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Objective: Outcomes are poor for patients with congenital adrenal hyperplasia (CAH), in part due to the supraphysiological glucocorticoid doses required to control adrenal androgen excess. Hydrocortisone (ie, cortisol) is the recommended glucocorticoid for treatment of CAH. However, the other endogenous glucocorticoid in humans, corticosterone, is actively transported out of metabolic tissues such as adipose tissue and muscle, so we hypothesized that corticosterone could control adrenal androgens while causing fewer metabolic adverse effects than hydrocortisone., Methods: Thirteen patients (8 female, 5 male) with CAH due to 21-hydroxylase deficiency completed a randomized placebo-controlled crossover study comparing 5 h intravenous infusions of either hydrocortisone, corticosterone or placebo. 6-6[2H]2-glucose and 1,1,2,3,3-[2H]5-glycerol were infused to measure glucose and glycerol kinetics, and blood samples were collected throughout. Subcutaneous abdominal adipose tissue biopsies were obtained at the end of each infusion., Results: During the infusion, corticosterone and hydrocortisone similarly reduced ACTH, 17α-hydroxyprogesterone, androstenedione, and testosterone (in females only) compared with placebo. Despite achieving circulating corticosterone concentrations ∼2.5-fold higher than hydrocortisone, by T + 300 min hydrocortisone but not corticosterone increased glucose and insulin concentrations and reduced 6-6-[2H]2-glucose clearance compared with placebo. Hydrocortisone increased mRNA levels of the glucocorticoid regulated transcript PER1 in adipose to a greater extent than corticosterone., Conclusions: Corticosterone acutely controls biochemical markers of androgen excess similarly to hydrocortisone but without inducing markers of glucocorticoid "toxicity" in CAH. These data demonstrate proof of concept that corticosterone may be a safer glucocorticoid replacement than current medications, although further research is required to assess the longer-term effects of corticosterone replacement., Competing Interests: Conflict of interest: None of the authors have any conflicts to disclose., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Demand for bear bile, a prized component of traditional Asian medicines, threaten Asiatic and sun bear population sustainability. While laws exist to prevent poaching and trafficking of bear parts and derivatives, smuggling persists with demand extending to surrogate species, including American black bears (Ursus americanus). Mitochondrial DNA (mtDNA) sequencing can identify products putatively containing biological bear material but can be undermined by PCR inhibitors in bile and a lack of sensitivity at trace levels. Quantitative PCR (qPCR) assays can be used to distinguish between closely related target species, while concomitantly evaluating inhibition and false negative results in low quality/quantity DNA applications. Herein, we develop a multiplexed qPCR assay to detect and differentiate among bear species, including highly diluted bile samples mixed within liquors as common dilutants. The assay detects as little as 10 locus copies/reaction of bear DNA with 95% confidence, distinguishing among sun, Asiatic and American black bears. Demonstrating the sensitivity and applicability of this assay in context of current bile mixture recipes, dilutions of 1:5,000 bile with ethanol, red wine, and spirits, all yielded clear quantifiable detections, where our data suggests as little as 1 drop of bile per 750 mL bottle of alcohol would still exceed the limits of detection (e.g., 1:15000 dilution or <0.05 mL bile per 750 mL bottle). Overall, this study provides a rapid, sensitive, and specific test to identify and distinguish among bear species commonly used for bile production to aid wildlife enforcement applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Microbiome diversity and diet composition concomitantly influence species health, fitness, immunity, and digestion. In environments where diet varies spatially and temporally, microbiome plasticity may promote rapid host adaptation to available resources. For northern ungulates in particular, metabarcoding of noninvasively collected fecal pellets presents unprecedented insights into their diverse ecological requirements and niches by clarifying the interrelationships of microbiomes, key to deriving nutrients, in context of altered forage availability in changing climates. Muskoxen ( Ovibos moschatus ) are Arctic-adapted species that experience fluctuating qualities and quantities of vegetation. Geography and seasonality have been noted to influence microbiome composition and diversity in muskoxen, yet it is unclear how their microbiomes intersect with diet. Following observations from other species, we hypothesized increasing diet diversity would result in higher microbiome diversity in muskoxen. We assessed diet composition in muskoxen using three common plant metabarcoding markers and explored correlations with microbiome data. Patterns of dietary diversity and composition were not fully concordant among the markers used, yet all reflected the primary consumption of willows and sedges. Individuals with similar diets had more similar microbiomes, yet in contrast to most literature, yielded negative relationships between microbiome and diet alpha diversity. This negative correlation may reflect the unique capacities of muskoxen to survive solely on high-fiber Arctic forage and provide insight into their resiliency to exploit changing dietary resources in a rapidly warming Arctic altering vegetation diversity., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Southeastern Canada is inhabited by an amalgam of hybridizing wolf-like canids, raising fundamental questions regarding their taxonomy, origins, and timing of hybridization events. Eastern wolves (Canis lycaon), specifically, have been the subject of significant controversy, being viewed as either a distinct taxonomic entity of conservation concern or a recent hybrid of coyotes (C. latrans) and grey wolves (C. lupus). Mitochondrial DNA analyses show some evidence of eastern wolves being North American evolved canids. In contrast, nuclear genome studies indicate eastern wolves are best described as a hybrid entity, but with unclear timing of hybridization events. To test hypotheses related to these competing findings we sequenced whole genomes of 25 individuals, representative of extant Canadian wolf-like canid types of known origin and levels of contemporary hybridization. Here we present data describing eastern wolves as a distinct taxonomic entity that evolved separately from grey wolves for the past ∼67,000 years with an admixture event with coyotes ∼37,000 years ago. We show that Great Lakes wolves originated as a product of admixture between grey wolves and eastern wolves after the last glaciation (∼8,000 years ago) while eastern coyotes originated as a product of admixture between "western" coyotes and eastern wolves during the last century. Eastern wolf nuclear genomes appear shaped by historical and contemporary gene flow with grey wolves and coyotes, yet evolutionary uniqueness remains among eastern wolves currently inhabiting a restricted range in southeastern Canada., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Responses to hormones that act through nuclear receptors are controlled by modulating hormone concentrations not only in the circulation but also within target tissues. The role of enzymes that amplify or reduce local hormone concentrations is well established for glucocorticoid and other lipophilic hormones; moreover, transmembrane transporters have proven critical in determining tissue responses to thyroid hormones. However, there has been less consideration of the role of transmembrane transport for steroid hormones. ATP-binding cassette (ABC) proteins were first shown to influence the accumulation of glucocorticoids in cells almost three decades ago, but observations over the past 10 years suggest that differential transport propensities of both exogenous and endogenous glucocorticoids by ABCB1 and ABCC1 transporters provide a mechanism whereby different tissues are preferentially sensitive to different steroids. This Review summarizes this evidence and the new insights provided for the physiology and pharmacology of glucocorticoid action, including new approaches to glucocorticoid replacement., (© 2022. Springer Nature Limited.)

