Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Prospective fecal microbiomic biomarkers for chronic wasting disease.

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the United States. Here, we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the USA compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer. Through machine-learning, these taxa and taxa pairs at each rank level could facilitate identification of around 70% of both the CWD-negative and the CWD-positive samples. Our results provide a potential tool for diagnostics and surveillance of CWD in the wild, as well as conceptual advances in our understanding of the disease.IMPORTANCEThis is a comprehensive study that tests the connection between the composition of the gut microbiome in deer in response to chronic wasting disease (CWD). We analyzed 50 fecal samples obtained from CWD-positive animals compared to 50 CWD-negative controls to identify CWD-dependent changes in the gut microbiome, matched with the analysis of fecal metabolites. Our results show promising trends suggesting that fecal microbial composition can directly correspond to CWD disease status. These results point to the microbial composition of the feces as a potential tool for diagnostics and surveillance of CWD in the wild, including non-invasive CWD detection in asymptomatic deer and deer habitats, and enable conceptual advances in our understanding of the disease.

SURVEILLANCE OF BATS IN THE UNITED STATES FOR SARS-COV-2 AND OTHER CORONAVIRUSES.

Bat coronaviruses (CoVs) are extremely prevalent throughout the globe and exhibit a wide range of genetic diversity. Currently, little is known about the susceptibility of New World bats to severe acute respiratory syndrome-2 (SARS-CoV-2), the causative agent of COVID-19. Also, there is limited information about the genetic diversity of other CoVs in the New World bats. The determination of genetic diversity of bat CoVs through continuous surveillance is essential to predict and mitigate the emergence of new CoVs and their impacts on the health of both humans and animals. In this study, 491 guano specimens collected from New World bats and 37 specimens collected from Old World bats during July 2020 to July 2021 were tested for SARS-COV-2 and other CoVs using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) panel and pan-coronavirus PCR that target a highly conserved region of CoVs. No evidence of SARS-CoV-2 was found in the tested specimens. An alpha CoV was detected in a single specimen from a big brown bat (Eptesicus fuscus). This information was used by wildlife agencies and rehabilitation facilities to permit the release of bats during the pandemic while mitigating the risk of spreading SARS-CoV-2 among North American bats and other wild animal populations.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Bat Red Blood Cells Express Nucleic Acid-Sensing Receptors and Bind RNA and DNA.

RBCs demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. However, little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. In this study, we show that bat RBCs express the nucleic acid-sensing TLRs TLR7 and TLR9 and bind the nucleic acid ligands, ssRNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens of humans are concealed in bats.

Age and season predict influenza A virus dynamics in urban gulls: consequences for natural hosts in unnatural landscapes.

Gulls are ubiquitous in urban areas due to a growing reliance on anthropogenic feeding sites, which has led to changes in their abundance, distribution, and migration ecology, with implications for disease transmission. Gulls offer a valuable model for testing hypotheses regarding the dynamics of influenza A virus (IAV) - for which gulls are a natural reservoir in urban areas. We sampled sympatric populations of Ring-billed (Larus delawarensis), Herring (L. argentatus), and Great Black-backed Gulls (L. marinus) along the densely populated Atlantic rim of North America to understand how IAV transmission is influenced by drivers such as annual cycle, host species, age, habitat type, and their interplay. We found that horizontal transmission, rather than vertical transmission, played an outsized role in the amplification of IAV due to the convergence of gulls from different breeding grounds and age classes. We detected overlapping effects of age and season in our prevalence model, identifying juveniles during autumn as the primary drivers of the seasonal epidemic in gulls. Gulls accumulated immunity over their lifespan, however short-term fluctuations in seroprevalence were observed, suggesting that migration may impose limits on the immune system to maintain circulating antibodies. We found that gulls in coastal urban habitats had higher viral prevalence than gulls captured inland, correlating with higher richness of waterbird species along the coast, a mechanism supported by our movement data. The peak in viral prevalence in newly fledged gulls that are capable of long-distance movement has important implications for the spread of pathogens to novel hosts during the migratory season as well as for human health as gulls increasingly utilize urban habitats.

Evaluation of a real-time RT-PCR panel for detection of SARS-CoV-2 in bat guano.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is an ongoing global health concern. The exact source of the virus has not been identified, but it is believed that this novel coronavirus originated in animals; bats in particular have been implicated as the primary reservoir of the virus. SARS-CoV-2 can also be transmitted from humans to other animals, including tigers, cats, and mink. Consequently, infected people who work directly with bats could transfer the virus to a wild North American bat, resulting in a new natural reservoir for the virus, and lead to new outbreaks of human disease. We evaluated a reverse-transcription real-time PCR panel for detection of SARS-CoV-2 in bat guano. We found the panel to be highly specific for SARS-CoV-2, and able to detect the virus in bat guano samples spiked with SARS-CoV-2 viral RNA. Our panel could be utilized by wildlife agencies to test bats in rehabilitation facilities prior to their release to the wild, minimizing the risk of spreading this virus to wild bat populations.

Concurrent Infection of Skunk Adenovirus-1, Listeria monocytogenes, and a Regionally Specific Clade of Canine Distemper Virus in One Gray Fox (Urocyon cinereoargenteus) and Concurrent Listeriosis and Canine Distemper in a Second Gray Fox.

