Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Influenza D virus utilizes both 9-O-acetylated N-acetylneuraminic and 9-O-acetylated N-glycolylneuraminic acids as functional entry receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

SARS-CoV-2 Outbreaks on Mink Farms-A Review of Current Knowledge on Virus Infection, Spread, Spillover, and Containment.

Many studies have been conducted to explore outbreaks of SARS-CoV-2 in farmed mink and their intra-/inter-species spread and spillover to provide data to the scientific community, protecting human and animal health. Studies report anthropozoonotic introduction, which was initially documented in April 2020 in the Netherlands, and subsequent inter-/intra-species spread of SARS-CoV-2 in farmed mink, likely due to SARS-CoV-2 host tropism capable of establishing efficient interactions with host ACE2 and the mink hosts' ability to enhance swift viral transmission due to their density, housing status, and occupational contacts. Despite the rigorous prevention and control measures adopted, transmission of the virus within and between animal species was efficient, resulting in the development of mink-associated strains able to jump back and forth among the mink hosts and other animal/human contacts. Current knowledge recognizes the mink as a highly susceptible animal host harboring the virus with or without clinical manifestations, furthering infection transmission as a hidden animal reservoir. A One Health approach is, thus, recommended in SARS-CoV-2 surveillance and monitoring on mink farms and of their susceptible contact animals to identify and better understand these potential animal hosts.

Understanding if the reward is worth the influenza risk: The true cost of showing pigs.

Influenza A virus transmission between pigs and humans has been reported periodically worldwide, and spillover events across the animal-human species barrier could lead to the next influenza pandemic. Swine exhibitions serve as a unique interface conducive to zoonotic disease transmission due to extensive commingling of pigs and humans for prolonged periods of time. The majority of zoonotic influenza A virus transmission in the United States has been linked to swine exhibitions, leading some to suggest additional controls for influenza A virus at the swine-human interface. Determining the value of the exhibition swine industry and gauging the financial impacts influenza A virus outbreaks could have on society, helps to inform adoption decisions of mitigation recommendations. This study estimates the total value of the exhibition swine industry in the United States and calculates the predicted costs of the most extreme mitigation strategy, cancelling swine exhibitions to reduce zoonotic influenza A virus transmission. Mixed methods, including a survey, were used to collect data and inform the study model. We estimated that the direct economic impact of the exhibition swine sector in 2018 was $1.2 billion. If pig shows were to be cancelled for one year, the estimated direct economic impact would be $357.1 million. A permanent, > 3-year ban on swine exhibitions would result in a $665 million economic impact, which is a 45% reduction from baseline. The direct economic impact of cancelling the swine show circuit could not be determined, as youth exhibitors may pursue alternative activities that cannot be precisely accounted for. However, the estimated loss to the swine industry justifies seeking enhanced mitigation to prevent disease transmission. Moreover, economic losses secondary to exhibition cancellations may explain hesitancy to participate in active influenza A virus surveillance efforts.

Evolution of influenza A viruses in exhibition swine and transmission to humans, 2013-2015.

AIMS: Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine-origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human-animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health. METHODS AND RESULTS: In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013-2015. Sixty-seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3-9.6, Clopper-Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year. CONCLUSIONS: These data indicate that multiple genotypes of swine-lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human-animal interface which are most likely to spillover into humans.

Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intra-host single nucleotide variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Swine industry stakeholders' perception on the use of water-based foam as an emergency mass depopulation method.

The U.S. pork supply chain is vulnerable to various internal and external threats and in need of prompt, comprehensive response plans. Under urgent circumstances, for example in the case of foreign disease incursions, swine farms will have to perform on-farm animal depopulation to prevent disease spread. Several animal depopulation methods including water-based foam (WBF) have been proposed and are under evaluation for feasibility in the field. However, the psychological/emotional impacts of applying depopulation methods for personnel managing and carrying on the tasks are not currently well understood. Thus, this study aimed to investigate WBF as an alternative for depopulation compared to existing methods approved by the American Veterinary Medical Association. Swine industry stakeholders were invited to voluntarily observe a WBF depopulation trial and to provide their self-reported perspectives before and after the observation. A survey was designed to explore key areas on expected and perceived method effectiveness, efficiency, and animal welfare considerations, as well as to evaluate short-term post-observation psychological impacts. Among 42 recruited stakeholders, 31.0% (13/42) were educators/researchers followed by animal health officials (26.2%, 11/42) and veterinarians (23.8%, 10/42), with an average of 11.7 ± 12.6 (n = 39) years of work experience. After the trial, respondents' positive perception of WBF depopulation increased specifically regarding the animal loading process being less stressful than restrained in-barn depopulation options (P = 0.003) and by the observation of fewer swine escape attempts and vocalizations than expected (P < 0.001). Respondents' positive perception of WBF also increased regarding to the time required to fill the trailer with foam, to stop hearing animal vocalization, and stop hearing animal movement, as the observed trial times were faster than their pre-observation estimates (P < 0.001). Additionally, 79.5% (31/39) of respondents agreed that the rapid destruction of animal populations had priority over animal welfare under urgent scenarios. Minor post-traumatic stress disorder-like (PTSD-like) symptoms from the observed trials were reported (26.7%, 4/15 respondents) one month after the observation. This study showed that the WBF depopulation process was perceived positively by swine stakeholders and may have limited short-term psychological impacts on personnel involved in animal depopulation.

