Non-invasive biomonitoring of polar bear feces can be used to estimate concentrations of metals of concern in traditional food.

The Arctic faces increasing exposure to environmental chemicals such as metals, posing health risks to humans and wildlife. Biomonitoring of polar bears (Ursus maritimus) can be used to quantify chemicals in the environment and in traditional foods consumed by the Inuit. However, typically, these samples are collected through invasive or terminal methods. The biomonitoring of feces could be a useful alternative to the current metal monitoring method within the Arctic. Here, we aim to 1) quantify the relationship between concentrations of metals in the feces and tissues (muscle, liver, and fat) of polar bears using predictive modeling, 2) develop an easy-to-use conversion tool for use in community-based monitoring programs to non-invasively estimate contaminant concentrations in polar bears tissues and 3) demonstrate the application of these models by examining potential exposure risk for humans from consumption of polar bear muscle. Fecal, muscle, liver, and fat samples were harvested from 49 polar bears through a community-based monitoring program. The samples were analyzed for 32 metals. Exploratory analysis indicated that mean metal concentrations generally did not vary by age or sex, and many of the metals measured in feces were positively correlated with the internal tissue concentration. We developed predictive linear regression models between internal (muscle, liver, fat) and external (feces) metal concentrations and further explored the mercury and methylmercury relationships for utility risk screening. Using the cross-validated regression coefficients, we developed a conversion tool that contributes to the One Health approach by understanding the interrelated health of humans, wildlife, and the environment in the Arctic. The findings support using feces as a biomonitoring tool for assessing contaminants in polar bears. Further research is needed to validate the developed models for other regions in the Arctic and assess the impact of environmental weathering on fecal metal concentrations.

The geographic distribution, and the biotic and abiotic predictors of select zoonotic pathogen detections in Canadian polar bears.

Increasing Arctic temperatures are facilitating the northward expansion of more southerly hosts, vectors, and pathogens, exposing naïve populations to pathogens not typical at northern latitudes. To understand such rapidly changing host-pathogen dynamics, we need sensitive and robust surveillance tools. Here, we use a novel multiplexed magnetic-capture and droplet digital PCR (ddPCR) tool to assess a sentinel Arctic species, the polar bear (Ursus maritimus; n = 68), for the presence of five zoonotic pathogens (Erysipelothrix rhusiopathiae, Francisella tularensis, Mycobacterium tuberculosis complex, Toxoplasma gondii and Trichinella spp.), and observe associations between pathogen presence and biotic and abiotic predictors. We made two novel detections: the first detection of a Mycobacterium tuberculosis complex member in Arctic wildlife and the first of E. rhusiopathiae in a polar bear. We found a prevalence of 37% for E. rhusiopathiae, 16% for F. tularensis, 29% for Mycobacterium tuberculosis complex, 18% for T. gondii, and 75% for Trichinella spp. We also identify associations with bear age (Trichinella spp.), harvest season (F. tularensis and MTBC), and human settlements (E. rhusiopathiae, F. tularensis, MTBC, and Trichinella spp.). We demonstrate that monitoring a sentinel species, the polar bear, could be a powerful tool in disease surveillance and highlight the need to better characterize pathogen distributions and diversity in the Arctic.

A new multiplexed magnetic capture-Droplet digital PCR tool for monitoring wildlife population health and pathogen surveillance.

Anthropogenic stressors are exacerbating the emergence and spread of pathogens worldwide. In regions like the Arctic, where ecosystems are particularly susceptible, marked changes are predicted in regional diversity, intensity, and patterns of infectious diseases. To understand such rapidly changing host-pathogen dynamics and mitigate the impacts of novel pathogens, we need sensitive disease surveillance tools. We developed and validated a novel multiplexed, magnetic capture, and ddPCR tool for the surveillance of multiple pathogens in polar bears, a sentinel species that is considered susceptible to climate change and other stressors with a pan-Arctic distribution. Through sequence-specific magnetic capture, we concentrated five target template sequences from three zoonotic bacteria (Erysipelothrix rhusiopathiae, Francisella tularensis, and Mycobacterium tuberculosis complex) and two parasitic (Toxoplasma gondii and Trichinella spp.) pathogens from large quantities (<100 g) of host tissue. We then designed and validated two multiplexed probe-based ddPCR assays for the amplification and detection of the low-concentration target DNA. Validations used 48 polar bear tissues (muscle and liver). We detected 14, 1, 3, 4, and 22 tissue positives for E. rhusiopathiae, F. tularensis, M. tuberculosis complex, T. gondii, and Trichinella spp., respectively. These multiplexed assays offer a rapid, specific tool for quantifying and monitoring the changing geographical and host distributions of pathogens relevant to human and animal health.

