susceptibility based on their locations and amino acid changes. The polymorphism at codon 242 is located in the C-terminal of the PrPC sequence that is cotranslationally cleaved off when the GPI-anchor is added. Thus, it is unlikely that the P242L polymor- phism plays a role in determining PrPC properties that influence F I G U R E 2   When the boreal woodland Chinchaga herd was omitted from statistical analyses, the difference in codon 138 between barren-ground and woodland caribou (left) and between barren-ground and woodland caribou of the mountatin ecotype (right) was statistically significant at a 5% significance level (p = .002 and .001, respectively). Tests were performed using a chi-squared test in the Genetics package in R Studio and graphs were generated in Graphpad Prism 8 Caribou subspecies GSV GNV GSM GNM SSV SNV SSM SNM Barren-ground (R. t. groenlandicus) 112 178 1 0 19 9 6 4 Woodland (R. t. caribou) 132 152 0 0 12 7 1 0 TA B L E 2   Number of individuals per haplotype group present in caribou herds in western Canada (codons 129/138/169; 146 is excluded as it is a synonymous substitution) 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 3836  |     ARIFIN et Al. the pathogenesis of CWD. The Y153F polymorphism is located within the first alpha-helix of PrPC (Wopfner et al., 1999). Tyrosine (Y) is identical to phenylalanine (F) and only differ in a hydroxyl group present in tyrosine. A previous study reported that the YYR (Tyrosine-Tyrosine-Arginine) motif at position 149–151 in human PrP, corresponding to 152–154 in cervids, is exposed in the PrPSc conformation and constitutes a PrPSc-specific epitope (Paramithiotis et al., 2003). The Y153F substitution might impact recognition by PrPSc-specific antibodies developed based on the YYR motif, but whether it actually plays a role in PrPC to PrPSc conversion is not known. The N→D polymorphism at codon 176, previously reported only in the Eurasian reindeer, was found in two NT barren-ground animals in this study. R. tarandus spp. is divided into two lineages based on microsatellite loci and mitochondrial DNA sequences (Yannic et al., 2014). The barren-ground populations belong to the Euro-Beringia clade that inhabit western Canada and Eurasia (Yannic et al., 2014). This shows the possibility that the Prnp gene variants are preserved within the lineage. Another explanation is the introduction of Eurasian reindeer into Alaska which were later herded into the Inuvik region in the 1930s, highlighting possibilities of introgression between caribou and reindeer in NT (Klein, 1980). Polymorphisms and their locations with respect to PrPC are visual- ized in Figure 3. Landscape features, such as major valleys and separation by riv- ers, have shown to be more accurate in describing caribou genetic structure than classification by subspecies and ecotype (Serrouya et al., 2012). We therefore examined genotypic differentiation at the herd level, finding Chinchaga was significantly different from most of the other herds at the 138 locus. Boreal woodland populations in the Chinchaga range, both in BC and Alberta, inhabit only the peatlands where lichen is abundant (Leech, Whittaker, & the Doig River First Nation, 2016; O’Leary, Saxena, & DeCoursey, 2002). Furthermore, the overlay with landscape motifs in Figure 1 indicates a possibility that the Chinchaga population in BC is located in a secluded habi- tat surrounded by higher elevation ground, which may result in re- duced gene flow. These provide possible reasons for the high 138N allele prevalence in the Chinchaga caribou herd, where geographic isolation limits genetic flow to and from other adjacent herds. A similarly high 138N prevalence has also been previously reported in the Chinchaga caribou population in Alberta (Cheng, Musiani, et al., 2017). Although there are likely many factors impacting free-ranging caribou populations, we propose that the high 138N allele preva- lence is advantageous should CWD expand to the Chinchaga pop- ulation, as caribou in this area are at high risk of extinction (Leech et al., 2016; O’Leary et al., 2002). Woodland boreal caribou in Saskatchewan are divided into two major populations, the northern SK1 range that inhabits the Boreal Shield and the southern SK2 range, further categorized into west, central and east, populating the Saskatchewan Boreal Plains (Saskatchewan Ministry of Environment, 2013). The SK2 range habitat overlaps with white-tailed deer, which are natural hosts of CWD (McLoughlin et al., 2019). Genetic information on these pop- ulations show that caribou in the SK2 range often migrate to the SK1 range but not vice versa, excepting high gene flow in the central range (Figure S2) (Priadka et al., 2018). Our results show that the majority of the SK1 range caribou harbor the wild-type 138SS Prnp genotype (97.5%), which is associated with susceptibility to natu- ral CWD infection routes (Güere et al., 2020; Mitchell et al., 2012; Moore et al., 2016). Should these herds contract CWD, they will increase transmission probability to woodland and barren-ground caribou populations in NT. In this study, barren-ground caribou have significantly higher 138N Prnp allele frequencies (36.8%) than wood- land caribou (27.9%), in particular of the mountain ecotype (22.7%), when the outlier Chinchaga herd was removed from the analyses. As barren-ground caribou migrate across vast areas of land (COSEWIC, 2016), they will pose a risk of widespread prion contamination if F I G U R E 3   Positions of caribou polymorphisms reported in this study within the prion protein sequence. GPI, glycosylphosphatidylinositol; HD, hydrophobic domain; OR, octapeptide repeat. *All animals were homozygous serine (S) at codon 225 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense      |  3837ARIFIN et Al. infected, eventually reaching the east and west coasts, and even Alaska. Climate change and industrial development have reduced the size of remaining caribou habitat leading to range retraction and increased contact between caribou and other cervids (especially white-tailed deer) and predator species, either directly or indi- rectly (Bauduin, McIntire, St-Laurent, & Cumming, 2018; Dawe & Boutin, 2016; Hebblewhite & Fortin, 2017; Hervieux et al., 2013; Leech et al., 2016; Tennant et al., 2020). Predators have been shown to pass prions in their faeces after consuming CWD-infected cervids (Krumm, Conner, Hobbs, Hunter, & Miller, 2010; Nichols, Fischer, Spraker, Kong, & VerCauteren, 2015), and mountain lions preferen- tially prey on CWD-infected deer (Krumm et al., 2010), which further increases the probability of prion dissemination. Should wild caribou come into contact with these prions, an additional risk factor is im- posed to their already Threatened status, as the disease has been shown to drive the decline of white-tailed and mule deer populations (DeVivo et al., 2017; Edmunds et al., 2016). Although CWD has not yet been detected in free-ranging caribou in Canada, transmission to these animals from infected captive or free-ranging cervids remains a concern for wildlife scientists and managers. Thus, CWD control measures and strategies for caribou are already part of current wild- life disease management guidelines (CWD map of positive tests, 2020; Gagnier, Laurion, & DeNicola, 2020; Williams, Miller, Kreeger, Kahn, & Thorne, 2002; Zimmer, Boxall, & Adamowicz, 2011). Our findings highlight herds with the highest susceptibility to CWD, assuming the 138N allele reduces susceptibility. Of the herds we examined, mountain caribou herds and those in the SK1 range in Saskatchewan are at the highest risk (Figure 1). We hope these find- ings help in mitigating CWD transmission to caribou by contributing to future decisions and planning of CWD surveillance and preventive measures in Canada. In conclusion, the 138N Prnp allele, associated with less sus- ceptibility to CWD, is found in caribou populations in Alaska (Happ et al., 2007), Alberta (Cheng, Musiani, et al., 2017), British Columbia, the Northwest Territories, Nunavut, Saskatchewan, and Yukon. It is more prevalent in