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.

Avian influenza A virus susceptibility, infection, transmission, and antibody kinetics in European starlings.

Avian influenza A viruses (IAVs) pose risks to public, agricultural, and wildlife health. Bridge hosts are spillover hosts that share habitat with both maintenance hosts (e.g., mallards) and target hosts (e.g., poultry). We conducted a comprehensive assessment of European starlings (Sturnus vulgaris), a common visitor to both urban and agricultural environments, to assess whether this species might act as a potential maintenance or bridge host for IAVs. First, we experimentally inoculated starlings with a wild bird IAV to investigate susceptibility and replication kinetics. Next, we evaluated whether IAV might spill over to starlings from sharing resources with a widespread IAV reservoir host. We accomplished this using a specially designed transmission cage to simulate natural environmental transmission by exposing starlings to water shared with IAV-infected mallards (Anas platyrhynchos). We then conducted a contact study to assess intraspecies transmission between starlings. In the initial experimental infection study, all inoculated starlings shed viral RNA and seroconverted. All starlings in the transmission study became infected and shed RNA at similar levels. All but one of these birds seroconverted, but detectable antibodies were relatively transient, falling to negative levels in a majority of birds by 59 days post contact. None of the contact starlings in the intraspecies transmission experiment became infected. In summary, we demonstrated that starlings may have the potential to act as IAV bridge hosts if they share water with IAV-infected waterfowl. However, starlings are unlikely to act as maintenance hosts due to limited, if any, intraspecies transmission. In addition, starlings have a relatively brief antibody response which should be considered when interpreting serology from field samples. Further study is needed to evaluate the potential for transmission from starlings to poultry, a possibility enhanced by starling's behavioral trait of forming very large flocks which can descend on poultry facilities when natural resources are scarce.

Intercontinental Movement of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4 Virus to the United States, 2021.

We detected Eurasian-origin highly pathogenic avian influenza A(H5N1) virus belonging to the Gs/GD lineage, clade 2.3.4.4b, in wild waterfowl in 2 Atlantic coastal states in the United States. Bird banding data showed widespread movement of waterfowl within the Atlantic Flyway and between neighboring flyways and northern breeding grounds.

SARS-CoV-2 Exposure in Escaped Mink, Utah, USA.

In August 2020, outbreaks of coronavirus disease were confirmed on mink farms in Utah, USA. We surveyed mammals captured on and around farms for evidence of infection or exposure. Free-ranging mink, presumed domestic escapees, exhibited high antibody titers, suggesting a potential severe acute respiratory syndrome coronavirus 2 transmission pathway to native wildlife.

Persistence of maternal antibodies to influenza A virus among captive mallards (Anas platyrhynchos).

Wild waterfowl are maintenance hosts of most influenza A virus (IAV) subtypes and are often the subjects of IAV surveillance and transmission models. While maternal antibodies have been detected in yolks and in nestlings for a variety of wild bird species and pathogens, the persistence of maternal antibodies to IAVs in mallard ducklings (Anas platyrhynchos) has not been previously investigated. Nonetheless, this information is important for a full understanding of IAV transmission dynamics because ducklings protected by maternal antibodies may not be susceptible to infection. In this study, we examined the transfer of IAV-specific maternal antibodies to ducklings. Blood samples were collected approximately every five days from ducklings hatched from hens previously infected with an H6 strain of IAV. Serum samples were tested for antibodies to IAV by an enzyme-linked immunosorbent assay. The median persistence of maternal antibodies in ducklings was 12.5 days (range: 4-33 days) post-hatch. The majority of ducklings (71%) had detectable maternal antibodies from 4 to 17 days post-hatch, while a small subset of individuals (29%) had detectable maternal antibodies for up to 21-33 days post-hatch. Antibody concentrations in hens near the time of egg laying were correlated with maternal antibody concentrations in the initial blood sample collected from ducklings (0-4 days post-hatch). Knowledge of the duration of maternal antibodies in ducklings will aid in the interpretation of IAV serological surveillance results and in the modeling of IAV transmission dynamics in waterfowl.

Low viral doses are sufficient to infect cottontail rabbits with avian influenza A virus.