Genotyping-in-thousands by sequencing (GT-seq) of noninvasive faecal and degraded samples: A new panel to enable ongoing monitoring of Canadian polar bear populations.

Genetic monitoring using noninvasive samples provides a complement or alternative to traditional population monitoring methods. However, next-generation sequencing approaches to monitoring typically require high quality DNA and the use of noninvasive samples (e.g., scat) is often challenged by poor DNA quality and contamination by nontarget species. One promising solution is a highly multiplexed sequencing approach called genotyping-in-thousands by sequencing (GT-seq), which can enable cost-efficient genomics-based monitoring for populations based on noninvasively collected samples. Here, we develop and validate a GT-seq panel of 324 single nucleotide polymorphisms (SNPs) optimized for genotyping of polar bears based on DNA from noninvasively collected faecal samples. We demonstrate (1) successful GT-seq genotyping of DNA from a range of sample sources, including successful genotyping (>50% loci) of 62.9% of noninvasively collected faecal samples determined to contain polar bear DNA; and (2) that we can reliably differentiate individuals, ascertain sex, assess relatedness, and resolve population structure of Canadian polar bear subpopulations based on a GT-seq panel of 324 SNPs. Our GT-seq data reveal spatial-genetic patterns similar to previous polar bear studies but at lesser cost per sample and through use of noninvasively collected samples, indicating the potential of this approach for population monitoring. This GT-seq panel provides the foundation for a noninvasive toolkit for polar bear monitoring and can contribute to community-based programmes - a framework which may serve as a model for wildlife conservation and management for species worldwide.

Regional contamination versus regional dietary differences: understanding geographic variation in brominated and chlorinated contaminant levels in polar bears.

The relative contribution of regional contamination versus dietary differences to geographic variation in polar bear (Ursus maritimus) contaminant levels is unknown. Dietary variation between Alaska, Canada, East Greenland, and Svalbard subpopulations was assessed by muscle nitrogen and carbon stable isotope (δ(15)N, δ(13)C) and adipose fatty acid (FA) signatures relative to their main prey (ringed seals). Western and southern Hudson Bay signatures were characterized by depleted δ(15)N and δ(13)C, lower proportions of C(20) and C(22) monounsaturated FAs and higher proportions of C(18) and longer chain polyunsaturated FAs. East Greenland and Svalbard signatures were reversed relative to Hudson Bay. Alaskan and Canadian Arctic signatures were intermediate. Between-subpopulation dietary differences predominated over interannual, seasonal, sex, or age variation. Among various brominated and chlorinated contaminants, diet signatures significantly explained variation in adipose levels of polybrominated diphenyl ether (PBDE) flame retardants (14-15%) and legacy PCBs (18-21%). However, dietary influence was contaminant class-specific, since only low or nonsignificant proportions of variation in organochlorine pesticide (e.g., chlordane) levels were explained by diet. Hudson Bay diet signatures were associated with lower PCB and PBDE levels, whereas East Greenland and Svalbard signatures were associated with higher levels. Understanding diet/food web factors is important to accurately interpret contaminant trends, particularly in a changing Arctic.

Fast carnivores and slow herbivores: differential foraging strategies among grizzly bears in the Canadian Arctic.

Categorizing animal populations by diet can mask important intrapopulation variation, which is crucial to understanding a species' trophic niche width. To test hypotheses related to intrapopulation variation in foraging or the presence of diet specialization, we conducted stable isotope analysis (δ(13)C, δ(15)N) on hair and claw samples from 51 grizzly bears (Ursus arctos) collected from 2003 to 2006 in the Mackenzie Delta region of the Canadian Arctic. We examined within-population differences in the foraging patterns of males and females and the relationship between trophic position (derived from δ(15)N measurements) and individual movement. The range of δ(15)N values in hair and claw (2.0-11.0‰) suggested a wide niche width and cluster analyses indicated the presence of three foraging groups within the population, ranging from near-complete herbivory to near-complete carnivory. We found no linear relationship between home range size and trophic position when the data were continuous or when grouped by foraging behavior. However, the movement rate of females increased linearly with trophic position. We used multisource dual-isotope mixing models to determine the relative contributions of seven prey sources within each foraging group for both males and females. The mean bear dietary endpoint across all foraging groups for each sex fell toward the center of the mixing polygon, which suggested relatively well-mixed diets. The primary dietary difference across foraging groups was the proportional contribution of herbaceous foods, which decreased for both males and females from 42-76 to 0-27% and 62-81 to 0-44%, respectively. Grizzlies of the Mackenzie Delta live in extremely harsh conditions and identifying within-population diet specialization has improved our understanding of varying habitat requirements within the population.

