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Plague is caused by a bacterial pathogen (Yersinia pestis) that can infect a wide range of mammal species, but its presence in wildlife is often underappreciated. Using a large-scale data set (n = 44,857) that details the extent of Y. pestis exposure in wildlife, we document exposure in 18 wildlife species, including coyotes (Canis latrans), bobcats (Lynx rufus), and black bears (Ursus americanus). Evidence of plague activity is widespread, with seropositive animals detected in every western state in the contiguous United States. Pathogen monitoring systems in wildlife that are both large scale and long-term are rare, yet they open the door for analyses on potential shifts in distribution that have occurred over time because of climate or land use changes. The data generated by these long-term monitoring programs, combined with recent advances in our understanding of pathogen ecology, offer a clearer picture of zoonotic pathogens and the risks they pose.

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.

Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations. IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Genetic reassortment between influenza A viruses (IAVs) facilitate emergence of pandemic strains, and swine are proposed as a “mixing vessel” for generating reassortants of avian and mammalian IAVs that could be of risk to mammals, including humans. However, how a transmissible reassortant emerges in swine are not well understood. Genomic analyses of 571 isolates recovered from nasal wash samples and respiratory tract tissues of a group of co-housed pigs (influenza-seronegative, avian H1N1 IAV–infected, and swine H3N2 IAV–infected pigs) identified 30 distinct genotypes of reassortants. Viruses recovered from lower respiratory tract tissues had the largest genomic diversity, and those recovered from turbinates and nasal wash fluids had the least. Reassortants from lower respiratory tracts had the largest variations in growth kinetics in respiratory tract epithelial cells, and the cold temperature in swine nasal cells seemed to select the type of reassortant viruses shed by the pigs. One reassortant in nasal wash samples was consistently identified in upper, middle, and lower respiratory tract tissues, and it was confirmed to be transmitted efficiently between pigs. Study findings suggest that, during mixed infections of avian and swine IAVs, genetic reassortments are likely to occur in the lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant., Author summary Genetic reassortments between avian and swine influenza viruses are likely to occur in the swine lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant; determination of tissue tropisms for potential reassortants between contemporary avian and swine influenza viruses would help identify transmissible reassortants with public health risks.

Given their free-ranging habits, feral swine could serve as reservoirs or spatially dynamic ‘mixing vessels’ for influenza A virus (IAV). To better understand virus shedding patterns and antibody response dynamics in the context of IAV surveillance amongst feral swine, we used IAV of feral swine origin to perform infection experiments. The virus was highly infectious and transmissible in feral swine, and virus shedding patterns and antibody response dynamics were similar to those in domestic swine. In the virus-inoculated and sentinel groups, virus shedding lasted ≤ 6 and ≤ 9 days, respectively. Antibody titres in inoculated swine peaked at 1 : 840 on day 11 post-inoculation (p.i.), remained there until 21 days p.i. and dropped to

Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During the period from 2010 to 2013, 8,239 serum samples were collected from feral swine across 35 U.S. states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 tested virus, whereas the other 23 did not cross-react with any tested virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine had been exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs. IMPORTANCE There are more than 5 million feral swine distributed across at least 35 states in the United States. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock, and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine-adapted IAVs. Our findings suggest that feral swine have been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

To determine whether, and to what extent, influenza A subtype H3 viruses were present in feral swine in the United States, we conducted serologic and virologic surveillance during October 2011–September 2012. These animals were periodically exposed to and infected with A(H3N2) viruses, suggesting they may threaten human and animal health.

