Natural selection on feralization genes contributed to the invasive spread of wild pigs throughout the United States.

Despite a long presence in the contiguous United States (US), the distribution of invasive wild pigs (Sus scrofa × domesticus) has expanded rapidly since the 1980s, suggesting a more recent evolutionary shift towards greater invasiveness. Contemporary populations of wild pigs represent exoferal hybrid descendants of domestic pigs and European wild boar, with such hybridization expected to enrich genetic diversity and increase the adaptive potential of populations. Our objective was to characterize how genetic enrichment through hybridization increases the invasiveness of populations by identifying signals of selection and the ancestral origins of selected loci. Our study focused on invasive wild pigs within Great Smoky Mountains National Park, which represents a hybrid population descendent from the admixture of established populations of feral pigs and an introduction of European wild boar to North America. Accordingly, we genotyped 881 wild pigs with multiple high-density single-nucleotide polymorphism (SNP) arrays. We found 233 markers under putative selection spread over 79 regions across 16 out of 18 autosomes, which contained genes involved in traits affecting feralization. Among these, genes were found to be related to skull formation and neurogenesis, with two genes, TYRP1 and TYR, also encoding for crucial melanogenesis enzymes. The most common haplotypes associated with regions under selection for the Great Smoky Mountains population were also common among other populations throughout the region, indicating a key role of putatively selective variants in the fitness of invasive populations. Interestingly, many of these haplotypes were absent among European wild boar reference genotypes, indicating feralization through genetic adaptation.

African swine fever at the wildlife-livestock interface: challenges for management and outbreak response within invasive wild pigs in the United States.

African swine fever (ASF) causes significant morbidity and mortality in both domestic and wild suids (Sus scrofa), and disease outbreaks convey profound economic costs to impacted industries due to death loss, the cost of culling exposed/infected animals as the primary disease control measure, and trade restrictions. The co-occurrence of domestic and wild suids significantly complicates ASF management given the potential for wild populations to serve as persistent sources for spillover. We describe the unique threat of African swine fever virus (ASFV) introduction to the United States from epidemiological and ecological perspectives with a specific focus on disease management at the wild-domestic swine interface. The introduction of ASF into domestic herds would require a response focused on containment, culling, and contact tracing. However, detection of ASF among invasive wild pigs would require a far more complex and intensive response given the challenges of detection, containment, and ultimately elimination among wild populations. We describe the state of the science available to inform preparations for an ASF response among invasive wild pigs, describe knowledge gaps and the associated studies needed to fill those gaps, and call for an integrated approach for preparedness that incorporates the best available science and acknowledges sociological attributes and the policy context needed for an integrated disease response.

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa).

Pseudorabies virus (PRV)-the causative agent of Aujeszky's disease-was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk-based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA-SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

Variance in offspring sex ratio and maternal allocation in a highly invasive mammal.

Skewed sex ratios at birth are widely reported in wild populations, however, the extent to which parents are able to modulate the sex ratio of offspring to maximize their own fitness remains unclear. This is particularly true for highly polytocous species as maximizing fitness may include trade-offs between sex ratio and the size and number of offspring in litters. In such cases, it may be adaptive for mothers to adjust both the number of offspring per litter and offspring sex to maximize individual fitness. Investigating maternal sex allocation in wild pigs (Sus scrofa) under stochastic environmental conditions, we predicted that under favorable conditions, high-quality mothers (larger and older) would produce male-biased litters and invest more in producing larger litters with more males. We also predicted sex ratio would vary relative to litter size, with a male-bias among smaller litters. We found evidence that increasing wild boar ancestry, maternal age and condition, and resource availability may weakly contribute to male-biased sex ratio, however, unknown factors not measured in this study are assumed to be more influential. High-quality mothers allocated more resources to litter production, but this relationship was driven by adjustment of litter size, not sex ratio. There was no relationship between sex ratio and litter size. Collectively, our results emphasized that adjustment of litter size appeared to be the primary reproductive characteristic manipulated in wild pigs to increase fitness rather than adjustment of offspring sex ratio.

A novel SNP assay reveals increased genetic variability and abundance following translocations to a remnant Allegheny woodrat population.

