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.

Adaptive risk-based targeted surveillance for foreign animal diseases at the wildlife-livestock interface.

Animal disease surveillance is an important component of the national veterinary infrastructure to protect animal agriculture and facilitates identification of foreign animal disease (FAD) introduction. Once introduced, pathogens shared among domestic and wild animals are especially challenging to manage due to the complex ecology of spillover and spillback. Thus, early identification of FAD in wildlife is critical to minimize outbreak severity and potential impacts on animal agriculture as well as potential impacts on wildlife and biodiversity. As a result, national surveillance and monitoring programs that include wildlife are becoming increasingly common. Designing surveillance systems in wildlife or, more importantly, at the interface of wildlife and domestic animals, is especially challenging because of the frequent lack of ecological and epidemiological data for wildlife species and technical challenges associated with a lack of non-invasive methodologies. To meet the increasing need for targeted FAD surveillance and to address gaps in existing wildlife surveillance systems, we developed an adaptive risk-based targeted surveillance approach that accounts for risks in source and recipient host populations. The approach is flexible, accounts for changing disease risks through time, can be scaled from local to national extents and permits the inclusion of quantitative data or when information is limited to expert opinion. We apply this adaptive risk-based surveillance framework to prioritize areas for surveillance in wild pigs in the United States with the objective of early detection of three diseases: classical swine fever, African swine fever and foot-and-mouth disease. We discuss our surveillance framework, its application to wild pigs and discuss the utility of this framework for surveillance of other host species and diseases.

Influenza A virus surveillance, infection and antibody persistence in snow geese (Anser caerulescens).

Some snow geese (Anser caerulescens) migrate between Eurasia and North America and exhibit high seroprevalence for influenza A viruses (IAVs). Hence, these birds might be expected to play a role in intercontinental dispersal of IAVs. Our objective in this manuscript was to characterize basic incidence and infection characteristics for snow geese to assess whether these birds are likely to significantly contribute to circulation of IAVs. Thus, we 1) estimated snow goose infection prevalence by summarizing > 5,000 snow goose surveillance records, 2) experimentally infected snow geese with a low pathogenic IAV (H4N6) to assess susceptibility and infection dynamics and 3) characterized long-term antibody kinetics. Infection prevalence based on surveillance data for snow geese was 7.88%, higher than the infection rates found in other common North American goose species. In the experimental infection study, only 4 of 7 snow geese shed viral RNA. Shedding in infected birds peaked at moderate levels (mean peak 102.62 EID50 equivalents/mL) and was exclusively associated with the oral cavity. Serological testing across a year post-exposure showed all inoculated birds seroconverted regardless of detectable shedding. Antibody levels peaked at 10 days post-exposure and then waned to undetectable levels by 6 months. In sum, while broad-scale surveillance results showed comparatively high infection prevalence, the experimental infection study showed only moderate susceptibility and shedding. Consequently, additional work is needed to assess whether snow geese might exhibit higher levels of susceptibility and shedding rates when exposed to other IAV strains.

Comparative susceptibility of eastern cottontails and New Zealand white rabbits to classical rabbit haemorrhagic disease virus (RHDV) and RHDV2.

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus.

SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus).

Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have angiotensin-converting enzyme 2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, exhibit social behavior, and can be abundant near urban centers. We evaluated 624 prepandemic and postpandemic serum samples from wild deer from four US states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples tested with a SARS-CoV-2 virus neutralization test showed high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

FOOT-AND-MOUTH DISEASE IN EXPERIMENTALLY INFECTED MULE DEER (ODOCOILEUS HEMIONUS).

The only known outbreak of foot-and-mouth disease (FMD) in wildlife in the US occurred in mule deer (Odocoileus hemionus) in California in 1924-25. There is little recorded information on the pathogenesis and epidemiology of the disease in deer in that outbreak. In this experimental study, we compared the susceptibility of mule deer to FMD virus (FMDV) serotype O to that of cattle (Bos taurus). We also determined the potential for intra- and interspecies transmission of FMDV serotype O in mule deer and cattle, and assessed conventional laboratory tests in their ability to detect FMDV in mule deer. Two mule deer and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. The inoculated steer and deer were kept in the same room with contact animals of both species. Exposed contact animals were moved to rooms with unexposed animals after becoming febrile. All mule deer (n=14) and cattle (n=6) developed clinical signs and lesions consistent with FMDV infection. Deer had a high prevalence of myocarditis and high mortality. Virus was transmitted between mule deer, from cattle to mule deer, and from mule deer to cattle. Virus and antibodies against nonstructural FMDV proteins in mule deer and cattle were detected by conventional laboratory tests. Virus shedding was detected by PCR and virus isolation up to 9 d postexposure in deer.