Our objective was to identify and evaluate mitochondrial diversity of wild pigs in the United States (U.S.). We obtained tissue samples from 81 individual pigs in 30 U.S. states and amplified a 403 base- pair region of mitochondrial DNA. We then downloaded overlapping sequences (n ¼904) from public repositories to create a global reference. We used parsimony and Bayesian techniques to evaluate phylogenetic relationships, and we used origins of published sequences from Eurasian wild boar to establish a phylogeographic reference. We then compared gene and nucleotide diversity measures for introduced pigs in North America with those of domestic swine, Eurasian wild boar, and feral pigs within broad-scale geographic groupings. We identified 15 haplotypes for introduced pigs, representing wild and domestic animals from>30 countries spanning the indigenous range of S. scrofa. Mitochondrial diversity measures and phylogenetic relationships indicated a strong association between introduced pigs and European domestic breeds, reflecting the known history of human colonization, trade, and settlement in the United States. Based on the geographic distribution ofhaplotypes in North America, we found that range expansion is a product of translocation from historical populations and introduction from new genetic sources. Finally, haplotype network analyses provided evidence of past demographic expansions within lineages, contributing to observed mtDNA variation among introduced pigs in North America. 2014 The Wildlife Society.

Brucellosis is a common infection of feral swine throughout the United States. With the recent expansion of feral swine populations across the country, this disease poses an increasing threat to agriculture and hunters. The standard approach to Brucella surveillance in feral swine has been serological testing, which gives an indication of past exposure and is a rapid method of determining populations where Brucella is present. More in-depth analyses require bacterial isolation to determine the Brucella species and biovar involved. Ultimately, for a comprehensive understanding of Brucella epizootiology in feral swine, incorporation of genotyping assays has become essential. Fortunately, the past decade has given rise to an array of genetic tools for assessing Brucella transmission and dispersal. This review aims to synthesize what is known about brucellosis in feral swine and will cover prospective genomic techniques that may be utilized to develop more complete understanding of the disease and its transmission history.

A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

Using filter paper to collect blood from wildlife for antibody analysis can be a powerful technique to simplify the collection, transport, and storage of blood samples. Despite these advantages, there are limited data that detail how long these samples can be stored and how storage conditions affect antibody longevity. We used blood samples collected on filter paper from coyotes experimentally infected with Yersinia pestis to determine optimum sample storage conditions over time. Blood samples collected on filter paper were stored for 454 d or more in four groups: 1) at ambient temperature and at ambient relative humidity, 2) at ambient temperature with desiccant, 3) at 4 C with desiccant, and 4) at −20 C with desiccant. Samples stored at 4 C or −20 C with desiccant had detectable antibody for a longer period of time than the samples stored at room temperature.

Distribution of oral rabies vaccine baits has been used as a strategy for managing rabies in the United States since the 1990s. Since that time, efforts have been made to improve baiting strategies with a focus on bait density to maximize both efficiency and cost effectiveness. An optimal rabies management strategy includes a vaccine bait preferred by the target species that is distributed at the minimal density needed to achieve population immunity to prevent rabies spread. The purpose of our pilot study was to examine the effect of 75, 150, and 300 baits/km2 vaccine bait densities on rabies virus neutralizing antibody (RVNA) seroprevalence in raccoons (Procyon lotor). Raboral V-RG® fishmeal polymer baits (Merial Inc. (now a part of Boehringer Ingelheim), Athens, Georgia) contain a tetracycline biomarker that was used to estimate bait consumption as another measure of intervention impact. Our results suggest that raccoon RVNA response increases as bait density increases, but the effect may not be sufficient to justify the cost except in the case of contingency actions or an epizootic. Non-target species, especially opossums (Didelphis virginianus) in certain areas, should be considered when determining an appropriate bait density to ensure sufficient baits are available for consumption by the target species.