Innate immune control of influenza virus interspecies adaptation.

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigated whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We found that IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Remarkably, influenza viruses passaged through Ifitm3-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Refinement of water-based foam depopulation procedures for finisher pigs during field conditions: Welfare implications and logistical aspects.

Water-based foam (WBF) depopulation is currently being researched as an alternative for rapid destruction of swine populations under emergency circumstances. Appropriate guidelines are needed to maintain method reliability and depopulation efficacy while minimizing animal distress under field conditions. Finisher pigs were depopulated using WBF with a 7.5-minute dwell time in two trials to evaluate the effect of; trial 1) foam fill level (1.5, 1.75, or 2.0 times the pig's head height) and trial 2) foam fill rate (slow, medium, or fast) on aversive pig responses (surface breaks, vocalization, and escape attempts) and time to cessation of cardiac activity. Activity and cardiac activity were recorded using subcutaneous bio-loggers for swine in trial 2. The average time to cessation of movement (COM) from the start of foam filling was compared for the foam fill rate groups using a generalized linear mixed effect model under Poisson distribution. Foam rate group was used as an independent variable, and replicates as a random effect. For trial 1, the average (mm:ss ± SD) time to fill completion was 01:18 ± 00:00, 00:47 ± 00:05, and 00:54 ± 00:05, for 1.5, 1.75, and 2.0 times the pig's head height, respectively. For trial 2, the average time to fill completion was 03:57 ± 00:32, 01:14 ± 00:23 and 00:44 ± 00:03, and the average time (mm:ss ± SE) to COM was 05:22 ± 00:21, 03:32 ± 00:14, and 03:11 ± 00:13 for slow, medium, and fast fill rate groups, respectively. A higher number of aversive pig responses were observed for the lowest foam fill level and slowest foam fill rate compared to increased fill levels and faster fill rates. For trial 2 the median (mm:ss ± IQR) time to fatal arrhythmia was 09:53 ± 02:48, 11:19 ± 04:04, and 10:57 ± 00:47 post-foam initiation for fast, medium, and slow foam rate groups, respectively. Time to cessation of cardiac activity was significantly shorter for the fast foam rate group compared to medium and slow foam rates groups (P = 0.04). For both trials, vocalizations were absent, and all pigs were unconscious following the 7.5-minute dwell time and no pigs needed a secondary euthanasia method. This WBF study showed that slower fill rates and low foam fill levels may extend the time until cessation of cardiac activity in swine during depopulation. A conservative recommendation with consideration of swine welfare during an emergency scenario would be a minimum foam fill level twice the pig's head height and a foam fill rate capable of covering pigs in foam within 60 s to minimize aversive responses and expedite cessation of cardiac activity.

Water-based medium-expansion foam depopulation of adult cattle.

Current options for depopulation of adult cattle are limited, have logistic constraints, and may not be practical on a large scale. Aspirated water-based foam (WBF) has been shown to be successful in depopulating poultry and swine but has yet to be tested in cattle. WBF is advantageous because necessary equipment can be readily available, easy to use, and presents minimal personnel risk. With the use of a modified rendering trailer in a field setting, we evaluated the efficacy of aspirated WBF for depopulation of adult cattle. Water-based medium-expansion foam was added to the trailer holding cattle to a depth of approximately 50 cm greater than head height. The study was conducted as a gated design and the initial trial was conducted using six anesthetized and six conscious animals for verification of the process and followed by four replicates each containing 18 conscious cattle. A total of 84 cattle were used, with a subset (n = 52) implanted with subcutaneous bio-loggers that recorded activity and electrocardiograms. Cattle were loaded onto the trailer and three gasoline-powered water pumps delivered foam into the trailer followed by a 15-min foam dwell period. Average (± SD) time to completely fill the trailer with foam was 84.8 ±â€…11.0 s. No animal vocalizations were heard during foam application or the dwell period, and all cattle were confirmed dead upon removal from the trailer after 15 min of immersion. Necropsies of a subset of cattle revealed foam extending to at least the tracheal bifurcation in all cattle and distal to this level in 67% (8/12) animals. Time to cessation of movement, which served as a proxy for loss of consciousness, was 2.5 ±â€…1.3 min and time to cardiac death was 8.5 ±â€…2.5 min as determined by data from animals carrying subcutaneous bio-loggers. The results of this study indicate that WBF is a rapid and effective method for depopulation of adult cattle with potential advantages in speed and carcass handling and disposal over current methods.