Muskoxen ( Ovibos moschatus ) are Arctic species within the Caprinae subfamily that are economically and culturally significant to northern Indigenous communities. Low genetic diversity from repeated genetic bottlenecks, coupled with the effects of Arctic warming (e.g., heat stress, changing forage, pathogen range expansions), present conservation concerns for this species. Reference genome assemblies enhance our ecological and evolutionary understanding of species (which in turn aid conservation efforts). Herein, we provide a full draft reference genome of muskox using Illumina Hiseq data and cross-species scaffolding. The final reference assembly yielded a genome of 2,621,890,883 bp in length, a scaffold N50 of ~13.2 million, and an annotation identifying ~19.3 k genes. The muskox genome assembly and annotation were then used to reconstruct a phylogenetic tree which estimated muskoxen diverged from other ungulate species~12 Mya. To gain insight into the demographic history of muskoxen we also performed pairwise sequentially Markovian coalescent (PSMC) that identified two population bottlenecks coinciding with major glaciation events contributing to the notoriously low genetic variation observed in muskoxen. Overall, this genome assembly provides a foundation for future population genomic studies, such as latitudinal analyses, to explore the capacity of muskoxen to adapt to rapidly changing environments.

Background: Cortisol and corticosterone both circulate in human plasma and, due to differing export by ATP-binding cassette (ABC) transporters, may exert differential cellular effects. ABCB1 (expressed in brain) exports cortisol not corticosterone while ABCC1 (expressed in adipose and skeletal muscle) exports corticosterone not cortisol. We hypothesised that ABCC1 inhibition increases corticosteroid receptor occupancy by corticosterone but not cortisol in humans., Methods: A randomised double-blind crossover study was conducted in 14 healthy men comparing placebo and ABCC1 inhibitor probenecid. Blood sampling, including from veins draining adipose and muscle, was undertaken before and after administration of mineralocorticoid receptor antagonist potassium canrenoate and glucocorticoid receptor antagonist mifepristone (RU486)., Results: During placebo, systemic plasma cortisol and corticosterone concentrations increased promptly after canrenoate. Cortisol uptake was detected from adipose but not muscle following canrenoate + RU486. Probenecid significantly increased systemic cortisol concentrations, and tended to increase corticosterone and ACTH concentrations, after combined receptor antagonism but had no effects on net glucocorticoid balance in either adipose or muscle. Using quantitative PCR in brain bank tissue, ABCC1 expression was 5-fold higher in human pituitary than hypothalamus and hippocampus. ABCB1 was more highly expressed in hypothalamus compared to pituitary., Conclusions: Although displacement of corticosterone and/or cortisol from receptors in adipose and skeletal muscle could not be measured with sufficient precision to detect effects of probenecid, ABCC1 inhibition induced a greater incremental activation of the hypothalamic-pituitary-adrenal axis after combined receptor blockade, consistent with ABCC1 exporting corticosterone from the pituitary and adding to the evidence that ABC transporters modulate tissue glucocorticoid sensitivity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest that is relevant to the subject matter or materials included in this work., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Skin is a key aspect of the immune system in the defence against pathogens. Skin pH regulates the activity of enzymes produced both by hosts and by microbes on host skin, thus implicating pH in disease susceptibility. Skin pH varies inter- and intra-specifically and is influenced by a variety of intrinsic and extrinsic variables. Increased skin alkalinity is associated with a predisposition to cutaneous infections in humans and dogs, and inter-specific and inter-individual variation in skin pH is implicated in differential susceptibility to some skin diseases. The cutaneous pH of bats has not been characterized but is postulated to play a role in susceptibility to white-nose syndrome (WNS), a fungal infection that has decimated several Nearctic bat species. We used non-invasive probes to measure the pH of bat flight membranes in five species with differing susceptibility to WNS. Skin pH ranged from 4.67 to 8.59 and varied among bat species, geographic locations, body parts, age classes, sexes and seasons. Wild Eptesicus fuscus were consistently more acidic than wild Myotis lucifugus, Myotis leibii and Perimyotis subflavus . Juvenile bats had more acidic skin than adults during maternity season but did not differ during swarming. Male M. lucifugus were more acidic than females during maternity season, yet this trend reversed during swarming. Bat skin was more acidic in summer compared to winter, a pattern also reported in humans. Skin pH was more acidic in captive than wild E. fuscus , suggesting environmental impacts on skin pH. The pH of roosting substrates affects skin pH in captive bats and may partially explain seasonal patterns in wild bats that use different roost types across seasons. Future research on the influence of pH on microbial pathogenic factors and skin barrier function may provide valuable insights on new therapeutic targets for treating bat skin conditions., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