Spatial and temporal trends of selected trace elements in liver tissue from polar bears (Ursus maritimus) from Alaska, Canada and Greenland.

Spatial trends and comparative changes in time of selected trace elements were studied in liver tissue from polar bears from ten different subpopulation locations in Alaska, Canadian Arctic and East Greenland. For nine of the trace elements (As, Cd, Cu, Hg, Mn, Pb, Rb, Se and Zn) spatial trends were investigated in 136 specimens sampled during 2005-2008 from bears from these ten subpopulations. Concentrations of Hg, Se and As were highest in the (northern and southern) Beaufort Sea area and lowest in (western and southern) Hudson Bay area and Chukchi/Bering Sea. In contrast, concentrations of Cd showed an increasing trend from east to west. Minor or no spatial trends were observed for Cu, Mn, Rb and Zn. Spatial trends were in agreement with previous studies, possibly explained by natural phenomena. To assess temporal changes of Cd, Hg, Se and Zn concentrations during the last decades, we compared our results to previously published data. These time comparisons suggested recent Hg increase in East Greenland polar bears. This may be related to Hg emissions and/or climate-induced changes in Hg cycles or changes in the polar bear food web related to global warming. Also, Hg:Se molar ratio has increased in East Greenland polar bears, which suggests there may be an increased risk for Hg(2+)-mediated toxicity. Since the underlying reasons for spatial trends or changes in time of trace elements in the Arctic are still largely unknown, future studies should focus on the role of changing climate and trace metal emissions on geographical and temporal trends of trace elements.

Trichinella nativa and Trichinella T6 in arctic foxes (Vulpes lagopus) from northern Canada.

Parasitic zoonotic nematodes of the genus Trichinella circulate in wildlife and domestic hosts worldwide through the ingestion of infected meat. Due to their role as scavengers and predators in terrestrial and marine arctic ecosystems, Arctic foxes (Vulpes lagopus) are ideal sentinels for the detection of Trichinella spp. In this study, we determined the prevalence, larval intensity, and species of Trichinella from 91 trapped Arctic foxes collected around the northern Canadian communities of Sachs Harbour (Ikaahuk) on Banks Island (n = 23), and Ulukhaktok and Cambridge Bay (Ikaluktutiak) on Victoria Island (n = 68). Using pepsin-HCl digestion, larvae of Trichinella spp. were recovered from the left forelimb muscle (flexor carpi ulnaris) in 19 of the 91 foxes (21% prevalence, 95% CI: 14-30%). For the first time in Arctic foxes in Canada, Trichinella species were identified using multiplex PCR that was followed up with PCR-RFLP to distinguish between T. nativa and T. chanchalensis. All infected foxes harbored T. nativa, and one fox was co-infected with Trichinella T6; the latter is a new host record. Age of the fox was significantly associated with Trichinella spp. infection and the odds of being infected were three times higher in foxes ≥2 years of age (p = 0.026), indicating cumulative exposure with age. While Arctic foxes are seldom harvested for human consumption, they serve as sentinel hosts of Trichinella spp., confirming the presence of the parasite in wildlife in the region.

Canadian polar bear population structure using genome-wide markers.

Predicting the consequences of environmental changes, including human-mediated climate change on species, requires that we quantify range-wide patterns of genetic diversity and identify the ecological, environmental, and historical factors that have contributed to it. Here, we generate baseline data on polar bear population structure across most Canadian subpopulations (n = 358) using 13,488 genome-wide single nucleotide polymorphisms (SNPs) identified with double-digest restriction site-associated DNA sequencing (ddRAD). Our ddRAD dataset showed three genetic clusters in the sampled Canadian range, congruent with previous studies based on microsatellites across the same regions; however, due to a lack of sampling in Norwegian Bay, we were unable to confirm the existence of a unique cluster in that subpopulation. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range.

Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo).

Understanding parasite diversity and distribution is essential in managing the potential impact of parasitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the standard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single copy orthologs of nuclear DNA indicated a common ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeographically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13).

Risk factors and prevalence of antibodies for Toxoplasma gondii in diaphragmatic fluid in wolverines (Gulo gulo) from the Northwest Territories, Canada.