BACKGROUND: Allegheny woodrats (Neotoma magister) are found in metapopulations distributed throughout the Interior Highlands and Appalachia. Historically these metapopulations persisted as relatively fluid networks, enabling gene flow between subpopulations and recolonization of formerly extirpated regions. However, over the past 45 years, the abundance of Allegheny woodrats has declined throughout the species' range due to a combination of habitat destruction, declining hard mast availability, and roundworm parasitism. In an effort to initiate genetic rescue of a small, genetically depauperate subpopulation in New Jersey, woodrats were translocated from a genetically robust population in Pennsylvania (PA) in 2015, 2016 and 2017. Herein, we assess the efficacy of these translocations to restore genetic diversity within the recipient population. RESULTS: We designed a novel 134 single nucleotide polymorphism panel, which was used to genotype the six woodrats translocated from PA and 82 individuals from the NJ population captured before and after the translocation events. These data indicated that a minimum of two translocated individuals successfully produced at least 13 offspring, who reproduced as well. Further, population-wide observed heterozygosity rose substantially following the first set of translocations, reached levels comparable to that of populations in Indiana and Ohio, and remained elevated over the subsequent years. Abundance also increased during the monitoring period, suggesting Pennsylvania translocations initiated genetic rescue of the New Jersey population. CONCLUSIONS: Our results indicate, encouragingly, that very small numbers of translocated individuals can successfully restore the genetic diversity of a threatened population. Our work also highlights the challenges of managing very small populations, such as when translocated individuals have greater reproductive success relative to residents. Finally, we note that ongoing work with Allegheny woodrats may broadly shape our understanding of genetic rescue within metapopulations and across heterogeneous landscapes.

Optimizing management of invasions in an uncertain world using dynamic spatial models.

Dispersal drives invasion dynamics of nonnative species and pathogens. Applying knowledge of dispersal to optimize the management of invasions can mean the difference between a failed and a successful control program and dramatically improve the return on investment of control efforts. A common approach to identifying optimal management solutions for invasions is to optimize dynamic spatial models that incorporate dispersal. Optimizing these spatial models can be very challenging because the interaction of time, space, and uncertainty rapidly amplifies the number of dimensions being considered. Addressing such problems requires advances in and the integration of techniques from multiple fields, including ecology, decision analysis, bioeconomics, natural resource management, and optimization. By synthesizing recent advances from these diverse fields, we provide a workflow for applying ecological theory to advance optimal management science and highlight priorities for optimizing the control of invasions. One of the striking gaps we identify is the extremely limited consideration of dispersal uncertainty in optimal management frameworks, even though dispersal estimates are highly uncertain and greatly influence invasion outcomes. In addition, optimization frameworks rarely consider multiple types of uncertainty (we describe five major types) and their interrelationships. Thus, feedbacks from management or other sources that could magnify uncertainty in dispersal are rarely considered. Incorporating uncertainty is crucial for improving transparency in decision risks and identifying optimal management strategies. We discuss gaps and solutions to the challenges of optimization using dynamic spatial models to increase the practical application of these important tools and improve the consistency and robustness of management recommendations for invasions.

Loci Associated With Antibody Response in Feral Swine (Sus scrofa) Infected With Brucella suis.

Feral swine (Sus scrofa) are a destructive invasive species widespread throughout the United States that disrupt ecosystems, damage crops, and carry pathogens of concern for the health of domestic stock and humans including Brucella suis-the causative organism for swine brucellosis. In domestic swine, brucellosis results in reproductive failure due to abortions and infertility. Contact with infected feral swine poses spillover risks to domestic pigs as well as humans, companion animals, wildlife, and other livestock. Genetic factors influence the outcome of infectious diseases; therefore, genome wide association studies (GWAS) of differential immune responses among feral swine can provide an understanding of disease dynamics and inform management to prevent the spillover of brucellosis from feral swine to domestic pigs. We sought to identify loci associated with differential antibody responses among feral swine naturally infected with B. suis using a case-control GWAS. Tissue, serum, and genotype data (68,516 bi-allelic single nucleotide polymorphisms) collected from 47 feral swine were analyzed in this study. The 47 feral swine were culture positive for Brucella spp. Of these 47, 16 were antibody positive (cases) whereas 31 were antibody negative (controls). Single-locus GWAS were performed using efficient mixed-model association eXpedited (EMMAX) methodology with three genetic models: additive, dominant, and recessive. Eight loci associated with seroconversion were identified on chromosome 4, 8, 9, 10, 12, and 18. Subsequent bioinformatic analyses revealed nine putative candidate genes related to immune function, most notably phagocytosis and induction of an inflammatory response. Identified loci and putative candidate genes may play an important role in host immune responses to B. suis infection, characterized by a detectable bacterial presence yet a differential antibody response. Given that antibody tests are used to evaluate brucellosis infection in domestic pigs and for disease surveillance in invasive feral swine, additional studies are needed to fully understand the genetic component of the response to B. suis infection and to more effectively translate estimates of Brucella spp. antibody prevalence among feral swine to disease control management action.