barren-ground than woodland caribou popu- lations, with statistical significance when the woodland Chinchaga herd was excluded from the analyses. This high 138N allele fre- quency in the Chinchaga range is probably due to landscape and geographic isolation. In the SK1 range in Saskatchewan, which over- laps with barren-ground caribou range, the 138N Prnp allele is pres- ent in much lower frequencies, thus increasing caribou susceptibility to contracting clinical CWD. This caribou population can serve as a gateway to possible CWD transmission from infected cervids, thus bringing into light the risk of CWD expansion into woodland and bar- ren-ground caribou in western North America. ACKNOWLEDG EMENTS We would like to acknowledge our collaborators Bryan Macbeth, Maeve Winchester and Helen Schwantje at the British Columbia Ministry of Forests, Lands, Natural Resource Operations, and Rural Development and Jane Harms at the Department of Environment, Yukon for providing caribou blood or tissue samples, as well as the Veterinary Medicine Faculty Teaching Facility at the University of Calgary. We acknowledge funding for this research from Genome Canada, Alberta Prion Research Institute and Alberta Agriculture and Forestry through Genome Alberta, and the University of Calgary. SG is supported by the Canada Research Chair program. PDM was supported by a Natural Sciences and Engineering Research Council (NSERC) Collaborative Research and Development grant. AUTHOR CONTRIBUTIONS M.I.A. wrote the paper. S.G. and M.I.A. designed the research. M.I.A., A.S., S.Y.S., and Y.H. performed the research. H.F., and P.D.M. provided samples. M.I.A., A.S., C.C., G.M., and S.G. analysed data. A.S., S.Y.S., H.F., P.D.M., C.C., G.M., and S.G. provided comments and edits to the manuscript. DATA ACCE SSIBILIT Y Caribou ID, genotype, herd and capture coordinates are listed in Table S2. Novel Prnp polymorphisms are submitted to Genbank with accession numbers MT361766 and MT361767. ORCID Catherine I Cullingham https://orcid.org/0000-0002-6715-0674 Sabine Gilch https://orcid.org/0000-0001-5923-3464 R E FE R E N C E S Aneichyk, T., Hendriks, W. T., Yadav, R., Shin, D., Gao, D., Vaine, C. A., … Talkowski, M. E. (2018). Dissecting the causal mechanism of X-linked dystonia-parkinsonism by integrating genome and transcriptome assembly. Cell, 172(5), 897–909.e21. https://doi.org/10.1016/j. cell.2018.02.011 Angers, R., Christiansen, J., Nalls, A. V., Kang, H.-E., Hunter, N., Hoover, E., … Telling, G. C. (2014). Structural effects of PrP polymorphisms on intra- and interspecies prion transmission. Proceedings of the National Academy of Sciences of the United States of America, 111(30), 11169– 11174. https://doi.org/10.1073/pnas.14047 39111 Baeten, L. A., Powers, B. E., Jewell, J. E., Spraker, T. R., & Miller, M. W. (2007). A Natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi). The Journal of Wildlife Diseases, 43(2), 309–314. https://doi.org/10.7589/0090-3558-43.2.309 Balachandran, A., Harrington, N. P., Algire, J., Soutyrine, A., Spraker, T. R., Jeffrey, M., … O-Rourke, K. I. (2010). Experimental oral transmission of chronic wasting disease to red deer (Cervus ela- phus elaphus): Early detection and late stage distribution of pro- tease-resistant prion protein. Canadian Veterinary Journal, 51(2), 169–178. Bartelt-Hunt, S. L., & Bartz, J. C. (2013). Behavior of prions in the environ- ment: Implications for prion biology. PLoS Pathogens, 9(2), e1003113. https://doi.org/10.1371/journ al.ppat.1003113 Bauduin, S., McIntire, E., St-Laurent, M.