Description of electroencephalographic data gathered using water-based medium-expansion foam as a depopulation method for nursery pigs.

The United States' swine industry is under constant threat of foreign animal diseases, which may emerge without warning due to the globalized transportation networks moving people, animals, and products. Therefore, having disease control and elimination protocols in place prior to pathogen introduction is paramount for business continuity and economic recovery. During extraordinary circumstances, it may become necessary to depopulate large populations of animals, including swine, as a disease containment measure. Currently approved depopulation methods for swine present significant logistical challenges when scaled to large populations or performed in field conditions. In the United States, water-based foam is currently approved for poultry depopulation, and recent field studies demonstrate water-based foam is an effective depopulation alternative for swine. While effective, the speed at which water-based foam induces loss of consciousness prior to death, a major welfare consideration, has not been adequately investigated. In this study, 12 nursery pigs were terminated using water-based medium-expansion foam to quantify the time to induce loss of consciousness and ultimately brain death. Each pig was implanted with subdermal electrodes to capture electroencephalographic data, placed in a body sling, and suspended in a plastic bulk container that was subsequently filled with water-based foam. Electroencephalographic data was recorded for 15 min, during which the pigs remained immersed in the water-based foam. Conservatively, average (± SD) time to unconsciousness and brain death was 1 min, 53 s ± 36 s and 3 min, 3 s ± 56 s, respectively. The relatively rapid loss of consciousness compared to other methods limits the amount of distress and is overall a positive finding for the welfare of the pigs that might be depopulated with water-based foam. The findings of this study add additional evidence supporting the use of water-based medium-expansion foam for an emergency depopulation of swine.

Shortening Duration of Swine Exhibitions to Reduce Risk for Zoonotic Transmission of Influenza A Virus.

Reducing zoonotic influenza A virus (IAV) risk in the United States necessitates mitigation of IAV in exhibition swine. We evaluated the effectiveness of shortening swine exhibitions to <72 hours to reduce IAV risk. We longitudinally sampled every pig daily for the full duration of 16 county fairs during 2014-2015 (39,768 nasal wipes from 6,768 pigs). In addition, we estimated IAV prevalence at 195 fairs during 2018-2019 to test the hypothesis that <72-hour swine exhibitions would have lower IAV prevalence. In both studies, we found that shortening duration drastically reduces IAV prevalence in exhibition swine at county fairs. Reduction of viral load in the barn within a county fair is critical to reduce the risk for interspecies IAV transmission and pandemic potential. Therefore, we encourage fair organizers to shorten swine shows to protect the health of both animals and humans.

Spatiotemporal changes in influenza A virus prevalence among wild waterfowl inhabiting the continental United States throughout the annual cycle.

Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.

Reliability of water-based medium-expansion foam as a depopulation method for nursery pigs and cull sows.

Animal disease preparedness plans including depopulation guidelines are paramount to prevent the spread of emerging infectious diseases but difficult to implement for swine under field conditions. However, water-based foam (WBF) is currently an approved and successfully deployed depopulation methodology in poultry. Therefore, the reliability of WBF as a depopulation method and the effectiveness and irreversibility of consciousness and consequential mortality in pigs of different ages was assessed across two trials. Trial 1 investigated the time to loss of consciousness and cessation of cardiac activity in nursery pigs (n = 72) at six different foam immersion time points (2.5, 5, 7.5, 10, 12.5 and 15 min) when placed in a 1.47 m3 (1.2 × 1.2 × 1.02 m, length × width × height) plastic bulk container. One pig per replicate was implanted with an ECG bio-logger. Irreversible loss of consciousness was observed after a 5-min immersion. The average (SD) time to development of a fatal arrhythmia from the initiation of the foam application was 7.3 min (1.82 s). Trial 2 aimed to validate the findings from Trial 1 in 75 larger cull sows across three replicates (n = 25). Sows were loaded into a 41-m3 sealed trailer (12.2 × 1.5 × 2.24 m), immersed in WBF and left undisturbed for 5 min post foam-filling completion. Six pigs in each replicate were implanted with an ECG bio-logger. A 5-min dwell time resulted in irreversible loss of consciousness and subsequent mortality in all cull sows. The average time (SD) to cessation of movement and fatal arrhythmia post foam-filling completion was 2.2 min (34.8 s) and 8.7 min (138.0 s), respectively. While a 5-min immersion in WBF induced irreversible loss of consciousness and death in both trials, a 7.5-min dwell time followed by observation for confirmation of death post WBF removal would be advisable for pigs of all sizes.

Evaluation of a Water-Based Medium-Expansion Foam Depopulation Method in Suckling and Finisher Pigs.

