Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska

For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine‐scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.     

Concluding Remarks

Rabies seems to persist throughout most arctic regions, and the northern parts of Norway, Sweden and Finland, is the only part of the Arctic where rabies has not been diagnosed in recent time. The arctic fox is the main host, and the same arctic virus variant seems to infect the arctic fox throughout the range of this species. The epidemiology of rabies seems to have certain common characteristics in arctic regions, but main questions such as the maintenance and spread of the disease remains largely unknown. The virus has spread and initiated new epidemics also in other species such as the red fox and the racoon dog. Large land areas and cold climate complicate the control of the disease, but experimental oral vaccination of arctic foxes has been successful. This article summarises the current knowledge and the typical characteristics of arctic rabies including its distribution and epidemiology.     

Combining genome‐wide association study and FST‐based approaches to identify targets of Borrelia‐mediated selection in natural rodent hosts

Recent advances in high‐throughput sequencing technologies provide opportunities to gain novel insights into the genetic basis of phenotypic trait variation. Yet to date, progress in our understanding of genotype–phenotype associations in nonmodel organisms in general and natural vertebrate populations in particular has been hampered by small sample sizes typically available for wildlife populations and a resulting lack of statistical power, as well as a limited ability to control for false‐positive signals. Here we propose to combine a genome‐wide association study (GWAS) and F_ST‐based approach with population‐level replication to partly overcome these limitations. We present a case study in which we used this approach in combination with genotyping‐by‐sequencing (GBS) single nucleotide polymorphism (SNP) data to identify genomic regions associated with Borrelia afzelii resistance or susceptibility in the natural rodent host of this Lyme disease‐causing spirochete, the bank vole (Myodes glareolus). Using this combined approach we identified four consensus SNPs located in exonic regions of the genes Slc26a4, Tns3, Wscd1 and Espnl, which were significantly associated with the voles’ Borrelia infectious status within and across populations. Functional links between host responses to bacterial infections and most of these genes have previously been demonstrated in other rodent systems, making them promising new candidates for the study of evolutionary host responses to Borrelia emergence. Our approach is applicable to other systems and may facilitate the identification of genetic variants underlying disease resistance or susceptibility, as well as other ecologically relevant traits, in wildlife populations.     

High‐throughput microsatellite genotyping in ecology: improved accuracy,efficiency, standardization and success with low‐quantity and degraded DNA

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high‐throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR‐amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low‐quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

A survey of single nucleotide polymorphisms identified from whole‐genome sequencing and their functional effect in the porcine genome,

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole‐genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire–Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss‐of‐function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss‐of‐function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses.     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

Haplotype‐based genotyping‐by‐sequencing in oat genome research

In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.     

Origins of the rabies viruses associated with an outbreak in Newfoundland during 2002-2003

After being free of rabies of terrestrial mammals since 1988, an outbreak of rabies occurred on the Island of Newfoundland in December 2002 and continued into the middle of 2003. Twenty-one cases, all due to the arctic fox strain of rabies virus, were reported. To explore the immediate origins of this outbreak, viruses from the Newfoundland epizootic were genetically compared to two other rabies viruses recovered in mid-2002 from Cartwright, a mainland coastal community near the Island. While all Island isolates from the 2002-03 outbreak were genetically very similar, consistent with a single introduction from the mainland, they were phylogenetically quite distant from the two samples from Cartwright. A broader-based study examined the relationships between the Island viruses and arctic fox strain viruses originating from across Canada over a period of 14 yr. This analysis indicated that the Newfoundland outbreak viruses were most similar to a variant identified in Labrador in 2004 but also widely distributed in northern Quebec both before and after the Newfoundland incursion. The eastern coastline of mainland Labrador has harbored a particularly large number of variants during the study period, some of which have not been detected elsewhere. A small number of Greenland isolates included in this study were dispersed within the clades of the Canadian samples rather than forming a discrete cluster, an observation that may underline the relative ease of movement of the rabies arctic lineage between these two countries as a result of animal movements over pack ice.     

The fluctuating world of a tundra predator guild: bottom‐up constraints overrule top‐down species interactions in winter