Optimal spatial prioritization of control resources for elimination of invasive species under demographic uncertainty.

Populations of invasive species often spread heterogeneously across a landscape, consisting of local populations that cluster in space but are connected by dispersal. A fundamental dilemma for invasive species control is how to optimally allocate limited fiscal resources across local populations. Theoretical work based on perfect knowledge of demographic connectivity suggests that targeting local populations from which migrants originate (sources) can be optimal. However, demographic processes such as abundance and dispersal can be highly uncertain, and the relationship between local population density and damage costs (damage function) is rarely known. We used a metapopulation model to understand how budget and uncertainty in abundance, connectivity, and the damage function, together impact return on investment (ROI) for optimal control strategies. Budget, observational uncertainty, and the damage function had strong effects on the optimal resource allocation strategy. Uncertainty in dispersal probability was the least important determinant of ROI. The damage function determined which resource prioritization strategy was optimal when connectivity was symmetric but not when it was asymmetric. When connectivity was asymmetric, prioritizing source populations had a higher ROI than allocating effort equally across local populations, regardless of the damage function, but uncertainty in connectivity structure and abundance reduced ROI of the optimal prioritization strategy by 57% on average depending on the control budget. With low budgets (monthly removal rate of 6.7% of population), there was little advantage to prioritizing resources, especially when connectivity was high or symmetric, and observational uncertainty had only minor effects on ROI. Allotting funding for improved monitoring appeared to be most important when budgets were moderate (monthly removal of 13-20% of the population). Our result showed that multiple sources of observational uncertainty should be considered concurrently for optimizing ROI. Accurate estimates of connectivity direction and abundance were more important than accurate estimates of dispersal rates. Developing cost-effective surveillance methods to reduce observational uncertainties, and quantitative frameworks for determining how resources should be spatially apportioned to multiple monitoring and control activities are important and challenging future directions for optimizing ROI for invasive species control programs.

Mixed ancestry from wild and domestic lineages contributes to the rapid expansion of invasive feral swine.

Invasive alien species are a significant threat to both economic and ecological systems. Identifying the processes that give rise to invasive populations is essential for implementing effective control strategies. We conducted an ancestry analysis of invasive feral swine (Sus scrofa, Linnaeus, 1758), a highly destructive ungulate that is widely distributed throughout the contiguous United States, to describe introduction pathways, sources of newly emergent populations and processes contributing to an ongoing invasion. Comparisons of high-density single nucleotide polymorphism genotypes for 6,566 invasive feral swine to a comprehensive reference set of S. scrofa revealed that the vast majority of feral swine were of mixed ancestry, with dominant genetic associations to Western heritage breeds of domestic pig and European populations of wild boar. Further, the rapid expansion of invasive feral swine over the past 30 years was attributable to secondary introductions from established populations of admixed ancestry as opposed to direct introductions of domestic breeds or wild boar. Spatially widespread genetic associations of invasive feral swine to European wild boar deviated strongly from historical S. scrofa introduction pressure, which was largely restricted to domestic pigs with infrequent, localized wild boar releases. The deviation between historical introduction pressure and contemporary genetic ancestry suggests wild boar-hybridization may contribute to differential fitness in the environment and heightened invasive potential for individuals of admixed domestic pig-wild boar ancestry.

Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa).

Wild pigs (Sus scrofa) are an invasive species descended from both domestic swine and Eurasian wild boar that was introduced to North America during the early 1500s. Wild pigs have since become the most abundant free-ranging exotic ungulate in the United States. Large and ever-increasing populations of wild pigs negatively impact agriculture, sport hunting, and native ecosystems with costs estimated to exceed $1.5 billion/year within the United States. Wild pigs are recognized as generalist feeders, able to exploit a broad array of locally available food resources, yet their feeding behaviors remain poorly understood as partially digested material is often unidentifiable through traditional stomach content analyses. To overcome the limitation of stomach content analyses, we developed a DNA sequencing-based protocol to describe the plant and animal diet composition of wild pigs. Additionally, we developed and evaluated blocking primers to reduce the amplification and sequencing of host DNA, thus providing greater returns of sequences from diet items. We demonstrate that the use of blocking primers produces significantly more sequencing reads per sample from diet items, which increases the robustness of ascertaining animal diet composition with molecular tools. Further, we show that the overall plant and animal diet composition is significantly different between the three areas sampled, demonstrating this approach is suitable for describing differences in diet composition among the locations.