The threat of foreign animal disease introduction through contaminated animal products, feed ingredients, and wildlife vectors have highlighted the need for additional approved methods for mass depopulation of swine under emergency scenarios, especially methods that can be applied to pigs across all production phases. The market disruption within the swine industry due to the SARS-CoV-2 pandemic has demonstrated this lack of preparation. The objective of this study was to validate water-based foam as a mass depopulation method for suckling (18 to 24 days of age) and finisher stage (63 to 100 days of age) pigs. Finisher pigs (n = 31, originally 32 but one finisher pig died prior to foaming), allocated as 9 triads and 1 set of 4 pigs, in 10 total replicates, and suckling pigs (n = 32), randomly allocated to two replicates, were completely covered in water-based medium-expansion foam for a 15-min dwell time in a bulk container. Container fill time for the trials were 6.5 ± 0.68 s and 5.3 ± 0.03 s for finisher and suckling pig replicates, respectively. Average (± SD) time for cessation of movement was 105 ± 39.1 s (s) for finisher pigs and 79.5 ± 10.5 s for suckling pigs. After completion of the 15-min dwell time in the foam, all pigs were confirmed dead upon removal from the container. The results from the present study suggest that the use of water-based foam can be an effective means of mass depopulation for suckling and finisher stage pigs, supporting previous research on the application to adult swine.

Technical Note: Validation of the effectiveness of electric stunning for euthanasia of mature swine (Sus scrofa domesticus).

Electrocution and the use of a penetrating captive bolt gun (PCBG) are both acceptable methods of euthanasia for market weight swine. Research has demonstrated that a PCBG is effective in both growing and mature swine. Given limited to no published research base on electrocution in mature swine, the objectives of the present study were to evaluate the efficacy of a two-stage (head only followed by head to heart, 10 s contact for each) mobile electric stunner (E-STUN, Hubert HAAS TBG 96N) and to assess euthanasia outcomes when comparing E-STUN with the frontal placement of a heavy-duty PCBG (Jarvis, In-line Cylinder Style) when applied to heavy-weight (>200 kg) mature boars and sows. Effectiveness of the E-STUN and PCBG was evaluated first in unconscious anesthetized mature swine (n = 7 boars and sows per treatment; average weight 282 ± 48 kg, n = 28) to reduce the risk of failure in a conscious animal and then in conscious mature swine (n = 3 boars and sows per treatment; average weight 282 ± 63 kg, n = 12). Data from both stages were combined for analyses. Treatment efficacy was defined as any pig that achieved cardiac and respiratory arrest within 10 min after treatment application. A three-point traumatic brain injury score (0 = normal; 1 = some abnormalities; and 2 = grossly abnormal, unrecognizable) was used to evaluate six neuroanatomical structures (cerebral cortex, cerebellum, hypothalamus, thalamus, pons, and brain stem), and the presence of intracranial hemorrhage was also noted. All animals were immediately rendered insensible with E-STUN and PCBG, and no difference was noted between treatments for the detection of corneal reflex following treatment application (P = 0.11). Rhythmic breathing was absent following the administration of either E-STUN or PCBG. When evaluating the time to last heartbeat, there was a significant interaction between sex and treatment. Boars euthanized via E-STUN had a 346.8-s decrease in time to last heartbeat compared with boars euthanized via PCBG (P < 0.001), and females euthanized via E-STUN had a 479.3-s decrease in time to last heartbeat compared with females euthanized via PCBG (P < 0.001). Intracranial hemorrhage was common for both methods, and visible disruption of neural tissue was evident due to the physical nature of the PCBG. This study demonstrated that a mobile E-STUN system is as effective as a heavy-duty PCBG in inducing insensibility and death and shows promise as an alternative method for euthanizing mature pigs on-farm.

Euthanasia is a moral obligation of all individuals working in the swine industry. A majority of acceptable methods have been validated for market weight pigs, while less attention has focused on heavy-weight mature boars and sows. The objectives of the current study were to evaluate the effectiveness of a mobile electric stunner (E-STUN) as a method of humane euthanasia in heavy-weight mature boars and sows and to assess the outcomes when compared with a penetrating captive bolt gun (PCBG) method. The efficacy of the treatment was defined as any pig that achieved cardiac and respiratory arrest within 10 min after treatment application. The amount of traumatic brain injury was evaluated across the brain, and the presence of intracranial hemorrhage was also noted. All animals were immediately rendered insensible, and rhythmic breathing was absent following either treatment application. Boars and sows had a decreased time to last heartbeat with the E-STUN when compared with the PCBG method. Intracranial hemorrhage was common for both methods, and visible disruption of brain tissue was evident due to the physical nature of the PCBG. This study demonstrated that a mobile E-STUN is as effective as a PCBG for humane euthanasia of heavy-weight mature swine.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.