Patterns of local adaptation can emerge in response to the selective pressures diseases exert on host populations as reflected in increased frequencies of respective, advantageous genotypes. Elucidating patterns of local adaptation enhance our understanding of mechanisms of disease spread and the capacity for species to adapt in context of rapidly changing environments such as the Arctic. Arctic rabies is a lethal disease that largely persists in northern climates and overlaps with the distribution of its natural host, arctic fox. Arctic fox populations display little neutral genetic structure across their North American range, whereas phylogenetically unique arctic rabies variants are restricted in their geographic distributions. It remains unknown if arctic rabies variants impose differential selection upon host populations, nor what role different rabies variants play in the maintenance and spread of this disease. Using a targeted, genotyping-by-sequencing assay, we assessed correlations of arctic fox immunogenetic variation with arctic rabies variants to gain further insight into the epidemiology of this disease. Corroborating past research, we found no neutral genetic structure between sampled regions, but did find moderate immunogenetic structuring between foxes predominated by different arctic rabies variants. FST outliers associated with host immunogenetic structure included SNPs within interleukin and Toll-like receptor coding regions (IL12B, IL5, TLR3 and NFKB1); genes known to mediate host responses to rabies. While these data do not necessarily reflect causation, nor a direct link to arctic rabies, the contrasting genetic structure of immunologically associated candidate genes with neutral loci is suggestive of differential selection and patterns of local adaptation in this system. These data are somewhat unexpected given the long-lived nature and dispersal capacities of arctic fox; traits expected to undermine local adaptation. Overall, these data contribute to our understanding of the co-evolutionary relationships between arctic rabies and their primary host and provide data relevant to the management of this disease., Competing Interests: The authors have declared that no competing interests exist.

Populations are exposed to different types and strains of pathogens across heterogeneous landscapes, where local interactions between host and pathogen may present reciprocal selective forces leading to correlated patterns of spatial genetic structure. Understanding these coevolutionary patterns provides insight into mechanisms of disease spread and maintenance. Arctic rabies (AR) is a lethal disease with viral variants that occupy distinct geographic distributions across North America and Europe. Red fox (Vulpes vulpes) are a highly susceptible AR host, whose range overlaps both geographically distinct AR strains and regions where AR is absent. It is unclear if genetic structure exists among red fox populations relative to the presence/absence of AR or the spatial distribution of AR variants. Acquiring these data may enhance our understanding of the role of red fox in AR maintenance/spread and inform disease control strategies. Using a genotyping-by-sequencing assay targeting 116 genomic regions of immunogenetic relevance, we screened for sequence variation among red fox populations from Alaska and an outgroup from Ontario, including areas with different AR variants, and regions where the disease was absent. Presumed neutral SNP data from the assay found negligible levels of neutral genetic structure among Alaskan populations. The immunogenetically-associated data identified 30 outlier SNPs supporting weak to moderate genetic structure between regions with and without AR in Alaska. The outliers included SNPs with the potential to cause missense mutations within several toll-like receptor genes that have been associated with AR outcome. In contrast, there was a lack of genetic structure between regions with different AR variants. Combined, we interpret these data to suggest red fox populations respond differently to the presence of AR, but not AR variants. This research increases our understanding of AR dynamics in the Arctic, where host/disease patterns are undergoing flux in a rapidly changing Arctic landscape, including the continued northward expansion of red fox into regions previously predominated by the arctic fox (Vulpes lagopus)., Competing Interests: The authors have declared that no competing interests exist.

Understanding how context (e.g., host species, environmental conditions) drives disease susceptibility is an essential goal of disease ecology. We hypothesized that in bat white-nose syndrome (WNS), species-specific host-pathogen interactions may partly explain varying disease outcomes among host species. We characterized bat and pathogen transcriptomes in paired samples of lesion-positive and lesion-negative wing tissue from bats infected with Pseudogymnoascus destructans in three parallel experiments. The first two experiments analyzed samples collected from the susceptible Nearctic Myotis lucifugus and the less-susceptible Nearctic Eptesicus fuscus , following experimental infection and hibernation in captivity under controlled conditions. The third experiment applied the same analyses to paired samples from infected, free-ranging Myotis myotis , a less susceptible, Palearctic species, following natural infection and hibernation (n = 8 sample pairs/species). Gene expression by P. destructans was similar among the three host species despite varying environmental conditions among the three experiments and was similar within each host species between saprophytic contexts (superficial growth on wings) and pathogenic contexts (growth in lesions on the same wings). In contrast, we observed qualitative variation in host response: M. lucifugus and M. myotis exhibited systemic responses to infection, while E. fuscus up-regulated a remarkably localized response. Our results suggest potential phylogenetic determinants of response to WNS and can inform further studies of context-dependent host-pathogen interactions.

Context: GDF15 is a stress-induced hormone acting in the hindbrain that activates neural circuitry involved in establishing aversive responses and reducing food intake and body weight in animal models. Anorexia, weight loss, nausea and vomiting are common manifestations of glucocorticoid deficiency, and we hypothesized that glucocorticoid deficiency may be associated with elevated levels of GDF15., Objective: To determine the impact of primary adrenal insufficiency (PAI) and glucocorticoid replacement on circulating GDF15 levels., Methods and Results: We measured circulating concentrations of GDF15 in a cohort of healthy volunteers and Addison's disease patients following steroid withdrawal. Significantly higher GDF15 (mean ± standard deviation [SD]) was observed in the Addison's cohort, 739.1 ± 225.8 pg/mL compared to healthy controls, 497.9 ± 167.7 pg/mL (P = 0.01). The effect of hydrocortisone replacement on GDF15 was assessed in 3 independent PAI cohorts with classical congenital adrenal hyperplasia or Addison's disease; intravenous hydrocortisone replacement reduced GDF15 in all groups. We examined the response of GDF15 to increasing doses of glucocorticoid replacement in healthy volunteers with pharmacologically mediated cortisol deficiency. A dose-dependent difference in GDF15 (mean ± SD) was observed between the groups with values of 491.0 ± 157.7 pg/mL, 427.0 ± 152.1 pg/mL and 360 ± 143.1 pg/mL, in the low, medium and high glucocorticoid replacement groups, respectively, P < .0001., Conclusions: GDF15 is increased in states of glucocorticoid deficiency and restored by glucocorticoid replacement. Given the site of action of GDF15 in the hindbrain and its effects on appetite, further study is required to determine the effect of GDF15 in mediating the anorexia and nausea that is a common feature of glucocorticoid deficiency., (© Endocrine Society 2019.)