Bourbon Virus in Wild and Domestic Animals, Missouri, USA, 2012-2013.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

Current status and future recommendations for feral swine disease surveillance in the United States.

USDA APHIS Wildlife Services (WS) responded to the threat of feral swine as a pathogen reservoir as early as 2004. To increase awareness and knowledge on that risk, WS began opportunistic sampling of animals harvested by its operational component to curtail swine damage to agriculture and property. Initially, pseudorabies and swine brucellosis were of most concern, as both serve as a potential threat to the domestic swine industry and the latter also possesses zoonotic implications. In 2006, classical swine fever, a foreign animal disease, became the main driver for feral swine pathogen surveillance. Subsequent years of surveillance identified numerous other disease risks inherent within populations of feral swine. Presently, feral swine surveillance falls under the purview of the APHIS National Feral Swine Damage Management Program, which began in 2014. In January 2018, a panel of animal disease experts representing industry, government, and academia were invited to Fort Collins, Colorado to discuss successes of this surveillance, identify any shortcomings or needs, and propose future feral swine surveillance. This manuscript serves to synthesize WS' surveillance and the future direction of these efforts.

Pseudorabies detected in hunting dogs in Alabama and Arkansas after close contact with feral swine (Sus scrofa).

BACKGROUND: Pigs (Sus scrofa) are the natural hosts of pseudorabies virus (PRV), also known as Aujeszky's disease. Infection in mammals, with the exception of humans, typically causes extreme itching, facial swelling, and excessive salivation, followed by death in non-suid species. The risk to susceptible mammals was assumed to decrease when PRV was eliminated from U.S. commercial swine in 2004, though the virus remains endemic in feral swine. Infected feral swine pose a threat to the disease-free status of the commercial swine industry, and to other animals, including dogs, that come in direct or indirect contact with them. Since dogs are commonly used for hunting feral swine, they are at high risk of exposure. CASE PRESENTATION: The following report describes the progression of pseudorabies infection in dogs in two states after exposure to feral swine. The first case occurred in a dog in Alabama after participation in a competitive wild hog rodeo. The second case occurred in multiple dogs in Arkansas after hunting feral swine, and subsequent consumption of the offal. The antibody prevalence of feral swine in the two states where the dogs were exposed is also examined. CONCLUSIONS: Dogs that are used for hunting feral swine are at high risk of exposure to pseudorabies because the disease is considered endemic in feral swine in the U.S.

LEPTOSPIRA ANTIBODIES DETECTED IN WILDLIFE IN THE USA AND THE US VIRGIN ISLANDS.

From 2011 to 2017, 4,534 serum samples from 13 wildlife species collected across the US and in one territory (US Virgin Islands) were tested for exposure to Leptospira serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. Of 1,759 canids, 1,043 cervids, 23 small Indian mongooses ( Herpestes auropunctatus), 1,704 raccoons ( Procyon lotor), and five striped skunks ( Mephitis mephitis), 27.0, 44.4, 30.4, 40.8, and 60%, respectively, were antibody positive for any of the six serovars. The most commonly detected serovars across all species were Bratislava and Grippotyphosa. Our results indicate that Leptospira titers are very common in a wide variety of wildlife species. These species may act as important reservoirs in the epidemiological cycle of the pathogen. Additional studies to determine the relationship between serologic evidence and shedding of the pathogen by wildlife are necessary to better understand the risk.

A Bead-Based Flow Cytometric Assay for Monitoring Yersinia pestis Exposure in Wildlife.

Yersinia pestis is the causative agent of plague and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores, which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semiautomated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1 Luminex plague assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for F1-specific IgG detection and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.

Serologic Evidence of Various Arboviruses Detected in White-Tailed Deer (Odocoileus virginianus) in the United States.