Global warming is predicted to change ecosystem functioning and structure in Arctic ecosystems by strengthening top‐down species interactions, i.e. predation pressure on small herbivores and interference between predators. Yet, previous research is biased towards the summer season. Due to greater abiotic constraints, Arctic ecosystem characteristics might be more pronounced in winter. Here we test the hypothesis that top‐down species interactions prevail over bottom‐up effects in Scandinavian mountain tundra (Northern Sweden) where effects of climate warming have been observed and top‐down interactions are expected to strengthen. But we test this ‘a priori’ hypothesis in winter and throughout the 3–4 yr rodent cycle, which imposes additional pulsed resource constraints. We used snowtracking data recorded in 12 winters (2004–2015) to analyse the spatial patterns of a tundra predator guild (arctic fox Vulpes lagopus, red fox Vulpes vulpes, wolverine Gulo gulo) and small prey (ptarmigan, Lagopus spp). The a priori top‐down hypothesis was then tested through structural equation modelling, for each phase of the rodent cycle. There was weak support for this hypothesis, with top‐down effects only discerned on arctic fox (weakly, by wolverine) and ptarmigan (by arctic fox) at intermediate and high rodent availability respectively. Overall, bottom‐up constraints appeared more influential on the winter community structure. Cold specialist predators (arctic fox and wolverine) showed variable landscape associations, while the boreal predator (red fox) appeared strongly dependent on productive habitats and ptarmigan abundance. Thus, we suggest that the unpredictability of food resources determines the winter ecology of the cold specialist predators, while the boreal predator relies on resource‐rich habitats. The constraints imposed by winters and temporary resource lows should therefore counteract productivity‐driven ecosystem change and have a stabilising effect on community structure. Hence, the interplay between summer and winter conditions should determine the rate of Arctic ecosystem change in the context of global warming.     

MHC class II DRB diversity in raccoons (<Emphasis Type="Italic">Procyon lotor</Emphasis>) reveals associations with raccoon rabies virus (Lyssavirus)

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.     

Sialic acid‐dependent interaction of group B streptococci with influenza virus‐infected cells reveals a novel adherence and invasion mechanism

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3‐linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3‐linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co‐sedimentation assay revealed that viruses with a preference for α2,3‐linked sialic acids bind to GBS in a sialic acid‐dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination‐inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre‐infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co‐infected by influenza viruses. This co‐infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3‐linked sialic acids and human viruses binding to this linkage type.     

Detection of individual ploidy levels with genotyping‐by‐sequencing (GBS) analysis

Ploidy levels sometimes vary among individuals or populations, particularly in plants. When such variation exists, accurate determination of cytotype can inform studies of ecology or trait variation and is required for population genetic analyses. Here, we propose and evaluate a statistical approach for distinguishing low‐level ploidy variants (e.g. diploids, triploids and tetraploids) based on genotyping‐by‐sequencing (GBS) data. The method infers cytotypes based on observed heterozygosity and the ratio of DNA sequences containing different alleles at thousands of heterozygous SNPs (i.e. allelic ratios). Whereas the method does not require prior information on ploidy, a reference set of samples with known ploidy can be included in the analysis if it is available. We explore the power and limitations of this method using simulated data sets and GBS data from natural populations of aspen (Populus tremuloides) known to include both diploid and triploid individuals. The proposed method was able to reliably discriminate among diploids, triploids and tetraploids in simulated data sets, and this was true for different levels of genetic diversity, inbreeding and population structure. Power and accuracy were minimally affected by low coverage (i.e. 2×), but did sometimes suffer when simulated mixtures of diploids, autotetraploids and allotetraploids were analysed. Cytotype assignments based on the proposed method closely matched those from previous microsatellite and flow cytometry data when applied to GBS data from aspen. An R package (gbs2ploidy) implementing the proposed method is available from CRAN.     

Elimination of rabies from red foxes in eastern Ontario

The province of Ontario (Canada) reported more laboratory confirmed rabid animals than any other state or province in Canada or the USA from 1958-91, with the exception of 1960-62. More than 95% of those cases occurred in the southern 10% of Ontario (approximately 100,000 km2), the region with the highest human population density and greatest agricultural activity. Rabies posed an expensive threat to human health and significant costs to the agricultural economy. The rabies variant originated in arctic foxes: the main vector in southern Ontario was the red fox (Vulpes vulpes), with lesser involvement of the striped skunk (Mephitis mephitis). The Ontario Ministry of Natural Resources began a 5 yr experiment in 1989 to eliminate terrestrial rabies from a approximately 30,000 km2 study area in the eastern end of southern Ontario. Baits containing oral rabies vaccine were dropped annually in the study area at a density of 20 baits/km2 from 1989-95. That continued 2 yr beyond the original 5 yr plan. The experiment was successful in eliminating the arctic fox variant of rabies from the whole area. In the 1980's, an average of 235 rabid foxes per year were reported in the study area. None have been reported since 1993. Cases of fox rabies in other species also disappeared. In 1995, the last bovine and companion animal cases were reported and in 1996 the last rabid skunk occurred. Only bat variants of rabies were present until 1999, when the raccoon variant entered from New York (USA). The success of this experiment led to an expansion of the program to all of southern Ontario in 1994. Persistence of terrestrial rabies, and ease of elimination, appeared to vary geographically, and probably over time. Ecological factors which enhance or reduce the long term survival of rabies in wild foxes are poorly understood.     