-H., & Cumming, S. G. (2018). Compensatory conservation measures for an endangered cari- bou population under climate change. Scientific Reports, 8, 16438. https://doi.org/10.1038/s4159 8-018-34822 -9 Benestad, S. L., Mitchell, G., Simmons, M., Ytrehus, B., & Vikøren, T. (2016). First case of chronic wasting disease in Europe in a Norwegian free-ranging reindeer. Veterinary Research, 47(1), 88. https://doi. org/10.1186/s1356 7-016-0375-4 Brayton, K. A., O’Rourke, K. I., Lyda, A. K., Miller, M. W., & Knowles, D. P. (2004). A processed pseudogene contributes to apparent mule 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://orcid.org/0000-0002-6715-0674 https://orcid.org/0000-0002-6715-0674 https://orcid.org/0000-0001-5923-3464 https://orcid.org/0000-0001-5923-3464 https://doi.org/10.1016/j.cell.2018.02.011 https://doi.org/10.1016/j.cell.2018.02.011 https://doi.org/10.1073/pnas.1404739111 https://doi.org/10.7589/0090-3558-43.2.309 https://doi.org/10.1371/journal.ppat.1003113 https://doi.org/10.1038/s41598-018-34822-9 https://doi.org/10.1186/s13567-016-0375-4 https://doi.org/10.1186/s13567-016-0375-4 3838  |     ARIFIN et Al. deer prion gene heterogeneity. Gene, 326, 167–173. https://doi. org/10.1016/j.gene.2003.10.022 Cheng, Y. C., Hannaoui, S., John, T. R., Dudas, S., Czub, S., & Gilch, S. (2017). Real-time quaking-induced conversion assay for detection of CWD prions in fecal material. Journal of Visualized Experiments, 127, e56373. https://doi.org/10.3791/56373 Cheng, Y. C., Musiani, M., Cavedon, M., & Gilch, S. (2017). High prev- alence of prion protein genotype associated with resistance to chronic wasting disease in one Alberta woodland caribou pop- ulation. Prion, 11(2), 136–142. https://doi.org/10.1080/19336 896.2017.1300741 COSEWIC (2016). COSEWIC assessment and status report on the cari- bou, Rangifer tarandus, Barren-ground population, in Canada (p. 123). Ottawa, ON: Committee on the Status of Endangered Wildlife in Canada. Retrieved from http://publi catio ns.gc.ca/colle ction s/colle ction_2017/eccc/CW69-14-746-2017-eng.pdf CWD map of positive tests|Chronic Wasting Disease (2020). Government of Saskatchewan. Retrieved from https://www.saska tchew an.ca/ resid ents/envir onmen t-publi c-healt h-and-safet y/wildl ife-issue s/ fish-and-wildl ife-disea ses/chron ic-wasti ng-disea se/cwd-map Dawe, K. L., & Boutin, S. (2016). Climate change is the primary driver of white-tailed deer (Odocoileus virginianus) range expansion at the northern extent of its range; land use is secondary. Ecology and Evolution, 6(18), 6435–6451. DeVivo, M. T., Edmunds, D. R., Kauffman, M. J., Schumaker, B. A., Binfet, J., Kreeger, T. J., … Cornish, T. E. (2017). Endemic chronic wasting disease causes mule deer population decline in Wyoming. PLoS One, 12(10), e0186512. https://doi.org/10.1371/journ al.pone.0186512 Edmunds, D. R., Kauffman, M. J., Schumaker, B. A., Lindzey, F. G., Cook, W. E., Kreeger, T. J., … Cornish, T. E. (2016). Chronic wasting disease drives population decline of White-Tailed deer. PLoS One, 11(8), e0161127. https://doi.org/10.1371/journ al.pone.0161127 Festa-Bianchet, M., Ray, J. C., Boutin, S., Côté, S. D., & Gunn, A. (2011). Conservation of caribou (Rangifer tarandus) in Canada: An uncertain future. Canadian Journal of Zoology, 89(5), 419–434. Gagnier, M., Laurion, I., & DeNicola, A. J. (2020). Control and surveil- lance operations to prevent chronic wasting disease establishment in free-ranging white-tailed deer in Québec, Canada. Animals (Basel), 10(2), 238. https://doi.org/10.3390/ani10 020283 Gilch, S., Chitoor, N., Taguchi, Y., Stuart, M., Jewell, J. E., & Schätzl, H. M. (2011). Chronic wasting disease. Topics in Current Chemistry, 305, 51–77. Giles, K., Woerman, A. L., Berry, D. B., & Prusiner, S. B. (2017). Bioassays and inactivation of prions. Cold Spring Harbor Perspectives in Biology, 9(8), a023499. https://doi.org/10.1101/cshpe rspect.a023499 Green, K. M., Browning, S. R., Seward, T. S., Jewell, J. E., Ross, D. L., Green, M. A., … Telling, G. C. (2008). The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation. Journal of General Virology, 89(2), 598–608. https://doi.org/10.1099/vir.0.83168 -0 Güere, M. E., Våge, J., Tharaldsen, H., Benestad, S. L., Vikøren, T., Madslien, K., … Tranulis, M. A. (2020). Chronic wasting disease as- sociated with prion protein gene (PRNP) variation in Norwegian wild reindeer (Rangifer tarandus). Prion, 14(1), 1–10. Haley, N. J., Mathiason, C. K., Carver, S., Zabel, M., Telling, G. C., & Hoover, E. A. (2011). Detection of chronic wasting disease prions in salivary, urinary, and intestinal tissues of deer: Potential mechanisms of prion shedding and transmission. Journal of Virology, 85(13), 6309– 6318. https://doi.org/10.1128/JVI.00425 -11 Hamir, A. N., Greenlee, J. J., Nicholson, E. M., Kunkle, R. A., Richt, J. A., Miller, J. M., & Hall, M. (2011). Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: Final report. Canadian Journal of Veterinary Research, 75(2), 152–156. Hannaoui, S., Amidian, S., Cheng, Y. C., Duque Velásquez, C., Dorosh, L., Law, S., … Gilch, S. (2017). Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency. PLoS Pathogens, 13(8), e1006553. https://doi. org/10.1371/journ al.ppat.1006553 Hannaoui, S., Schatzl, H. M., & Gilch, S. (2017). Chronic wasting disease: Emerging prions and their potential risk. PLoS Pathogens, 13(11), e1006619. https://doi.org/10.1371/journ al.ppat.1006619 Happ, G. M., Huson, H. J., Beckmen, K. B., & Kennedy, L. J. (2007). Prion protein genes in caribou from Alaska. Journal of Wildlife Diseases, 43(2), 224–228. https://doi.org/10.7589/0090-3558-43.2.224 Hebblewhite, M., & Fortin, D. (2017). Canada fails to protect its caribou. Science, 358(6364), 730–731. Henderson, D. M., Denkers, N. D., Hoover, C. E., Garbino, N., Mathiason, C. K., & Hoover, E. A. (2015). Longitudinal detection of prion shed- ding in saliva and urine by chronic wasting disease-infected deer by real-time quaking-induced conversion. Journal of Virology, 89(18), 9338–9347. https://doi.org/10.1128/JVI.01118 -15 Hervieux, D., Hebblewhite, M., DeCesare, N. J., Russell, M., Smith, K., Robertson, S., & Boutin, S. (2013). Widespread declines in woodland caribou (Rangifer tarandus caribou) continue in Alberta. Canadian Journal of Zoology, 91(12), 872–882. Johnson, C. J., Herbst, A., Duque-Velasquez, C., Vanderloo, J. P., Bochsler, P., Chappell, R., & McKenzie, D. (2011). Prion protein polymorphisms affect chronic wasting disease progression. PLoS One, 6(3), e17450. https://doi.org/10.1371/journ al.pone.0017450 Kahn, S., Dubé, C., Bates, L., & Balachandran, A. (2004). Chronic wast- ing disease in Canada: Part 1. Canadian Veterinary Journal, 45(5), 397–404. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., … Drummond, A. (2012). Geneious Basic: An integrated and ex- tendable desktop software platform for the organization and analy- sis of sequence data. Bioinformatics, 28(12), 1647–1649. https://doi. org/10.1093/bioin forma tics/bts199 Klein, D. R. (1980). Conflicts between domestic reindeer and their wild counterparts: A review of Eurasian and North American experience. Arctic, 33(3), 739–756. Krumm, C. E., Conner, M. M., Hobbs, N. T., Hunter, D. O., & Miller, M. W. (2010). Mountain lions prey selectively on prion-infected mule deer. Biology Letters, 6(2), 209–211. https://doi.org/10.1098/ rsbl.2009.0742 Kurt, T. D., & Sigurdson, C. J. (2016). Cross-species transmission of CWD prions. Prion, 10(1), 83–91. https://doi.org/10.1080/19336 896.2015.1118603 Kuznetsova, A., McKenzie, D., Cullingham, C., & Aiken, J. M. (2020). Long- term incubation PrPCWD with soils affects prion recovery but not in- fectivity. Pathogens (Basel Switzerland), 9(4). https://doi.org/10.3390/ patho gens9 040311 Leech, S. M., Whittaker, C. & the Doig River First Nation (2016). Madziih (caribou) Tsáá? ché ne dane traditional knowledge and restoration study. Report prepared for DFN and the David Suzuki Foundation by the Firelight Group December 2016. p. 60. Linden, R., Martins, V. R., Prado, M. A. M., Cammarota, M., Izquierdo, I., & Brentani, R. R. (2008). Physiology of the prion protein. Physiologial Reviews, 88(2), 673–728. https://doi.org/10.1152/physr ev.00007.2007 Mallory, C. D., & Boyce, M. S. (2019). Prioritization of landscape connec- tivity for the conservation of Peary caribou. Ecology and Evolution, 9(4), 2189–2205. https://doi.org/10.1002/ece3.4915 McLoughlin, P. D., Superbie, C., Stewart, K., Tomchuk, P., Neufeld, B., Barks, D., … Johnstone, J. F. (2019). Population and habitat ecology of boreal caribou and their predators in the Saskatchewan boreal shield. Final report. Department of Biology, University of Saskatchewan, Saskatoon. p. 238. 