Toxoplasma gondii, a zoonotic food borne parasite that can infect almost all warm-blooded animals including people, and ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization (FAO/WHO, 2014). Exposure to T. gondii has been reported in wildlife and people in the Canadian North, despite low densities of feline definitive hosts. The ecology of this host-parasite system could be affected by changing climate and landscape in boreal and sub-Arctic regions, and surveillance data are critically needed. Wolverines are an economically and culturally important species in northern Canada due to their valuable fur. Fluid obtained from diaphragmatic muscle of 127 wolverines (Gulo gulo) were tested for antibodies to T. gondii using an enzyme linked immunosorbent assay (ELISA). A seroprevalence of 62% (Confidence Interval (CI): 53-71%) was observed. This result indicates high levels of exposure, likely either through environmental contamination with T. gondii oocysts shed by infected wild felids, or consumption of carcasses/offal of other intermediate hosts containing tissue cysts with bradyzoites in tissues. We examined factors associated with seropositivity, including age, sex, harvest location, harvest location with respect to treeline, and body condition index. Adult (≥2 years) wolverines had 5.2 times higher odds of being sero-positive than juvenile (<1 years) wolverines. The highest seroprevalence was observed in wolverines from Sahtu and South Slave regions. Proportion of sero-positive wolverines harvested above and below the tree line was not significantly different (60% vs 65%). Age was the only significant predictor of T. gondii exposure in wolverines (using logistic regression analysis); further studies should target larger sample sizes. This study is an example of how fluid from diaphragmatic muscle can be used for screening for T. gondii antibodies in wolverines. The diaphragm, commonly collected for screening for another food borne parasite, Trichinella, in wildlife harvested for human consumption, can be used for screening of T. gondii exposure in wildlife. Due to their predatory and scavenging lifestyle and high trophic level, wolverines could serve as a sentinel species for T. gondii.

Trichinella pseudospiralis in a wolverine (Gulo gulo) from the Canadian North.

Species of Trichinella are a globally distributed assemblage of nematodes, often with distinct host ranges, which include people, domestic, and wild animals. Trichinella spp. are important in northern Canada, where dietary habits of people and methods of meat preparation (drying, smoking, fermenting as well as raw) increase the risk posed by these foodborne zoonotic parasites. Outbreaks in the arctic and subarctic regions of Canada and the United States are generally attributed to T. nativa (T2) or the T6 genotype, when genetic characterization is performed. We report the discovery of Trichinella pseudospiralis (T4), a non-encapsulated species, in a wolverine (Gulo gulo) from the Northwest Territories of Canada. This parasite has been previously reported elsewhere from both mammals and carnivorous birds, but our findings represent new host and geographic records for T. pseudospiralis. Multiplex PCR and sequencing of fragments of Cytochrome Oxidase Subunit I (COI) and D3 rDNA confirmed the identification. Phylogenetically, Canadian isolates linked with each other and others derived from Palearctic or Neotropical regions, but not elsewhere in the Nearctic (continental USA). We suggest that migratory birds might have played a role in the dispersal of this pathogen 1000's of km to northwestern Canada. Wolverines are not typically consumed by humans, and thus should not pose a direct food safety risk for trichinellosis. However, the current finding suggests that they may serve as an indicator of a broader distribution for T. pseudospiralis. Along with infection risk already recognized for T. nativa and Trichinella T6, our observations emphasize the need for further studies using molecular diagnostics and alternative methods to clarify if this is a solitary case, or if T. pseudospiralis and other freeze susceptible species of Trichinella (such as T. spiralis) circulate more broadly in wildlife in Canada, and elsewhere.

A Transdisciplinary Approach to Brucella in Muskoxen of the Western Canadian Arctic 1989-2016.

Brucella serostatus was evaluated in 3189 muskoxen sampled between 1989 and 2016 from various locations of the Canadian Arctic archipelago and mainland, near the communities of Sachs Harbour and Ulukhaktok, Northwest Territories, and Cambridge Bay and Kugluktuk, Nunavut. Brucella antibodies were found only in muskoxen sampled around Cambridge Bay, both on southern Victoria Island and on the adjacent mainland (Kent Peninsula). Consistent with participatory epidemiology data documented from local harvesters describing increased Brucella-like syndromes (swollen joints and lameness) and a decreased proportion of juveniles, the apparent Brucella seroprevalence in the sampled muskoxen of the Cambridge Bay area increased from 0.9% (95% CI 0.3-2.1) in the period of 1989-2001 to 5.6% (95% CI 3.3-8.9) in 2010-2016. The zoonotic bacteria Brucella suis biovar 4 was also cultured from tissues of muskoxen sampled on Victoria Island near Ulukhaktok in 1996 (n = 1) and Cambridge Bay in 1998, 2014, and 2016 (n = 3). Overall, our data demonstrate that B. suis biovar 4 is found in muskoxen that are harvested for food and by guided hunts on Victoria Island and Kent Peninsula, adding an important public health dimension to this study. Robust participatory epidemiology data on muskox health and diseases greatly enhanced the interpretation of our Cambridge Bay data and, combined with the serological and microbiological data, provide compelling evidence that the prevalence of B. suis biovar 4 has increased in this area since the late 1990s. This study enhances the available knowledge on Brucella exposure and infection in muskoxen and provides an example of how scientific knowledge and local knowledge can work together to better understand disease status in wildlife.