Mitochondrial genome of an Allegheny Woodrat (Neotoma magister).

The Allegheny woodrat (Neotoma magister) is endemic to the eastern United States. Population numbers have decreased rapidly over the last four decades due to habitat fragmentation, disease-related mortality, genetic isolation and inbreeding depression; however, effective management is hampered by limited genetic resources. To begin addressing this need, we sequenced and assembled the entire Allegheny woodrat mitochondrial genome. The genome assembly is 16,310 base pairs in length, with an overall base composition of 34% adenine, 27% thymine, 26% cytosine and 13% guanine. This resource will facilitate our understanding of woodrat population genetics and behavioral ecology.

EVALUATION OF ANTHELMINTIC FISHMEAL POLYMER BAITS FOR THE CONTROL OF BAYLISASCARIS PROCYONIS IN FREE-RANGING RACCOONS (PROCYON LOTOR).

Baylisascaris procyonis is a common gastrointestinal parasite of raccoons (Procyon lotor) and is a zoonotic helminth with the potential to cause severe or fatal infection. Raccoons thrive in human-dominated landscapes, and the fecal-oral transmission pathway and lack of effective treatment make B. procyonis a serious threat to public health. The distribution of medicinal baits has emerged as a socially acceptable and cost-effective method for managing disease in free-ranging wildlife. We assessed the suitability of a mass-producible anthelmintic bait for B. procyonis mitigation by evaluating the willingness of free-ranging raccoons to consume anthelmintic baits and determining whether bait consumption successfully cleared B. procyonis infections from raccoons. Anthelmintic baits were modified from standard fishmeal polymer baits, the food attractant commonly used in oral rabies vaccine baits, with the introduction of 220 mg of pyrantel pamoate into the fishmeal mixture. We captured 16 naturally infected raccoons, presented one anthelmintic bait, and monitored B. procyonis infection over 90 d by screening feces for eggs. Treatment cleared B. procyonis infections for nine of 12 raccoons that consumed >10 g of the 15 g bait. We used remote cameras to monitor in situ patterns of bait consumption for anthelmintic baits relative to standard baits. Both anthelmintic and standard baits were rapidly consumed, with no differences in the rate of consumption between bait types. However, after bait contact, raccoons demonstrated a greater willingness to consume standard baits while ignoring anthelmintic baits more frequently (P = 0.06). Initial trials of anthelmintic baits show promise, although refinement in both dose and palatability is needed. At mass production scales, the addition of pyrantel pamoate to fishmeal polymer baits would be inexpensive, potentially making anthelmintic baits a viable management option when coupled with an oral rabies vaccine or used independently for B. procyonis mitigation.

Development of an automated dispenser for the delivery of medicinal or vaccine-laden baits to raccoons (Procyon lotor).

Medicinal baits are distributed to manage zoonotic diseases, including raccoon (Procyon lotor) rabies, but efficient distribution strategies are needed for suburban environments. We developed an automated dispenser that transfers fishmeal polymer baits at user-specified intervals from a magazine to a receptacle fitted with a filter that exploits raccoon dexterity to limit consumption by nontarget species. We introduce the concept of automated dispensers and describe bait removal success rates for raccoons versus nontarget species. We monitored visitation with remote cameras after deploying a dispenser, programmed to present two baits per night, in three disjunct forest patches in northwest Indiana. Raccoons removed 72% of baits; nontarget, white-footed mice (Peromyscus leucopus) removed 11%; Virginia opossums (Didelphis virginiana) removed 9%. Bait removal success varied significantly between raccoons (76%) and opossums (21%), improving bait delivery specificity relative to hand baiting. Accumulation of baits in receptacles resulted in excess (more than one) bait consumption (39% of baits consumed by raccoons were excess), suggesting design improvements are needed to present additional baits only after previous baits have been consumed. Automated dispensers successfully sustained bait availability throughout the operational period. Subsequent research is needed to determine whether a sustained availability of baits achieved with automated dispensers is more effective for the treatment of raccoons in suburban environments than traditional distribution methods.

Reduction of Baylisascaris procyonis eggs in raccoon latrines, suburban Chicago, Illinois, USA.

Baylisascaris procyonis, a common roundworm of raccoons, causes severe or fatal human infections, often in suburban areas. To evaluate the effectiveness of a baiting strategy requiring minimal labor, we distributed medicated baits near raccoon latrines in suburban Chicago, Illinois, USA. This strategy lowered B. procyonis prevalence in raccoons, possibly reducing risk to humans.