White-tailed deer (Odocoileus virginianus) are an abundant mammal with a wide geographic distribution in the United States, which make them good sentinels for monitoring arboviral activity across the country. Exposure to various arboviruses has been detected in white-tailed deer, typically in conjunction with another diagnostic finding. To better assess the exposure of white-tailed deer to seven arboviruses, we tested 1,508 sera collected from 2010 to 2016 for antibodies to eastern equine encephalitis (2.5%), Powassan (4.2%), St. Louis encephalitis, (3.7%), West Nile (6.0%), Maguari (19.4%), La Crosse (30.3%), and bluetongue (7.8%) viruses. At least one arbovirus was detected in 51.3%, and exposure to more than one arbovirus was identified in 17.6% of the white-tailed deer sampled.

Antibodies to Various Zoonotic Pathogens Detected in Feral Swine (Sus scrofa) at Abattoirs in Texas, USA.

The zoonotic risk posed to employees by slaughtering feral swine (Sus scrofa) at two abattoirs in Texas was assessed by testing feral swine serum samples for exposure to influenza A virus, Leptospira, Trichinella spiralis, and Toxoplasma gondii. Blood was collected from a total of 376 feral swine between the two facilities during six separate collection periods in 2015. Antibodies to one or more serovars of Leptospira were identified in 48.9% of feral swine tested, with Bratislava and Pomona as the most commonly detected serovars, and antibodies to influenza A virus were detected in 14.1% of feral swine. Antibodies to T. gondii and T. spiralis were identified in 9.0 and 3.5%, respectively, of feral swine tested. Our results suggest that abattoir employees should be aware of the potential for exposure to various zoonotic pathogens when slaughtering feral swine, wear appropriate personal protective equipment, and participate in medical monitoring programs to ensure detection and prompt treatment. In addition, consumers of feral swine should cook the meat to the appropriate temperature and wash hands and kitchen surfaces thoroughly after preparing meat.

LIMITED ANTIBODY EVIDENCE OF EXPOSURE TO MYCOBACTERIUM BOVIS IN FERAL SWINE (SUS SCROFA) IN THE USA.

Bovine tuberculosis is a chronic disease of cattle ( Bos taurus ) caused by the bacterium Mycobacterium bovis . Efforts have been made in the US to eradicate the disease in cattle, but spillover into wildlife and subsequent spillback have impeded progress in some states. In particular, infection in white-tailed deer ( Odocoileus virginianus ) has been followed by infection in cattle in some Midwestern states. Infection has also been documented in feral swine ( Sus scrofa ) on the Hawaiian island of Molokai and in various European countries, but no large-scale survey of antibody exposure to the bacteria has been conducted in feral swine in the US. We tested 488 sera from feral swine collected near previously documented outbreaks of bovine tuberculosis in cattle and captive cervids, in addition to 2,237 feral swine sera collected across the US from 1 October 2013 to 30 September 2014. While all but one of the samples were antibody negative, the results are important for establishing baseline negative data since feral swine are capable reservoirs and could be implicated in future outbreaks of the disease.

Surveillance for highly pathogenic H5 avian influenza virus in synanthropic wildlife associated with poultry farms during an acute outbreak.

In November 2014, a Eurasian strain H5N8 highly pathogenic avian influenza virus was detected in poultry in Canada. Introduced viruses were soon detected in the United States and within six months had spread to 21 states with more than 48 million poultry affected. In an effort to study potential mechanisms of spread of the Eurasian H5 virus, the United States Department of Agriculture coordinated several epidemiologic investigations at poultry farms. As part of those efforts, we sampled synanthropic birds and mammals at five infected and five uninfected poultry farms in northwest Iowa for exposure to avian influenza viruses. Across all farms, we collected 2,627 samples from 648 individual birds and mammals. House mice were the most common mammal species captured while house sparrows, European starlings, rock pigeons, swallows, and American robins were the most commonly captured birds. A single European starling was positive for Eurasian H5 viral RNA and seropositive for antibodies reactive to the Eurasian H5 virus. Two American robins were also seropositive. No mammal species showed evidence of infection. These results indicate synanthropic species merit further scrutiny to better understand potential biosecurity risks. We propose a set of management practices aimed at reducing wildlife incursions.

FOOT-AND-MOUTH DISEASE IN A SMALL SAMPLE OF EXPERIMENTALLY INFECTED PRONGHORN (ANTILOCAPRA AMERICANA).