As part of the USA’s National Strategy for Pandemic Influenza, an Interagency Strategic Plan for the Early Detection of Highly Pathogenic H5N1 Avian Influenza in Wild Migratory Birds was developed and implemented. From 1 April 2006 through 31 March 2009, 261 946 samples from wild birds and 101 457 wild bird fecal samples were collected in the USA; no highly pathogenic avian influenza was detected. The United States Department of Agriculture, and state and tribal cooperators accounted for 213 115 (81%) of the wild bird samples collected; 31, 27, 21 and 21% of the samples were collected from the Atlantic, Pacific, Central and Mississippi flyways, respectively. More than 250 species of wild birds in all 50 states were sampled. The majority of wild birds (86%) were dabbling ducks, geese, swans and shorebirds. The apparent prevalence of low pathogenic avian influenza viruses during biological years 2007 and 2008 was 9.7 and 11.0%, respectively. The apparent prevalence of H5 and H7 subtypes across all species sampled were 0.5 and 0.06%, respectively. The pooled fecal samples (n = 101 539) positive for low pathogenic avian influenza were 4.0, 6.7 and 4.7% for biological years 2006, 2007 and 2008, respectively. The highly pathogenic early detection system for wild birds developed and implemented in the USA represents the largest coordinated wildlife disease surveillance system ever conducted. This effort provided evidence that wild birds in the USA were free of highly pathogenic avian influenza virus (given the expected minimum prevalence of 0.001%) at the 99.9% confidence level during the surveillance period.

Sera from 936 mammalian mesopredators (Virginia opossums, gray foxes, striped skunks, hooded skunks, raccoons, a bobcat, and a red fox) were collected during 2003 and 2004 in California, Arizona, Texas, Louisiana, Ohio, and Wyoming and screened for flavivirus-specific antibodies by an epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA). Serum samples positive for antibodies against flaviviruses were screened for West Nile virus (WNV)-specific antibodies by blocking ELISA and selectively confirmed with plaque-reduction neutralization tests. High prevalence rates were observed in raccoons (45.6%) and striped skunks (62.9%). The high WNV antibody prevalence noted in mesopredators, their peridomestic tendencies, and their overall pervasiveness make these species potentially useful sentinels for monitoring flaviviruses in defined areas.

Trichinella spp. and Toxoplasma gondii are important zoonotic parasites that infect warm blooded animals and humans worldwide. Among domesticated food animals, pigs are the main host for Trichinella spiralis. Pigs, chickens, sheep, and goats are known to be infected with T. gondii at varying rates, depending on husbandry. Infections in wildlife with these parasites are generally higher than in domesticated species. Feral swine act as reservoirs of infection in the sylvatic ecosystem for Trichinella spp. and T. gondii, acting as sources of infection for peridomestic carnivores whose home ranges overlap with domestic pigs. Feral swine can have direct contact with non-biosecure domestic pigs, presenting opportunity for direct disease transmission through cannibalistic behavior. Determination of the prevalence of Trichinella spp. and T. gondii infection in feral swine is needed to understand the risk of transmission of these parasites to domestic pigs. A cross-sectional serological survey was conducted between 2006 and 2010 to estimate the antibody prevalence of Trichinella spp. and T. gondii and risk factors associated with infection in feral swine in the USA. Serum samples were tested from 3247 feral pigs from 32 states; results are reported from 26 states. Maximum entropy ecological niche modeling and spatial scan statistic were utilized to predict the geographic range and to examine clusters of infection of Trichinella spp. and T. gondii in feral pigs. The seroprevalence of antibodies to Trichinella spp. and T. gondii was 3.0% and 17.7%, respectively. Species distribution modeling indicated that the most probable distribution areas for both parasites was similar, concentrated primarily in the South and the Midwest regions of the USA. A follow up survey conducted during 2012-2013 revealed that 2.9% of 984 sampled feral swine were seropositive for Trichinella spp., and 28.4% were seropositive for T. gondii. Three hundred and thirty (330) tongues were collected from the 984 sampled animals during 2012-2013; 1.81% were tissue positive for T. spiralis muscle larvae; no other genotypes were found. The potential exists for introduction of these pathogens into domestic herds of non-biosecure domestic pigs as a result of increasing overlap of the range of feral pigs with non-biosecure domestic pigs production facilities in the USA.

Avian influenza is a viral disease that primarily infects wild and domestic birds, but it also can be transmitted to a variety of mammals. In 2006, the United States of America Departments of Agriculture and Interior designed a large-scale, interagency surveillance effort that sought to determine if highly pathogenic avian influenza viruses were present in wild bird populations within the United States of America. This program, combined with the Canadian and Mexican surveillance programs, represented the largest, coordinated wildlife disease surveillance program ever implemented. Here we analyze data from 197,885 samples that were collected from over 200 wild bird species. While the initial motivation for surveillance focused on highly pathogenic avian influenza, the scale of the data provided unprecedented information on the ecology of avian influenza viruses in the United States, avian influenza virus host associations, and avian influenza prevalence in wild birds over time. Ultimately, significant advances in our knowledge of avian influenza will depend on both large-scale surveillance efforts and on focused research studies.