Influenza A viruses (IAVs) have been reported in wild lagomorphs in environments where they share resources with waterfowl. Recent studies have conclusively shown that a North American lagomorph, cottontail rabbits (Sylvilagus sp.), become infected following exposure to IAVs and can shed significant quantities of virus. However, the minimum infectious dose and the efficiency of various routes of infection have not been evaluated. Thirty-six cottontail rabbits were used in a dose response study assessing both the oral and nasal routes of infection. The nasal route of infection proved to be the most efficient, as all cottontail rabbits shed viral RNA following inoculation with doses as low as 102 EID50. The oral route of infection was less efficient, but still produced infection rates of ≥ 50% at relatively low doses (i.e., 103 and 104 EID50). These results suggest that cottontail rabbits are highly susceptible to IAVs at low exposure doses that have been routinely observed in environments contaminated by waterfowl. Furthermore, this study supports earlier observations that cottontail rabbits may pose a biosecurity risk to poultry operations, as a virus-contaminated water source or contaminated environment, even at low viral titers, could be sufficient to initiate viral replication in cottontail rabbits.

Transmission of H6N2 wild bird-origin influenza A virus among multiple bird species in a stacked-cage setting.

Live bird markets are common in certain regions of the U.S. and in other regions of the world. We experimentally tested the ability of a wild bird influenza A virus to transmit from index animals to naïve animals at varying animal densities in stacked cages in a simulated live bird market. Two and six mallards, five and twelve quail, and six and nine pheasants were used in the low-density and high-density stacks of cages, respectively. Transmission did not occur in the high-density stack of cages likely due to the short duration and relatively low levels of shedding, a dominance of oral shedding, and the lack of transmission to other mallards in the index cage. In the low-density stack of cages, transmission occurred among all species tested, but not among all birds present. Oral and cloacal shedding was detected in waterfowl but only oral shedding was identified in the gallinaceous birds tested. Overall, transmission was patchy among the stacked cages, thereby suggesting that chance was involved in the deposition of shed virus in key locations (e.g., food or water bowls), which facilitated transmission to some birds.

Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Susceptibility of rock doves to low-pathogenic avian influenza A viruses.

Following a 2008 outbreak of North American low-pathogenic H5N8 influenza A virus at an upland gamebird farm, we sero-sampled rock doves (pigeons, Columba livia) at the outbreak site and conducted experimental inoculations of wild-caught pigeons using the H5N8 virus and another low-pathogenic virus (H4N6). While 13% of pigeons at the outbreak site were seropositive, none were positive for exposure to H5, and one was positive for N8. Challenged pigeons exhibited low susceptibility and limited viral RNA excretion for both viruses tested, but at least one individual had RNA loads indicative of the potential for viral transmission to other birds.

Wildlife contact rates at artificial feeding sites in Texas.

Given the popularity of feeding white-tailed deer (Odocoileus virginianus) in Texas and the increasing amount of corn that is distributed, more information is needed on the impacts of this activity on non-target wildlife. Our objectives were to report visitation, intra- and interspecific contact, and contact rates of wildlife at artificial feeding sites in Texas. Our study was conducted at three sites in Kleberg and Nueces counties, Texas. We trapped animals from February to April and August to September, 2009 and marked animals with passive integrated transponder (PIT) tags. At each site and season, we placed one feeder system containing a PIT tag reader within 600 m of trap locations. Readers detected PIT tags from a distance of 25 cm. We determined a contact event to occur when two different PIT tags were detected by feeder systems within 5 s. We recorded 62,719 passes by raccoons (Procyon lotor), 103,512 passes by collared peccaries (Pecari tajacu), 2,923 passes by feral swine (Sus scrofa), 1,336 passes by fox squirrels (Sciurus niger), and no passes by opossums (Didelphis virginiana) at feeder systems. For site-season combinations in which contact events occurred, we found intraspecific contact rates (contacts per day) for raccoons, collared peccaries, and feral swine to be 0.81-124.77, 0.69-38.08, and 0.0-0.66, respectively. Throughout our study we distributed ~2,625 kg of whole kernel corn, which resulted in 6,351 contact events between marked wildlife (2.4 contacts per kg of corn). If 136 million kg of corn is distributed in Texas annually, we would expect >5.2 billion unnatural contact events between wildlife would result from this activity each year in Texas. Consequently, we do not believe that it is wise for natural resource managers to maintain artificial feeding sites for white-tailed deer or other wildlife due to pathogen transmission risks.

Environmental transmission of influenza A virus in mallards.