2013–2018. Mitchell, G. B., Sigurdson, C. J., O’Rourke, K. I., Algire, J., Harrington, N. P., Walther, I., … Balachandran, A. (2012). Experimental oral trans- mission of chronic wasting disease to reindeer (Rangifer tarandus 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.1016/j.gene.2003.10.022 https://doi.org/10.1016/j.gene.2003.10.022 https://doi.org/10.3791/56373 https://doi.org/10.1080/19336896.2017.1300741 https://doi.org/10.1080/19336896.2017.1300741 http://publications.gc.ca/collections/collection_2017/eccc/CW69-14-746-2017-eng.pdf http://publications.gc.ca/collections/collection_2017/eccc/CW69-14-746-2017-eng.pdf https://www.saskatchewan.ca/residents/environment-public-health-and-safety/wildlife-issues/fish-and-wildlife-diseases/chronic-wasting-disease/cwd-map https://www.saskatchewan.ca/residents/environment-public-health-and-safety/wildlife-issues/fish-and-wildlife-diseases/chronic-wasting-disease/cwd-map https://www.saskatchewan.ca/residents/environment-public-health-and-safety/wildlife-issues/fish-and-wildlife-diseases/chronic-wasting-disease/cwd-map https://doi.org/10.1371/journal.pone.0186512 https://doi.org/10.1371/journal.pone.0161127 https://doi.org/10.3390/ani10020283 https://doi.org/10.1101/cshperspect.a023499 https://doi.org/10.1099/vir.0.83168-0 https://doi.org/10.1128/JVI.00425-11 https://doi.org/10.1371/journal.ppat.1006553 https://doi.org/10.1371/journal.ppat.1006553 https://doi.org/10.1371/journal.ppat.1006619 https://doi.org/10.7589/0090-3558-43.2.224 https://doi.org/10.1128/JVI.01118-15 https://doi.org/10.1371/journal.pone.0017450 https://doi.org/10.1093/bioinformatics/bts199 https://doi.org/10.1093/bioinformatics/bts199 https://doi.org/10.1098/rsbl.2009.0742 https://doi.org/10.1098/rsbl.2009.0742 https://doi.org/10.1080/19336896.2015.1118603 https://doi.org/10.1080/19336896.2015.1118603 https://doi.org/10.3390/pathogens9040311 https://doi.org/10.3390/pathogens9040311 https://doi.org/10.1152/physrev.00007.2007 https://doi.org/10.1152/physrev.00007.2007 https://doi.org/10.1002/ece3.4915      |  3839ARIFIN et Al. tarandus). PLoS One, 7(6), e39055. https://doi.org/10.1371/journ al.pone.0039055 Moore, S. J., Kunkle, R., Greenlee, M. H. W., Nicholson, E., Richt, J., Hamir, A., … Greenlee, J. (2016). Horizontal transmission of chronic wasting disease in reindeer. Emerging Infectious Diseases, 22(12), 2142–2145. https://doi.org/10.3201/eid22 12.160635 Nalls, A. V., McNulty, E., Powers, J., Seelig, D. M., Hoover, C., Haley, N. J., … Mathiason, C. K. (2013). Mother to offspring transmission of chronic wasting disease in reeves’ muntjac deer. PLoS One, 8(8), e71844. https://doi.org/10.1371/journ al.pone.0071844 National Museum of Canada (1962). Revision of the reindeer and caribou, genus Rangifer. (Bulletin National Museum of Canada; No. 177). Nichols, T. A., Fischer, J. W., Spraker, T. R., Kong, Q., & VerCauteren, K. C. (2015). CWD prions remain infectious after passage through the digestive system of coyotes (Canis latrans). Prion, 9(5), 367–375. O’Leary, D., Saxena, A., & DeCoursey, C. (2002). Biophysical inventory of chinchaga wildland park (p. 72). Valleyview, AB: Alberta Community Development Parks and Portected Areas. O'Rourke, K. I., Spraker, T. R., Hamburg, L. K., Besser, T. E., Brayton, K. A., & Knowles, D. P. (2004). Polymorphisms in the prion precursor func- tional gene but not the pseudogene are associated with susceptibil- ity to chronic wasting disease in white-tailed deer. Journal of General Virology, 85(5), 1339–1346. https://doi.org/10.1099/vir.0.79785 -0 Paramithiotis, E., Pinard, M., Lawton, T., LaBoissiere, S., Leathers, V. L., Zou, W.-Q., … Cashman, N. R. (2003). A prion protein epitope selec- tive for the pathologically misfolded conformation. Nature Medicine, 9(7), 893–899. https://doi.org/10.1038/nm883 Pirisinu, L., Tran, L., Chiappini, B., Vanni, I., Di Bari, M., Vaccari, G., … Benestad, S. L. (2018). Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway. Emerging Infectious Diseases, 24(12), 2210–2218. http://dx.doi.org/10.3201/eid24 12.18 0702 Priadka, P., Manseau, M., Trottier, T., Hervieux, D., Galpern, P., McLoughlin, P. D., & Wilson, P. J. (2018). Partitioning drivers of spa- tial genetic variation for a continuously distributed population of bo- real caribou: Implications for management unit delineation. Ecology and Evolution, 14, e4682. https://doi.org/10.1002/ece3.4682 Pritzkow, S., Morales, R., Lyon, A., Concha-Marambio, L., Urayama, A., & Soto, C. (2018). Efficient prion disease transmission through com- mon environmental materials. Journal of Biological Chemistry, 293(9), 3363–3373. https://doi.org/10.1074/jbc.M117.810747 Prusiner, S. B. (1998). Prions. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13363–13383. Raymond, G. J., Bossers, A., Raymond, L. D., O’Rourke, K. I., McHolland, L. E., Bryant, P. K. III, … Caughey, B. (2000). Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease. The EMBO Journal, 19(17), 4425–4430. https://doi. org/10.1093/emboj/ 19.17.4425 Rhyan, J. C., Miller, M. W., Spraker, T. R., McCollum, M., Nol, P., Wolfe, L. L., … O'Rourke, K. I. (2011). Failure of fallow deer (Dama dama) to develop chronic wasting disease when exposed to a contam- inated environment and infected mule deer (Odocoileus hemi- onus). Journal of Wildlife Diseases, 47(3), 739–744. https://doi. org/10.7589/0090-3558-47.3.739 Robinson, A. L., Williamson, H., Güere, M. E., Tharaldsen, H., Baker, K., Smith, S. L., … Houston, F. (2019). Variation in the prion protein gene (PRNP) sequence of wild deer in Great Britain and mainland Europe. Veterinary Research, 50(1), 59. https://doi.org/10.1186/ s1356 7-019-0675-6 Robinson, S. J., Samuel, M. D., O’Rourke, K. I., & Johnson, C. J. (2012). The role of genetics in chronic wasting disease of North American cervids. Prion, 6(2), 153–162. https://doi.org/10.4161/pri.19640 Safar, J. G., Lessard, P., Tamgüney, G., Freyman, Y., Deering, C., Letessier, F., … Prusiner, S. B. (2008). Transmission and detection of prions in feces. Journal of Infectious Diseases, 198(1), 81–89. https://doi. org/10.1086/588193 Saskatchewan Ministry of Environment (2013). Conservation strategy for boreal woodland caribou (Rangifer tarandus caribou) in Saskatchewan. (Fish and Wildlife Technical Report 2014). Retrieved from http:// publi catio ns.gov.sk.ca/docum ents/66/89807 -Engli sh.pdf Serrouya, R., Paetkau, D., McLellan, B. N., Boutin, S., Campbell, M., & Jenkins, D. A. (2012). Population size and major valleys explain mi- crosatellite variation better than taxonomic units for caribou in western Canada. Molecular Ecology, 21(11), 2588–2601. https://doi. org/10.1111/j.1365-294X.2012.05570.x Sigurdson, C. J., Williams, E. S., Miller, M. W., Spraker, T. R., O’Rourke, K. I., & Hoover, E. A. (1999). Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus). Journal of General Virology, 80(10), 2757–2764. https:// doi.org/10.1099/0022-1317-80-10-2757 Somerville, R. A., Fernie, K., Smith, A., Bishop, K., Maddison, B. C., Gough, K. C., & Hunter, N. (2019). BSE infectivity survives burial for five years with only limited spread. Archives of Virology, 164(4), 1135– 1145. https://doi.org/10.1007/s0070 5-019-04154 -8 Tennant, J. M., Li, M., Henderson, D. M., Tyer, M. L., Denkers, N. D., Haley, N. J., … Hoover, E. A. (2020). Shedding and stability of CWD prion seeding activity in cervid feces. PLoS One, 15(3), e0227094. https://doi.org/10.1371/journ al.pone.0227094 Thomas, D. C., & Gray, D. R. (2002). Update COSEWIC status report on the woodland caribou rangifer tarandus caribou in Canada. In COSEWIC assessment and update status report on the the Woodland Caribou Rangifer tarandus caribou in Canada. Ottawa, ON: Committee on the Status of Endangered Wildlife in Canada. pp. 1–98. Retrieved from http://www.sarar egist ry.gc.ca/virtu al_sara/files/ cosew ic/sr_woodl and_carib ou_e.pdf. Vikøren, T., Våge, J., Madslien, K. I., Røed, K. H., Rolandsen, C. M., Tran, L., … Benestad, S. L. (2019). First detection of chronic wasting dis- ease in a wild red deer (Cervus elaphus) in Europe. Journal of Wildlife Diseases, 55(4), 970. https://doi.org/10.7589/2018-10-262 Wik, L., Mikko, S., Klingeborn, M., Stéen, M., Simonsson, M., & Linné, T. (2012). Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia. Prion, 6(3), 256–260. Williams, E. S., Miller, M. W., Kreeger, T. J., Kahn, R. H., & Thorne, E. T. (2002). Chronic wasting disease of deer and elk: A review with rec- ommendations for management. The Journal of Wildlife Management, 66(3), 551. http://dx.doi.org/10.2307/3803123 Williams, E. S., & Young, S. (1980). Chronic wasting disease of captive mule deer: A spongiform encephalopathy. Journal of Wildlife Diseases, 16(1), 89–98. https://doi.org/10.7589/0090-3558-16.1.89 Williams, E. S., & Young, S. (1982). Spongiform encephalopathy of rocky mountain elk. Journal of Wildlife Diseases, 18(4), 465–471. https://doi. org/10.7589/0090-3558-18.4.465 Williams, E. S., & Young, S. (1993). Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni). Veterinary Pathology, 30(1), 36–45. Wopfner, F., Weidenhöfer, G., Schneider, R., von Brunn, A., Gilch, S., Schwarz, T. F., … Schatzl, H. M. (1999). Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. Journal of Molecular Biology, 289(5), 1163–1178. Yannic, G., Pellissier, L., Ortego, J., Lecomte, N., Couturier, S., Cuyler, C., … Côté, S. D. (2014). Genetic diversity in caribou linked to past and future climate change. Nature Climate Change, 4(2), 132–137. https:// doi.org/10.1038/nclim ate2074 Zimmer, N., Boxall, P. C., Adamowicz, W. L. (Vic). (2011). The impact of chronic wasting disease and its management on hunter perceptions, opinions, and behaviors in Alberta, Canada. Journal of Toxicology and 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.1371/journal.pone.0039055 https://doi.org/10.1371/journal.pone.0039055 https://doi.org/10.3201/eid2212.160635 https://doi.org/10.1371/journal.pone.0071844 https://doi.org/10.1099/vir.0.79785-0 https://doi.org/10.1038/nm883 http://dx.doi.org/10.3201/eid2412.180702 http://dx.doi.org/10.3201/eid2412.180702 https://doi.org/10.1002/ece3.4682 https://doi.org/10.1074/jbc.M117.810747 https://doi.org/10.1093/emboj/19.17.4425 https://doi.org/10.1093/emboj/19.17.4425 https://doi.org/10.7589/0090-3558-47.3.739 https://doi.org/10.7589/0090-3558-47.3.739 https://doi.org/10.1186/s13567-019-0675-6 https://doi.org/10.1186/s13567-019-0675-6 https://doi.org/10.4161/pri.19640 https://doi.org/10.1086/588193 https://doi.org/10.1086/588193 http://publications.gov.sk.ca/documents/66/89807-English.pdf http://publications.gov.sk.ca/documents/66/89807-English.pdf https://doi.org/10.1111/j.1365-294X.2012.05570.x https://doi.org/10.1111/j.1365-294X.2012.05570.x https://doi.org/10.1099/0022-1317-80-10-2757 https://doi.org/10.1099/0022-1317-80-10-2757 https://doi.org/10.1007/s00705-019-04154-8 https://doi.org/10.1371/journal.pone.0227094 http://www.sararegistry.gc.ca/virtual_sara/files/cosewic/sr_woodland_caribou_e.pdf http://www.sararegistry.gc.ca/virtual_sara/files/cosewic/sr_woodland_caribou_e.pdf https://doi.org/10.7589/2018-10-262 http://dx.doi.org/10.2307/3803123 https://doi.org/10.7589/0090-3558-16.1.89 https://doi.org/10.7589/0090-3558-18.4.465 https://doi.org/10.7589/0090-3558-18.4.465 https://doi.org/10.1038/nclimate2074 https://doi.org/10.1038/nclimate2074 3840  |     ARIFIN et Al. Environmental Health, Part A, 74(22-24), 1621–1635. http://dx.doi. org/10.1080/15287 394.2011.618988 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Arifin MI, Staskevicius A, Shim SY, et al. Large-scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility. Mol Ecol. 2020;29:3830–3840. https://doi. org/10.1111/mec.15602 1365294x, 2020, 20, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/m ec.15602 by C anadian A griculture L ibrary, W iley O nline L ibrary on [23/04/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://dx.doi.org/10.1080/15287394.2011.618988 http://dx.doi.org/10.1080/15287394.2011.618988 https://doi.org/10.1111/mec.15602 https://doi.org/10.1111/mec.15602