Samples were collected in 35 states as part of a national monitoring system to detect multiple diseases in feral swine (Sus scrofa). During March 2009 through December 2010, we collected serum samples from 4,479 feral swine from 13 states, and 159 animals tested were seropositive for brucellosis. No difference in likelihood of infection was found between males and females, but adults were more likely than sub-adults or juveniles to be exposed to brucellosis. Feral swine sampled during winter months also were more likely to be seropositive than animals sampled during other seasons. Apparent prevalence varied among states, and seropositive animals often were clustered in specific counties within a state. We recommend improved diagnostics and stricter regulations on movement of feral swine both intra- and inter-state to minimize further spread of the disease and to decrease the risk of reintroduction of brucellosis into livestock.

A captive striped skunk (Mephitis mephitis) study was conducted between Feb- ruary and June 2004 at the United States Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services National Wildlife Research Center, Fort Col- lins, Colorado, USA. The main objective was to determine the percentage of adult striped skunks that were marked after consuming placebo oral rabies vaccine (ORV) baits con- taining 100 mg of an experimental microen- capsulated (coated microparticle) tetracycline hydrochloride biomarker. Biomarkers were identified in the canine teeth and mandibles of five of five skunks that consumed an ORV bait. A second objective was to determine if the microencapsulated tetracycline was resistant to photochemical conversion from tetracycline to epitetracycline. After 15 days of exposure, conversion from tetracycline to epitetracycline concentration in the microencapsulated prod- uct (mean 1.9% conversion, SD51.24) was significantly less (P50.006) than the pure-grade tetracycline powder (mean 7.5% conversion, SD51.37). Results support the use of micro- encapsulated tetracycline hydrochloride as a biomarker in circumstances where the use of conventional powdered tetracycline hydrochlo- ride is not feasible due to ORV bait design constraints. Oral rabies vaccine (ORV) baits typical- ly contain tetracycline hydrochloride as a biomarker (Johnston and Tinline, 2002). The tetracycline biomarker becomes in- corporated into the teeth and bones of animals that consume the ORV baits (Johnston et al., 1999). These permanent marks in the teeth allow ORV program coordinators to evaluate baiting success by trapping animals within an ORV zone, extracting a tooth from each individual for tetracycline analysis, and determining the number of target animals that have consumed one or more baits. Discrepan- cies between biomarker and vaccination levels in the target population allow ORV programs to discover potential problems with a particular vaccine lot, or the ORV bait itself. Biomarker data can also be used to optimize ORV program strategies by comparing bait consumption in the target species to variation in baiting density and flight line intervals (Johnston and Tinline

Author(s): Johnston, John J.; Hurley, Jerome C.; Primus, Thomas M.; Schmit, B. S.; DeLiberto, Thomas J. | Abstract: The U.S. Department of Agriculture distributes more than 10 million tetracycline-containing rabies vaccine baits to control the spread of wildlife-vectored rabies to humans, pets, and livestock. To estimate the percent of target species consuming the baits, raccoons were collected in baited areas and teeth were analyzed for the presence of the tetracycline biomarker. Several incidents of low biomarker detection rates prompted an investigation of the stability of the biomarker in the baits. These studies indicated that a portion of the tetracycline was converted to epitetracycline. Additionally, significant quantities of both compounds were trapped in the polymer which is homogeneously distributed throughout the bait. This situation is likely responsible for low biomarker detection rates. To alleviate this problem, we developed an alternative bait matrix which permits increased stability and bioavailability of the tetracycline biomarker. This new bait matrix increased the availability of the tetracycline marker from 25.2 to 87.3% and decreased epitetracycline formation from 12.4 to 3.6%.