Influence of carbon and lipid sources on variation of mercury and other trace elements in polar bears (Ursus maritimus).

In the present study, the authors investigated the influence of carbon and lipid sources on regional differences in liver trace element (As, Cd, Cu, total Hg, Mn, Pb, Rb, Se, and Zn) concentrations measured in polar bears (Ursus maritimus) (n = 121) from 10 Alaskan, Canadian Arctic, and East Greenland subpopulations. Carbon and lipid sources were assessed using δ(13) C in muscle tissue and fatty acid (FA) profiles in subcutaneous adipose tissue as chemical tracers. A negative relationship between total Hg and δ(13) C suggested that polar bears feeding in areas with higher riverine inputs of terrestrial carbon accumulate more Hg than bears feeding in areas with lower freshwater input. Mercury concentrations were also positively related to the FA 20:1n-9, which is biosynthesized in large amounts in Calanus copepods. This result raises the hypothesis that Calanus glacialis are an important link in the uptake of Hg in the marine food web and ultimately in polar bears. Unadjusted total Hg, Se, and As concentrations showed greater geographical variation among polar bear subpopulations compared with concentrations adjusted for carbon and lipid sources. The Hg concentrations adjusted for carbon and lipid sources in Bering-Chukchi Sea polar bear liver tissue remained the lowest among subpopulations. Based on these findings, the authors suggest that carbon and lipid sources for polar bears should be taken into account when one is assessing spatial and temporal trends of long-range transported trace elements.

Flame retardants and legacy contaminants in polar bears from Alaska, Canada, East Greenland and Svalbard, 2005-2008.

Flame retardants and legacy contaminants were analyzed in adipose tissue from 11 circumpolar polar bear (Ursus maritimus) subpopulations in 2005-2008 spanning Alaska east to Svalbard. Although 37 polybrominated diphenyl ethers (PBDEs), total-(α)-hexabromocyclododecane (HBCD), 2 polybrominated biphenyls (PBBs), pentabromotoluene, pentabromoethylbenzene, hexabromobenzene, 1,2-bis(2,4,6-tribromophenoxy(ethane) and decabromodiphenyl ethane were screened, only 4 PBDEs, total-(α-)HBCD and BB153 were consistently found. Geometric mean ΣPBDE (4.6-78.4 ng/g lipid weight (lw)) and BB153 (2.5-81.1 ng/g lw) levels were highest in East Greenland (43.2 and 39.2 ng/g lipid weight (lw), respectively), Svalbard (44.4 and 20.9 ng/g lw) and western (38.6 and 30.1 ng/g lw) and southern Hudson Bay (78.4 and 81.1 ng/g lw). Total-(α)-HBCD levels (<0.3-41.1 ng/g lw) were lower than ΣPBDE levels in all subpopulations except in Svalbard, consistent with greater European HBCD use versus North American pentaBDE product use. ΣPCB levels were high relative to flame retardants as well as other legacy contaminants and increased from west to east (1797-10,537 ng/g lw). ΣCHL levels were highest among legacy organochlorine pesticides and relatively spatially uniform (765-3477 ng/g lw). ΣDDT levels were relatively low and spatially variable (31.5-206 ng/g lw). However, elevated proportions of p,p'-DDT to ΣDDT in Alaska and Beaufort Sea relative to other subpopulations suggested fresh inputs from vector control use in Asia and/or Africa. Comparing earlier circumpolar polar bear studies, ΣPBDE, total-(α)-HBCD, p,p'-DDE and ΣCHL levels consistently declined, whereas levels of other legacy contaminants did not. International regulations have clearly been effective in reducing levels of several legacy contaminants in polar bears relative to historical levels. However, slow or stalling declines of certain historic pollutants like PCBs and a complex mixture of "new" chemicals continue to be of concern to polar bear health and that of their arctic marine ecosystems.