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.

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.

Not playing by the rules: Unusual patterns in the epidemiology of parasites in a natural population of feral horses (Equus caballus) on Sable Island, Canada.

Sable Island, Nova Scotia, Canada hosts one of few natural populations of feral horses (Equus caballus) never exposed to anthelmintics. Coproculture revealed cyathostomes, Strongylus equinus, S. edentatus, and S. vulgaris, with S. equinus (unusually) dominating in adult horses and cyathostomes dominating in young horses (<3 years of age). We examined 35 horses found dead in the springs of 2017 and 2018, as well as fecal samples from live horses in spring (n = 45) and summer 2018 (n = 236) using McMaster fecal flotation and Baermann larval sedimentation on fresh samples, and modified Wisconsin flotation and sucrose gradient immunofluorescent assay for Giardia and Cryptosporidium on frozen samples. Mean strongyle fecal egg counts were 666 eggs per gram (EPG) in dead horses, 689 EPG in live horses in spring, and 1105 EPG in summer; domestic horses are usually treated at counts exceeding 200 EPG. Adult horses (unusually) had patent infections with the lungworm Dictyocaulus arnfieldi and ascarids (Parascaris spp.), and in spring, dead horses had 5 times higher odds of having patent ascarid infections than live horses, likely due to malnutrition and corresponding immunodeficiency. Fecal prevalence and intensity of D. arnfieldi and Parascaris spp. were significantly higher in young horses, and in spring versus summer. A higher proportion of fecal samples were positive for strongyle and ascarid eggs using a centrifugal flotation technique on previously frozen feces, as compared to a passive flotation method on fresh feces. Eggs of the tapeworm Paranoplocephala mamillana were present in fecal samples from 28% of live, and 42% of dead, horses in spring. This research represents several new geographic records (S. edentatus, D. arnfieldi, and Eimeria leuckarti), provides insight into unusual patterns of parasite epidemiology in a nutrition-limited environment, and has conservation and biosecurity implications for this unique equine population, as well as for parasite management in domestic horses.