Modeling control of rabies outbreaks in red fox populations to evaluate culling,vaccination, and vaccination combined with fertility control

A predictive model of spread and control of rabies in red fox (Vulpes vulpes) populations was used to evaluate efficacy of culling, oral vaccination, and oral vaccination and fertility control (V + FC) as rabies control strategies. In addition, effects of season, fox population density, and a delay in starting control were modeled. At fox densities of 0.5 fox families/km2 or greater, a single oral vaccination campaign with bait uptake rates of less than 50% resulted in ineffective rabies control. An uptake rate of at least 80% was required to give a better than 80% chance of eliminating rabies. Vaccination was least effective at controlling rabies if applied 1 or 2 mo before the foxes gave birth. Seasonal timing of poison or V + FC had little effect on efficacy, which was always more successful than the oral vaccination alone. The longer the delay between the simulated start of the rabies infection and the application of a single vaccination campaign, the less successful was the control, particularly at the higher fox densities tested. At a fox density of 0.25 families/km2, all the strategies were equally successful at eliminating rabies. At higher fox densities V + FC was slightly less successful than culling, whereas vaccination-only was considerably less successful. The sole use of vaccination is not considered a viable control method for areas with high fox densities. The model suggests that an area of culling centered on the disease focus, plus an outer ring of vaccine or V + FC, could be the best strategy to control a point-source wildlife rabies outbreak.     

Raccoons (Procyon lotor) in Germany as potential reservoir species for Lyssaviruses

Raccoons can be found almost everywhere in Germany since their first successful introduction in 1934. Although the animal is a well-known reservoir species for rabies in the USA, during the last European fox rabies epizootic, only a few rabid raccoons were reported from Germany. In recent years, the raccoon population density has increased tremendously, especially in (semi) urban settings. Presently, Germany is free of terrestrial wildlife rabies. To assess the potential risk that the raccoon population in Germany could act as a reservoir species upon reemergence of rabies, the susceptibility of the local raccoon population was investigated. Wild-caught animals were inoculated with the most likely lyssavirus variants to infect the local population. It was shown that the raccoons were fully susceptible for a dog and raccoon rabies virus isolate. Also, five of six raccoons inoculated with a fox rabies virus isolate showed clinical signs. However, none of the raccoons infected with European Bat Lyssavirus type 1 succumbed to rabies; meanwhile, all these raccoons seroconverted. It is concluded that the highest risk for the raccoon population in Germany to become infected with lyssaviruses is through the importation of rabies infected dogs.     

Arctic Fox Responses to Tourism Activity

As the interest for nature-based tourism activities increases, it is important to provide evidence-based guidelines for wildlife-human interactions to minimize the disturbance caused to wildlife. In Fennoscandia, the endangered arctic fox (Vulpes lagopus) is subject to increasing tourism interest and some regions recommend a minimum approach distance of 300 m, but the guidelines have not been scientifically validated. We conducted experimental human approaches towards arctic fox den sites to study activity and behavioral changes in response to the approaching observer. The first arctic foxes hid when approached within 300 m, but many had increased their vigilance already at the start distance of 500 m. At approximately 200 m, the hiding probability increased rapidly at dens disturbed and undisturbed by tourism activities. Arctic foxes at disturbed dens allowed the observer to approach more closely before they increased their vigilance and before they hid compared to foxes at undisturbed dens. We confirm that a minimum distance of 300 m might be sufficient for most arctic foxes to refrain from hiding, but a longer distance would be required to avoid causing any disturbance. We recommend a minimum approach distance of ≥300 m to be implemented in all Fennoscandian regions inhabited by the arctic fox. © 2020 The Wildlife Society.     

A versatile Rapture (RAD‐Capture) platform for genotyping marine turtles

Advances in high‐throughput sequencing (HTS) technologies coupled with increased interdisciplinary collaboration are rapidly expanding capacity in the scope and scale of wildlife genetic studies. While existing HTS methods can be directly applied to address some evolutionary and ecological questions, certain research goals necessitate tailoring methods to specific study organisms, such as high‐throughput genotyping of the same loci that are comparable over large spatial and temporal scales. These needs are particularly common for studies of highly mobile species of conservation concern like marine turtles, where life history traits, limited financial resources and other constraints require affordable, adaptable methods for HTS genotyping to meet a variety of study goals. Here, we present a versatile marine turtle HTS targeted enrichment platform adapted from the recently developed Rapture (RAD‐Capture) method specifically designed to meet these research needs. Our results demonstrate consistent enrichment of targeted regions throughout the genome and discovery of candidate variants in all species examined for use in various conservation genetics applications. Accurate species identification confirmed the ability of our platform to genotype over 1,000 multiplexed samples and identified areas for future methodological improvement such as optimization for low initial concentration samples. Finally, analyses within green turtles supported the ability of this platform to identify informative SNPs for stock structure, population assignment and other applications over a broad geographic range of interest to management. This platform provides an additional tool for marine turtle genetic studies and broadens capacity for future large‐scale initiatives such as collaborative global marine turtle genetic databases.