Ranaviruses have been associated with chelonian mortality. In Canada, the first two cases of ranavirus were detected in turtles in 2018 in Ontario, although a subsequent survey of its prevalence failed to detect additional positive cases. To confirm the prevalence of ranavirus in turtles in Ontario, we used a more sensitive method to investigate if lower level persistent infection was present in the population. Here we report results via a combination of qPCR, PCR, Sanger sequencing and genome sequencing from turtles from across Ontario, with no clinical signs of illness. We found 2 positives with high viral load and 5 positives with low viral load. Histopathology found subtle histological changes. DNA sequences identified two types of frog virus 3 (FV3), and genome sequencing identified a ranavirus similar to wild-type FV3. Our results show that the virus has been present in Ontario's turtles as subclinical infections., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sue Jacqueline Carstairs is employed as the Executive and Medical Director of the Ontario Turtle Conservation Centre (Kawartha Turtle Trauma Centre). The other authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There is a recognized disconnect in priority and synergy between academic and practitioners in forensic science. In this work, we personally reflect on our experiences in conducting research studies that directly involve academic and practitioner stakeholders. We believe, amongst others, that this "gap" can be mitigated through regular and productive communication. We also emphasize the need to create stronger and national research strategies which identifies the current and pressing needs of enforcement officials in order to bring these needs directly to academia. As part of this, researchers should actively seek to make sure what they study will be relevant within the discipline. Our reflection is geared on direct feedback from an entomological study in large scale sampling of blowflies and workshops in bloodstain pattern analysis using a forensic blood substitute., (Copyright © 2019 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

The Canadian Arctic is an extreme environment with low floral and faunal diversity characterized by major seasonal shifts in temperature, moisture, and daylight. Muskoxen ( Ovibos moschatus ) are one of few large herbivores able to survive this harsh environment. Microbiome research of the gastrointestinal tract may hold clues as to how muskoxen exist in the Arctic, but also how this species may respond to rapid environmental changes. In this study, we investigated the effects of season (spring/summer/winter), year (2007-2016), and host genetic structure on population-level microbiome variation in muskoxen from the Canadian Arctic. We utilized 16S rRNA gene sequencing to characterize the fecal microbial communities of 78 male muskoxen encompassing two population genetic clusters. These clusters are defined by Arctic Mainland and Island populations, including the following: (a) two mainland sampling locations of the Northwest Territories and Nunavut and (b) four locations of Victoria Island. Between these geographic populations, we found that differences in the microbiome reflected host-associated genetic cluster with evidence of migration. Within populations, seasonality influenced bacterial diversity with no significant differences between years of sampling. We found evidence of pathogenic bacteria, with significantly higher presence in mainland samples. Our findings demonstrate the effects of seasonality and the role of host population-level structure in driving fecal microbiome differences in a large Arctic mammal., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTVs have been reported only in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within open reading frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (<100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread. IMPORTANCE Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade., (Copyright © 2019 American Society for Microbiology.)

The measurement of insects is an important component of many entomological applications, including forensic evidence, where larvae size is used as a proxy for developmental stage, and hence time since colonization/death. Current methods for measuring insects are confounded by varying preservation techniques, biased and non-standardized measurements, and often a lack of sample size given practical constraints. Towards enhanced accuracy and precision in measuring live insects to help avoid these variables, and that allows for different measurements to be analyzed, we developed a non-invasive, digital method using widely available free analytical software to measure live blow fly larvae. Using crime scene photographic equipment currently standard in investigation protocols, we measured the live length of 282 Phormia regina larvae. Repeated measurements of maggots, for all instars, were performed for several orientations and images. Most accurate measurements were obtained when maggots were oriented in their natural full extension. Killed specimens resulted in greater length measurements (Mean 1.79 ± 1.11 mm) when compared to live length. Herein, we report a technically simple, fast, and accurate measurement technique adapted for field and lab-based measurements, as well as, a simple linear equation for conversion of live length to standard killed length measurements. We propose this method be utilized for the standardization of forensic entomological evidence collection and development model creation., (© 2019 The Authors.)

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Blow fly (Diptera: Calliphoridae) larvae are commonly used in forensic cases to determine postmortem intervals using development rates and successional changes in community composition. Studies are conducted from different regions to provide these data. We wanted to know how widely applicable these data are. We examined whether urbanized landscapes have distinct urban blow fly communities or whether the community composition in urbanized areas is simply a variation of that found in the surrounding habitat or ecozone. Using liver baited traps, we sampled 7,272 flies from 32 sites across Canada and used mapping analysis to assess urban and rural landcover classifications, and compared urban and rural species abundance and composition. Blow fly species communities from urban areas across Canada were made up of similar species and differed from the communities found in nearby rural sites. Trapping at rural sites caught more blow flies compared with urban sites (mean flies/site 59.5 and 12.4). Of the 14 species caught, 8 were caught at urban sites, 61% of these being Cynomya cadaverina Robineau-Desvoidy, 14% Phormia regina Meigen, and 11% Lucilia sericata (Meigen). In rural sites, all 14 species were caught, 41% of specimens caught were P. regina, 21% C. cadaverina, 10% Calliphora vomitoria (Linnaeus), with only 4% L. sericata. These data suggest that regional studies are appropriate for forensic entomology applications in urban landscapes, given the similar trends across Canada, less so for wilderness or rural landscapes., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Frog virus 3 (FV3) and FV3-like ranaviruses can infect a variety of cold-blooded aquatic species and present a primary threat to amphibians across the globe. Previous studies of FV3-like viruses have largely investigated higher-level phylogenetic distinctions of these pathogens via portions of the conserved major capsid protein (MCP), and the putative virulence gene vIF-2α. Few studies, however, have investigated the spatial distribution of FV3 variants at the population level3-data that can be used to further understand the spatial epidemiology of this disease. In this study, we sequenced the MCP and vIF-2α of 127 FV3-positive amphibians sampled from Canadian water bodies in Ontario, northeastern Alberta, and southern Northwest Territories to explore whether intraspecific genetic variation exists within FV3. There was a lack of variation at the 2 markers across these regions, suggesting that there is a lack of FV3 sequence diversity in Canada, which may hint at a single source of infection that has spread. However, an undocumented variant termed Wood Buffalo ranavirus (WBRV) was detected in samples from 3 sites in Alberta and Northwest Territories that clustered within the FV3-like lineage with 99.3% sequence homology for MCP. For vIF-2α, all sequences were the expected truncated variant except for 6 samples in Ontario. These latter sequences were suggestive of recombination with common midwife toad virus (CMTV). The lack of variation suggests that higher-resolution genome analyses will be required to further explore the spatial spread and intraspecific variation of the disease.

Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.

Current understanding of in vivo human brown adipose tissue (BAT) physiology is limited by a reliance on positron emission tomography (PET)/computed tomography (CT) scanning, which has measured exogenous glucose and fatty acid uptake but not quantified endogenous substrate utilization by BAT. Six lean, healthy men underwent 18 fluorodeoxyglucose-PET/CT scanning to localize BAT so microdialysis catheters could be inserted in supraclavicular BAT under CT guidance and in abdominal subcutaneous white adipose tissue (WAT). Arterial and dialysate samples were collected during warm (∼25°C) and cold exposure (∼17°C), and blood flow was measured by 133 xenon washout. During warm conditions, there was increased glucose uptake and lactate release and decreased glycerol release by BAT compared with WAT. Cold exposure increased blood flow, glycerol release, and glucose and glutamate uptake only by BAT. This novel use of microdialysis reveals that human BAT is metabolically active during warm conditions. BAT activation substantially increases local lipolysis but also utilization of other substrates such as glutamate., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Linked article : https://doi.org/10.1002/ece3.4034.

Pseudogymnoascus destructans is the causal agent of bat white-nose syndrome (WNS), which is devastating some North American bat populations. Previous transcriptome studies provided insight regarding the molecular mechanisms involved in WNS; however, it is unclear how different environmental parameters could influence pathogenicity. This information could be useful in developing management strategies to mitigate the negative impacts of P. destructans on bats. We cultured three P. destructans isolates from Atlantic Canada on two growth media (potato dextrose agar and Sabouraud dextrose agar) that differ in their nitrogen source, and at two separate incubation temperatures (4 C and 15 C) that approximate the temperature range of bat hibernacula during the winter and a temperature within its optimal mycelial growth range. We conducted RNA sequencing to determine transcript levels in each sample and performed differential gene expression (DGE) analyses to test the influence of growth medium and incubation temperature on gene expression. We also compared our in vitro results with previous RNA-sequencing data sets generated from P. destructans growing on the wings of a susceptible host, Myotis lucifugus. Our findings point to a critical role for substrate and incubation temperature in influencing the P. destructans transcriptome. DGE analyses suggested that growth medium plays a larger role than temperature in determining P. destructans gene expression and that although the psychrophilic fungus responds to different nitrogen sources, it may have evolved for continued growth at a broad range of low temperatures. Further, our data suggest that down-regulation of the RNA-interference pathway and increased fatty acid metabolism are involved in the P. destructans-bat interaction. Finally, we speculate that to reduce the activation of host defense responses, P. destructans minimizes changes in the expression of genes encoding secreted proteins during bat colonization.

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing (HTS) technologies, such as genotype-by-sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization-based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to "capture" specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300-kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on-target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer-read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune-related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.

White-nose syndrome (WNS) has devastated populations of hibernating bats in eastern North America, leading to emergency conservation listings for several species including the previously ubiquitous little brown myotis ( Myotis lucifugus ). However, some bat populations near the epicenter of the WNS panzootic appear to be stabilizing after initial precipitous declines, which could reflect a selective immunogenetic sweep. To investigate the hypothesis that WNS exerts significant selection on the immunome of affected bat populations, we developed a novel, high-throughput sequence capture assay targeting 138 adaptive, intrinsic, and innate immunity genes of putative adaptive significance, as well as their respective regulatory regions (~370 kbp of genomic sequence/individual). We used the assay to explore baseline immunogenetic variation in M. lucifugus and to investigate whether particular immune genes/variants are associated with WNS susceptibility. We also used our assay to detect 1,038 putatively neutral single nucleotide polymorphisms and characterize contemporary population structure, providing context for the identification of local immunogenetic adaptation. Sequence capture provided a cost-effective, "all-in-one" assay to test for neutral genetic and immunogenetic structure and revealed fine-scale, baseline immunogenetic differentiation between sampling sites <600 km apart. We identified functional immunogenetic variants in M. lucifugus associated with WNS susceptibility. This study lays the foundations for future investigations of rangewide immunogenetic adaptation to WNS in M. lucifugus and provides a blueprint for studies of evolutionary rescue in other host-pathogen systems.

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.

Mitigation of emerging infectious diseases that threaten global biodiversity requires an understanding of critical host and pathogen responses to infection. For multihost pathogens where pathogen virulence or host susceptibility is variable, host-pathogen interactions in tolerant species may identify potential avenues for adaptive evolution in recently exposed, susceptible hosts. For example, the fungus Pseudogymnoascus destructans causes white-nose syndrome (WNS) in hibernating bats and is responsible for catastrophic declines in some species in North America, where it was recently introduced. Bats in Europe and Asia, where the pathogen is endemic, are only mildly affected. Different environmental conditions among Nearctic and Palearctic hibernacula have been proposed as an explanation for variable disease outcomes, but this hypothesis has not been experimentally tested. We report the first controlled, experimental investigation of response to P. destructans in a tolerant, European species of bat (the greater mouse-eared bat, Myotis myotis ). We compared body condition, disease outcomes and gene expression in control (sham-exposed) and exposed M. myotis that hibernated under controlled environmental conditions following treatment. Tolerant M. myotis experienced extremely limited fungal growth and did not exhibit symptoms of WNS. However, we detected no differential expression of genes associated with immune response in exposed bats, indicating that immune response does not drive tolerance of P. destructans in late hibernation. Variable responses to P. destructans among bat species cannot be attributed solely to environmental or ecological factors. Instead, our results implicate coevolution with the pathogen, and highlight the dynamic nature of the "white-nose syndrome transcriptome." Interspecific variation in response to exposure by the host (and possibly pathogen) emphasizes the importance of context in studies of the bat-WNS system, and robust characterization of genetic responses to exposure in various hosts and the pathogen should precede any attempts to use particular bat species as generalizable "model hosts."

Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and Phormia regina (Meigen) (Diptera: Calliphoridae) are morphologically similar blow fly species commonly used for estimating postmortem intervals. Field collection and storage of adults can result in color changes, in particular on calypters and palps; often collected specimens show damage such as wing fray or fungal growth. We measured the frons width: total head width ratio using photographs (ImageJ version 1.49) to differentiate these two species. Both sexes were distinguishable to species, with the greatest difference between males: 12.34% P. terraenovae versus 1.62% P. regina, less so for females: 40.25% P. terraenovae, versus 33.65% P. regina. Incorporating this feature into future blow fly keys would help with distinguishing field-caught specimens when other features are obstructed., (© 2016 American Academy of Forensic Sciences.)

The processes leading to genetic isolation influence a population's local extinction risk, and should thus be identified before conservation actions are implemented. Natural or human-induced circumstances can result in historical or contemporary barriers to gene flow and/or demographic bottlenecks. Distinguishing between these hypotheses can be achieved by comparing genetic diversity and differentiation in isolated vs. continuous neighboring populations. In Ontario, American black bears (Ursus americanus) are continuously distributed, genetically diverse, and exhibit an isolation-by-distance structuring pattern, except on the Bruce Peninsula (BP). To identify the processes that led to the genetic isolation of BP black bears, we modelled various levels of historical and contemporary migration and population size reductions using forward simulations. We compared simulation results with empirical genetic indices from Ontario black bear populations under different levels of geographic isolation, and conducted additional simulations to determine if translocations could help achieve genetic restoration. From a genetic standpoint, conservation concerns for BP black bears are warranted because our results show that: i) a recent demographic bottleneck associated with recently reduced migration best explains the low genetic diversity on the BP; and ii) under sustained isolation, BP black bears could lose between 70% and 80% of their rare alleles within 100 years. Although restoring migration corridors would be the most effective method to enhance long-term genetic diversity and prevent inbreeding, it is unrealistic to expect connectivity to be re-established. Current levels of genetic diversity could be maintained by successfully translocating 10 bears onto the peninsula every 5 years. Such regular translocations may be more practical than landscape restoration, because areas connecting the peninsula to nearby mainland black bear populations have been irreversibly modified by humans, and form strong barriers to movement.

American badgers ( Taxidea taxus jacksoni) at the periphery of the species' range in Ontario, Canada, are listed as endangered because of an estimated population size of <200 mature individuals. The main threats faced by this population include habitat loss and road mortality. However, on 18 November 2013, a radio-implanted badger was found nonresponsive in an agricultural field with signs consistent with canine distemper virus infection, which was subsequently confirmed. This prompted our investigation into the occurrence of pathogens in this endangered carnivore to better quantify the level of risk infectious disease poses to population persistence. We examined serum samples from nine live-trapped individuals and 27 whole badger specimens submitted for postmortem examination. We found evidence of exposure to canine distemper virus, canine parvovirus, and leptospires. However, infection associated with disease was not the leading cause of mortality. Future research into the effects of disease on kit survival and a comprehensive understanding of disease severity and spread from reservoir populations (e.g., raccoons [ Procyon lotor ] and striped skunks [ Mephitis mephitis ]) to badgers will be of particular importance to the conservation of this endangered population.

Small and isolated populations often exhibit low genetic diversity due to drift and inbreeding, but may simultaneously harbour adaptive variation. We investigate spatial distributions of immunogenetic variation in American badger subspecies ( Taxidea taxus ), as a proxy for evaluating their evolutionary potential across the northern extent of the species' range. We compared genetic structure of 20 microsatellites and the major histocompatibility complex (MHC DRB exon 2) to evaluate whether small, isolated populations show low adaptive polymorphism relative to large and well-connected populations. Our results suggest that gene flow plays a prominent role in shaping MHC polymorphism across large spatial scales, while the interplay between gene flow and selection was stronger towards the northern peripheries. The similarity of MHC alleles within subspecies relative to their neutral genetic differentiation suggests that adaptive divergence among subspecies can be maintained despite ongoing gene flow along subspecies boundaries. Neutral genetic diversity was low in small relative to large populations, but MHC diversity within individuals was high in small populations. Despite reduced neutral genetic variation, small and isolated populations harbour functional variation that likely contribute to the species evolutionary potential at the northern range. Our findings suggest that conservation approaches should focus on managing adaptive variation across the species range rather than protecting subspecies per se.

Complete panmixia across the entire range of a species is a relatively rare phenomenon; however, this pattern may be found in species that have limited philopatry and frequent dispersal. American white pelicans (Pelecanus erythrorhyncos) provide a unique opportunity to examine the role of long-distance dispersal in facilitating gene flow in a species recently reported as panmictic across its broad breeding range. This species is also undergoing a range expansion, with new colonies arising hundreds of kilometers outside previous range boundaries. In this study, we use a multiple stable isotope (δ2H, δ13C, δ15N) approach to examine feather isotopic structuring at 19 pelican colonies across North America, with the goal of establishing an isotopic basemap that could be used for assigning individuals at newly established breeding sites to source colonies. Within-colony isotopic variation was extremely high, exceeding 100‰ in δ2H within some colonies (with relatively high variation also observed for δ13C and δ15N). The high degree of within-site variation greatly limited the utility of assignment-based approaches (42% cross-validation success rate; range: 0-90% success). Furthermore, clustering algorithms identified four likely isotopic clusters; however, those clusters were generally unrelated to geographic location. Taken together, the high degree of within-site isotopic variation and lack of geographically-defined isotopic clusters preclude the establishment of an isotopic basemap for American white pelicans, but may indicate that a high incidence of long-distance dispersal is facilitating gene flow, leading to genetic panmixia.