IMPORTANCE: Wild birds are the natural reservoir hosts of influenza A viruses. Highly pathogenic strains of influenza A viruses pose risks to wild birds, poultry, and human health. Thus, understanding how these viruses are transmitted between birds is critical. We conducted an experiment where we experimentally infected mallards which are ducks that are commonly exposed to influenza viruses. We exposed several contact ducks to the experimentally infected duck to estimate the probability that a contact duck would become infected from either exposure to the virus shed directly from the infected duck or shared water contaminated with the virus from the infected duck. We found that environmental transmission from contaminated water best predicted the probability of transmission to naïve contact ducks, relatively low levels of virus in the water were sufficient to cause infection, and the probability of a naïve duck becoming infected varied over time.

Epidemiologic and Genomic Evidence for Zoonotic Transmission of SARS-CoV-2 among People and Animals on a Michigan Mink Farm, United States, 2020.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility's overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.

Strength in numbers: Avian influenza A virus transmission to poultry from a flocking passerine.

The effects of flock size of European starlings (Sturnus vulgaris) was experimentally manipulated to assess the potential of influenza A virus (IAV; H4N6) transmission from a flocking passerine to bobwhite quail (Colinus virginianus) through shared food and water resources to mimic starling intrusions into free-range and backyard poultry operations. Of the three starling flock sizes tested (n = 30, n = 20 and n = 10), all successfully transmitted the virus to all or most of the quail in each animal room (6/6, 6/6 and 5/6) by the end of the experimental period, as determined by seroconversion and/or viral RNA shedding. Although starlings have been shown to be inconsistent shedders of IAVs and when they do replicate and subsequently shed virus they typically do so at low to moderate levels, this study has provided evidence that relatively small flocks (i.e., 10 or possibly a smaller number) of this species can collectively transmit the virus to a highly susceptible gallinaceous bird species. Future work should assess if starlings can transmit IAVs to additional poultry species commonly found in backyard or free-range settings.

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.

Plague Exposure in Mammalian Wildlife Across the Western United States.

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.

Biodetection of a specific odor signature in mallard feces associated with infection by low pathogenic avian influenza A virus.

Outbreaks of avian influenza virus (AIV) infection included the spread of highly pathogenic AIV in commercial poultry and backyard flocks in the spring of 2015. This resulted in estimated losses of more than $8.5 million from federal government expenditures, $1.6 billion from direct losses to produces arising from destroyed turkey and chicken egg production, and economy-wide indirect costs of $3.3 billion from impacts on retailers and the food service industries. Additionally, these outbreaks resulted in the death or depopulation of nearly 50 million domestic birds. Domesticated male ferrets (Mustela putorius furo) were trained to display a specific conditioned behavior (i.e. active scratch alert) in response to feces from AIV-infected mallards in comparison to feces from healthy ducks. In order to establish that ferrets were identifying samples based on odors associated with infection, additional experiments controlled for potentially confounding effects, such as: individual duck identity, housing and feed, inoculation concentration, and day of sample collection (post-infection). A final experiment revealed that trained ferrets could detect AIV infection status even in the presence of samples from mallards inoculated with Newcastle disease virus or infectious laryngotracheitis virus. These results indicate that mammalian biodetectors are capable of discriminating the specific odors emitted from the feces of non-infected versus AIV infected mallards, suggesting that the health status of waterfowl can be evaluated non-invasively for AIV infection via monitoring of volatile fecal metabolites. Furthermore, in situ monitoring using trained biodetectors may be an effective tool for assessing population health.

An Opportunistic Survey Reveals an Unexpected Coronavirus Diversity Hotspot in North America.

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.

A Review of Avian Influenza A Virus Associations in Synanthropic Birds.

Avian influenza A viruses (IAV) have received significant attention due to the threat they pose to human, livestock, and wildlife health. In this review, we focus on what is known about IAV dynamics in less common avian species that may play a role in trafficking IAVs to poultry operations. Specifically, we focus on synanthropic bird species. Synanthropic species, otherwise known as peridomestic, are species that are ecologically associated with humans and anthropogenically modified landscapes, such as agricultural and urban areas. Aquatic birds such as waterfowl and shorebirds are the species most commonly associated with avian IAVs, and are generally considered the reservoir or maintenance hosts in the natural ecology of these viruses. Waterfowl and shorebirds are occasionally associated with poultry facilities, but are uncommon or absent in many areas, especially large commercial operations. In these cases, spillover hosts that share resources with both maintenance hosts and target hosts such as poultry may play an important role in introducing wild bird viruses onto farms. Consequently, our focus here is on what is known about IAV dynamics in synanthropic hosts that are commonly found on both farms and in nearby habitats, such as fields, lakes, wetlands, or riparian areas occupied by waterfowl or shorebirds.