Allegheny woodrat (Neotoma magister) captive propagation to promote recovery of declining populations.

The Allegheny woodrat (Neotoma magister) is endemic to the eastern United States with local distributions restricted to rocky habitats within deciduous forests. Over the last 40 years, woodrats have declined precipitously due to an array of human-mediated pressures. There is growing interest in the captive propagation of woodrats as a tool to promote in situ conservation, but their solitary social structure, territorial behavior, and low fecundity present challenges for the attainment of levels of ex situ reproduction sufficient to support reintroduction programs. In 2009 we established a captive breeding program with 12 wild-caught individuals (4.8) collected from Indiana and Pennsylvania. Restricting breeding to wild-caught individuals, over 26 months we produced 19 litters comprised of 43 pups (26.17), of which 40 (24.16) survived to weaning. In sum, wild-caught individuals readily habituated to the captive environment and the low fecundity of woodrats was offset by high survival rates for both adults and juveniles. Therefore, when managed appropriately, captive Allegheny woodrat populations should be capable of supporting the release of surplus individuals to augment in situ conservation measures.

Use of experimental translocations of Allegheny woodrat to decipher causal agents of decline.

Translocations are an important tool for wildlife conservation, although progress in the field of reintroduction biology has been hindered by the ad hoc and opportunistic nature of many translocations. We used an experimental translocation to elucidate the role of raccoon roundworm (Baylisascaris procyonis) and inbreeding depression in the decline of the Allegheny woodrat (Neotoma magister), an endangered species. We translocated woodrats from genetically diverse populations in the core of the species range to 4 previously occupied sites (reintroductions) and 2 sites supporting genetically depauperate populations (reinforcements) in Indiana (U.S.A.). In 2 reintroduction sites and 1 reinforcement site, we distributed anthelmintic baits to passively deworm raccoons and reduce the risk of woodrat exposure to roundworms. The remaining sites served as controls. We used raccoon latrine surveys and fecal flotation to monitor temporal variability in roundworm prevalence and effect of treatment. We used live trapping and microsatellite genotyping to monitor the demographic and genetic response of translocated populations over the following 54 months. At the conclusion of the study, 4 of 6 translocations were successfully maintaining abundance through local recruitment. The distribution of anthelmintic baits reduced levels of roundworm contamination, but levels of contamination were also low in 2 of 3 control sites. Reintroductions failed at control sites, one of which was due to high roundworm exposure. The other failed control reintroduction was likely attributable to demographic stochasticity and limited reproductive potential following initial mortality within the first 4 months. In both control and treatment reinforcements, increases in both allelic richness and heterozygosity were accompanied by increases in abundance, which is suggestive of genetic rescue. Our results demonstrate that mitigation of roundworm exposure through the distribution of anthelmintic baits can facilitate woodrat recovery and that diversity within genetically depauperate populations can be restored through the introduction of a limited number of individuals.

Reducing Baylisascaris procyonis roundworm larvae in raccoon latrines.

Baylisascaris procyonis roundworms, a parasite of raccoons, can infect humans, sometimes fatally. Parasite eggs can remain viable in raccoon latrines for years. To develop a management technique for parasite eggs, we tested anthelmintic baiting. The prevalence of eggs decreased at latrines, and larval infections decreased among intermediate hosts, indicating that baiting is effective.

Optimization of Raccoon latrine surveys for quantifying exposure to Baylisascaris procyonis.

Raccoon (Procyon lotor) latrine surveys are an efficient and cost-effective method to quantify Baylisascaris procyonis exposure risk for intermediate hosts. Nevertheless, current methodologies may introduce bias by homogenizing scats collected within latrines and estimating exposure risk at the latrine scale. The objective of this study was to examine the potential for raccoon latrine size to bias estimates of risk of exposure when utilizing current methodologies that pool scats prior to analysis. We accomplished this by evaluating correlation of the presence of B. procyonis eggs among scats collected within latrines. Specimens were collected in southern Indiana during November and December of 2006. Raccoon scats collected within latrines exhibited no correlation with the presence of B. procyonis eggs. Accordingly, to eliminate the bias introduced by pooling scats within latrines, scats rather than latrines should be treated as the sampling unit for estimating risk of exposure for intermediate hosts. Further, evaluation of exposure risk based on individual scats has the potential to greatly reduce the time and expense associated with obtaining precise estimates of B. procyonis exposure risk.