Elucidating the adaptive genetic potential of wildlife populations to environmental selective pressures is fundamental for species conservation. Genes of the major histocompatibility complex (MHC) are highly polymorphic, and play a key role in the adaptive immune response against pathogens. MHC polymorphism has been linked to balancing selection or heterogeneous selection promoting local adaptation. However, spatial patterns of MHC polymorphism are also influenced by gene flow and drift. Wolverines are highly vagile, inhabiting varied ecoregions that include boreal forest, taiga, tundra, and high alpine ecosystems. Here, we investigated the immunogenetic variation of wolverines in Canada as a surrogate for identifying local adaptation by contrasting the genetic structure at MHC relative to the structure at 11 neutral microsatellites to account for gene flow and drift. Evidence of historical positive selection was detected at MHC using maximum likelihood codon-based methods. Bayesian and multivariate cluster analyses revealed weaker population genetic differentiation at MHC relative to the increasing microsatellite genetic structure towards the eastern wolverine distribution. Mantel correlations of MHC against geographical distances showed no pattern of isolation by distance (IBD: r = -0.03, p = 0.9), whereas for microsatellites we found a relatively strong and significant IBD (r = 0.54, p = 0.01). Moreover, we found a significant correlation between microsatellite allelic richness and the mean number of MHC alleles, but we did not observe low MHC diversity in small populations. Overall these results suggest that MHC polymorphism has been influenced primarily by balancing selection and to a lesser extent by neutral processes such as genetic drift, with no clear evidence for local adaptation. This study contributes to our understanding of how vulnerable populations of wolverines may respond to selective pressures across their range.

Forensic entomology involves the use of insects and arthropods to assist a spectrum of medico-criminal investigations that range from identifying cases of abuse, corpse movements, and most commonly, post mortem interval estimates. Many of these applications focus on the use of blowflies given their predicable life history characteristics in their larval stages. Molecular tools have become increasingly important in the unambiguous identification of larval blowfly species, however, these same tools have the potential to broaden the array of molecular applications in forensic entomology to include individual identifications and population assignments. Herein, we establish a microsatellite profiling system for the blowfly, Phormiaregina (Diptera: Calliphoridae). The goal being to create a system to identify the population genetic structure of this species and subsequently establish if these data are amenable to identifying corpse movements based on the geographic distribution of specific genetic clusters of blowflies. Using next generation sequencing technology, we screened a partial genomic DNA sequence library of P.regina, searching for di-, tetra-, and penta-nucleotide microsatellite loci. We identified and developed primers for 84 highly repetitive segments of DNA, of which 14 revealed consistent genotypes and reasonable levels of genetic variation (4-26 alleles/locus; heterozygosity ranged from 0.385 to 0.909). This study provides the first step in assessing the utility of microsatellite markers to track the movements and sources of corpses via blowflies., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Local adaptation is necessary for population survival and depends on the interplay between responses to selective forces and demographic processes that introduce or retain adaptive and maladaptive attributes. Host-parasite systems are dynamic, varying in space and time, where both host and parasites must adapt to their ever-changing environment in order to survive. We investigated patterns of local adaptation in raccoon populations with varying temporal exposure to the raccoon rabies virus (RRV). RRV infects approximately 85% of the population when epizootic and has been presumed to be completely lethal once contracted; however, disease challenge experiments and varying spatial patterns of RRV spread suggest some level of immunity may exist. We first assessed patterns of local adaptation in raccoon populations along the eastern seaboard of North America by contrasting spatial patterns of neutral (microsatellite loci) and functional, major histocompatibility complex (MHC) genetic diversity and structure. We explored variation of MHC allele frequencies in the light of temporal population exposure to RRV (0-60 years) and specific RRV strains in infected raccoons. Our results revealed high levels of MHC variation (66 DRB exon 2 alleles) and pronounced genetic structure relative to neutral microsatellite loci, indicative of local adaptation. We found a positive association linking MHC genetic diversity and temporal RRV exposure, but no association with susceptibility and resistance to RRV strains. These results have implications for landscape epidemiology studies seeking to predict the spread of RRV and present an example of how population demographics influence the degree to which populations adapt to local selective pressures., (© 2014 John Wiley & Sons Ltd.)

Background: Frog virus 3 (FV3) is the type species of the genus Ranavirus, and in the past few decades, FV3 infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia. The reasons for the pathogenicity of FV3 are not well understood., Findings: We investigated three FV3 isolates designated SSME, wt-FV3, and aza-Cr, and reported that our wt-FV3 and aza-Cr strains showed similar levels of virulence, while SSME was the least virulent in an in vivo study with Lithiobates pipiens tadpoles. Using 454 GS-FLX sequencing technology, we sequenced SSME and compared it to the published wt-FV3 genome. SSME had multiple amino acid deletions in ORFs 49/50L, 65L, 66L, and 87L, which may explain its reduced virulence. We also investigated repeat regions and found that repeat copy number differed between isolates, with only one group of 3 isolates and 1 pair of isolates being identical at all 3 locations., Conclusions: In this study we have shown that genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select repeat locations. These genomic areas with deletions/insertions may represent regions that affect virulence, and therefore require investigation. Furthermore, we have identified repeat regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions.

Biogeochemical markers in ecology have provided a useful means for indicating geographic origin and movement patterns of species on various temporal and spatial scales. We used trace element analysis to resolve spatial and habitat-specific environmental gradients in elemental distributions that could be used to infer geographic origin and habitat association in a model terrestrial carnivore: American badger (Taxidea taxus jacksoni). To accomplish this, we generated element base-maps using spatial principal component analysis, and assessed habitat-specific signatures using multivariate statistics from soil element concentrations in southwestern Ontario, Canada. Using canonical correlation analysis (CCA) we also test whether element variability in the claw keratin of a terrestrial carnivore could be explained by the chemical variability in the soils of the local environment. Results demonstrated that trace element signatures in soils vary locally with land use practices and soil texture type and broadly with the underlying geology. CCA results suggest that chemical profiles in claws can be linked to the surrounding chemical environment, providing evidence that geographic patterns in mammalian movement can be discerned on the basis of claw chemistry. From this, we conclude that geographic assignment of individuals based on element profiles in their tissues and referenced against soil elemental distributions would be coarse (at a spatial scale of 100-1000 km, depending on the chemical heterogeneity of the landscape), but could be used to assess origin of highly mobile animals or habitat association of individuals. Compared to stable isotope analysis, the assessment of trace elements can provide a much greater level of detail in backcasting animal movement pathways., (© 2013. Published by Elsevier B.V. All rights reserved.)

Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular reservoir host. Active management of these host vectors is needed to minimize the negative impacts of this disease, and an understanding of the immune response to RV infection aids strategies for host vaccination. Current knowledge of immune responses to RV infection comes primarily from rodent models in which an innate immune response triggers activation of several genes and signalling pathways. It is unclear, however, how well rodent models represent the immune response of natural hosts. This study investigates the innate immune response of a primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The extent and temporal course of this response during RRV infection was analysed using genes predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor-3; TNF receptor). We found that RRV activated components of the innate immune system, with changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that natural reservoirs of rabies may not mimic the immune response triggered in rodent models, highlighting the need for further studies of infection in primary hosts.

Interglacial-glacial cycles of the Quaternary are widely recognized in shaping phylogeographic structure. Patterns from cold adapted species can be especially informative - in particular, uncovering additional glacial refugia, identifying likely recolonization patterns, and increasing our understanding of species' responses to climate change. We investigated phylogenetic structure of the wolverine, a wide-ranging cold adapted carnivore, using a 318 bp of the mitochondrial DNA control region for 983 wolverines (n=209 this study, n=774 from GenBank) from across their full Holarctic distribution. Bayesian phylogenetic tree reconstruction and the distribution of observed pairwise haplotype differences (mismatch distribution) provided evidence of a single rapid population expansion across the wolverine's Holarctic range. Even though molecular evidence corroborated a single refugium, significant subdivisions of population genetic structure (0.01< ΦST <0.99, P<0.05) were detected. Pairwise ΦST estimates separated Scandinavia from Russia and Mongolia, and identified five main divisions within North America - the Central Arctic, a western region, an eastern region consisting of Ontario and Quebec/Labrador, Manitoba, and California. These data are in contrast to the nearly panmictic structure observed in northwestern North America using nuclear microsatellites, but largely support the nuclear DNA separation of contemporary Manitoba and Ontario wolverines from northern populations. Historic samples (c. 1900) from the functionally extirpated eastern population of Quebec/Labrador displayed genetic similarities to contemporary Ontario wolverines. To understand these divergence patterns, four hypotheses were tested using Approximate Bayesian Computation (ABC). The most supported hypothesis was a single Beringia incursion during the last glacial maximum that established the northwestern population, followed by a west-to-east colonization during the Holocene. This pattern is suggestive of colonization occurring in accordance with glacial retreat, and supports expansion from a single refugium. These data are significant relative to current discussions on the conservation status of this species across its range.

ONRAB is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB in captive animals, we sought to examine persistence and shedding of ONRAB in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB vaccine baits. Samples were screened for the presence and quantity of ONRAB DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB greater than 10(3)EU/mL of ONRAB DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB in animals is short-lived and the likelihood of horizontal transmission to other organisms is low., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species., (© 2012 Blackwell Publishing Ltd.)

Variation within major histocompatibility complex (MHC) genes is important in recognizing pathogens and initiating an immune response. These genes are relevant in enhancing our understanding of how species cope with rapid environmental changes and concomitant fluctuations in selective pressures such as invasive, infectious diseases. Disease-based models suggest that diversity at MHC is maintained through balancing selection arising from the coevolution of hosts and pathogens. Despite intensive balancing selection, sequence motifs or even identical MHC alleles can be shared across multiple species; three potential mechanisms have been put forth to explain this phenomenon: common ancestry, convergent evolution, and random chance. To understand the processes that maintain MHC similarity across divergent species, we examined the variation at two orthologous MHC-DRB genes in widespread North American Musteloid species, striped skunks (Mephitis mephitis), and raccoons (Procyon lotor). These species are often sympatric and exposed to a similar suite of diseases (e.g., rabies, canine distemper, and parvovirus). Given their exposure to similar selective pressures from pathogens, we postulated that similar DRB alleles may be present in both species. Our results indicated that similar motifs are present within both species, at functionally relevant polymorphic sites. However, based on phylogenetic analyses that included previously published MHC sequences of several closely related carnivores, the respective MHC-DRB alleles do not appear to have been maintained through common ancestry and unlikely through random chance. Instead, the similarities observed between the two mesocarnivore species may rather be due to evolutionary convergence.

We examined the mitochondrial genetic structure of American white pelicans (Pelecanus erythrorhynchos) to: 1) verify or refute whether American white pelicans are panmictic and 2) understand if any lack of genetic structure is the result of contemporary processes or historical phenomena. Sequence analysis of mitochondrial DNA control region haplotypes of 367 individuals from 19 colonies located across their North American range revealed a lack of population genetic or phylogeographic structure. This lack of structure was unexpected because: 1) Major geographic barriers such as the North American Continental Divide are thought to limit dispersal; 2) Differences in migratory behavior are expected to promote population differentiation; and 3) Many widespread North American migratory bird species show historic patterns of differentiation resulting from having inhabited multiple glacial refugia. Further, high haplotype diversity and many rare haplotypes are maintained across the species' distribution, despite frequent local extinctions and recolonizations that are expected to decrease diversity. Our findings suggest that American white pelicans have a high effective population size and low natal philopatry. We suggest that the rangewide panmixia we observed in American white pelicans is due to high historical and contemporary gene flow, enabled by high mobility and a lack of effective physical or behavioral barriers.

In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

The major histocompatibility complex (MHC) presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor). Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus) and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250 bp) and DRB exon 2 (228 bp). MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4-15.8% divergence) and translated into 1 to 21 (1.3-27.6% divergence) amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005), indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host.