There is limited information on the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American wildlife and none concerning pronghorn ( Antilocapra americana ). In an experimental study of 13 pronghorn and six steers ( Bos taurus ), we compared the susceptibility of pronghorn to FMD virus (FMDV) strain O, with that of cattle ( Bos taurus ). We also determined the potential for intra- and interspecies transmission of FMDV strain O in pronghorn and cattle, assessed the application of conventional laboratory tests in their suitability to detect FMDV infection in pronghorn, and evaluated the potential role of pronghorn as efficient long-term carriers of FMDV. After acclimation to containment at Plum Island Animal Disease Center, two pronghorn and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were housed with contact animals. When contact-exposed animals developed fever they were placed in rooms with previously unexposed animals. All inoculated and exposed cattle and pronghorn developed clinical disease typical of FMD. Pronghorn developed severe foot lesions and mild to moderate oral lesions, primarily on the tongue. Duration of clinical signs in both species was 2-3 wk with foot abnormalities evident to the end of the study (51 d postexposure). Other lesions included pancreatitis, myositis of the tongue, and secondary lesions including pleuritis, pneumonia, decubital ulcers, and tenosynovitis. Virus transmission occurred between pronghorn, from cattle to pronghorn, and from pronghorn to cattle. Conventional laboratory tests detected virus and antibodies against nonstructural and structural FMDV proteins in pronghorn and cattle. Virus was present in some animals for 1 wk but was not detectable by virus isolation or PCR at 3 wk postinfection or afterward.

Feral swine virome is dominated by single-stranded DNA viruses and contains a novel Orthopneumovirus which circulates both in feral and domestic swine.

Feral swine are known reservoirs for various pathogens that can adversely affect domestic animals. To assess the viral ecology of feral swine in the USA, metagenomic sequencing was performed on 100 pooled nasal swabs. The virome was dominated by small, ssDNA viruses belonging to the families Circoviridae, Anelloviridae and Parvovirinae. Only four RNA viruses were identified: porcine kobuvirus, porcine sapelovirus, atypical porcine pestivirus and a novel Orthopneumovirus, provisionally named swine orthopneumovirus (SOV). SOV shared ~90 % nucleotide identity to murine pneumonia virus (MPV) and canine pneumovirus. A modified, commercially available ELISA for MPV found that approximately 30 % of both feral and domestic swine sera were positive for antibodies cross-reactive with MPV. Quantitative reverse transcription-PCR identified two (2 %) and four (5.0 %) positive nasal swab pools from feral and domestic swine, respectively, confirming that SOV circulates in both herds.

Dietary magnesium and copper affect survival time and neuroinflammation in chronic wasting disease.

Chronic wasting disease (CWD), the only known wildlife prion disease, affects deer, elk and moose. The disease is an ongoing and expanding problem in both wild and captive North American cervid populations and is difficult to control in part due to the extreme environmental persistence of prions, which can transmit disease years after initial contamination. The role of exogenous factors in CWD transmission and progression is largely unexplored. In an effort to understand the influence of environmental and dietary constituents on CWD, we collected and analyzed water and soil samples from CWD-negative and positive captive cervid facilities, as well as from wild CWD-endozootic areas. Our analysis revealed that, when compared with CWD-positive sites, CWD-negative sites had a significantly higher concentration of magnesium, and a higher magnesium/copper (Mg/Cu) ratio in the water than that from CWD-positive sites. When cevidized transgenic mice were fed a custom diet devoid of Mg and Cu and drinking water with varied Mg/Cu ratios, we found that higher Mg/Cu ratio resulted in significantly longer survival times after intracerebral CWD inoculation. We also detected reduced levels of inflammatory cytokine gene expression in mice fed a modified diet with a higher Mg/Cu ratio compared to those on a standard rodent diet. These findings indicate a role for dietary Mg and Cu in CWD pathogenesis through modulating inflammation in the brain.

Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease.

The purpose of our study was to describe the progressive accumulation of the abnormal conformer of the prion protein (PrP(CWD)) and spongiform degeneration in a single section of brain stem in Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease (CWD). A section of obex from 85 CWD-positive elk was scored using the presence and abundance of PrP(CWD) immunoreactivity and spongiform degeneration in 10 nuclear regions and the presence and abundance of PrP(CWD) in 10 axonal tracts, the subependymal area of the fourth ventricle, and the thin subpial astrocytic layer (glial limitans). Data was placed in a formula to generate an overall obex score. Data suggests that PrP(CWD) immunoreactivity and spongiform degeneration has a unique and relatively consistent pattern of progression throughout a section of obex. This scoring technique utilizing a single section of obex may prove useful in future work for estimating the presence and abundance of PrP(CWD) in peripheral tissues and the nervous system in elk with CWD.