One free-ranging Gray fox (Urocyon cinereoargenteus) underwent autopsy following neurologic disease, with findings including morbilliviral inclusions and associated lesions in numerous tissues, adenoviral intranuclear inclusions in bronchial epithelial cells, and septic pleuropneumonia, hepatitis, splenitis, and meningoencephalitis. Molecular diagnostics on fresh lung identified a strain within a distinct clade of canine distemper that is currently unique to wildlife in New England, as well as the emerging multi-host viral pathogen skunk adenovirus-1. Bacterial culture of fresh liver resulted in a pure growth of Listeria monocytogenes, with whole genome sequencing indicating that the isolate had a vast array of antimicrobial resistance and virulence-associated genes. One year later, a second fox was euthanized for inappropriate behavior in a residential area, and diagnostic workup revealed canine distemper and septic L. monocytogenes, with the former closely related to the distemper virus found in the previous fox and the latter divergent from the L. monocytogenes from the previous fox.

Atypical Dermatophytosis in 12 North American Porcupines (Erethizon dorsatum) from the Northeastern United States 2010-2017.

Twelve wild North American porcupines (Erethizon dorsatum) out of a total of 44 of this species examined in an 8-year period were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on seven and five animals, respectively. Atypical dermatophytosis was diagnosed in all cases. Lesions consisted of diffuse severe epidermal hyperkeratosis and mild hyperplasia with mild lymphoplasmacytic dermatitis and no folliculitis. Dermatophytes were noted histologically as hyphae and spores in hair shafts, and follicular and epidermal keratin. Trichophyton sp. was grown in 5/6 animals where culture was performed, with a molecular diagnosis of Arthroderma benhamiae/Trichophyton mentagrophytes in these five cases. Metagenomic analysis of formalin-fixed paraffin-embedded tissue samples from three cases identified fungi from 17 orders in phyla Basidiomycota and Ascomycota. Alteration of therapy from ketaconazole, which was unsuccessful in four out of five early cases, to terbinafine or nitraconazole led to the resolution of disease and recovery to release in four subsequent animals. In all, six animals were euthanized or died due to dermatopathy, no cases resolved spontaneously, and six cases were resolved with therapy. The work we present demonstrates an atypical lesion and anatomical distribution due to dermatophytosis in a series of free-ranging wild porcupines and the successful development of novel techniques for extracting and sequencing nucleic acids from fungus in archival formalin-fixed paraffin-embedded animal tissue.

Infection of eight mesocarnivores in New Hampshire and Vermont with a distinct clade of canine distemper virus in 2016-2017.

Three fishers (Martes pennanti), 2 gray foxes (Urocyon cinereoargenteus), 1 mink (Neovison vison), 1 skunk (Mephitis mephitis), and 1 raccoon (Procyon lotor), from Vermont and New Hampshire, had lesions on autopsy consistent with canine distemper virus (CDV) infections diagnosed in a 12-mo period in 2016-2017. Lesions of CDV infection were most commonly noted in the lungs (8 of 8 animals), urothelium (5 of 8), biliary tract (5 of 8), gastrointestinal tract (4 of 7), and brain (4 of 6). Splenic lesions were seen in 3 animals. The diagnosis was confirmed via immunohistochemistry and virus isolation. Viral genotyping indicated that all 8 animals were infected with a distinct clade of CDV that has only been reported in wildlife in New England, and this clade of viruses is distinct from vaccine strains. During the 12 mo when these cases occurred, no other CDV clade was identified in any other wildlife or domesticated animal submitted from the 2 states.

Experimental Infection of Common Eider Ducklings with Wellfleet Bay Virus, a Newly Characterized Orthomyxovirus.

Wellfleet Bay virus (WFBV), a novel orthomyxovirus in the genus Quaranjavirus, was first isolated in 2006 from carcasses of common eider (Somateria mollissima) during a mortality event in Wellfleet Bay (Barnstable County, Massachusetts, USA) and has since been repeatedly isolated during recurrent mortality events in this location. Hepatic, pancreatic, splenic, and intestinal necrosis was observed in dead eiders. We inoculated 6-week-old common eider ducklings with WFBV in an attempt to recreate the naturally occurring disease. Approximately 25% of inoculated eiders had onset of clinical disease and required euthanasia; an additional 18.75% were adversely affected based on net weight loss during the trial. Control ducklings did not become infected and did not have clinical disease. Infected ducklings with clinical disease had pathologic lesions consistent with those observed during natural mortality events. WFBV was reisolated from 37.5% of the inoculated ducklings. Ducklings surviving to 5 days postinoculation developed serum antibody titers to WFBV.

Detection of Wellfleet Bay Virus Antibodies in Sea Birds of the Northeastern USA.

Wellfleet Bay virus (WFBV) is a recently described orthomyxovirus isolated from the tissues of Common Eiders (Somateria mollissima) collected during recurrent mortality events on Cape Cod, Massachusetts, US. Coastal Massachusetts is the only location where disease or mortality associated with this virus has been detected in wild birds, and a previous seroprevalence study found a significantly higher frequency of viral exposure in eiders from this location than from other areas sampled in North America. This suggests that coastal Massachusetts is an epicenter of WFBV exposure, but the reason for this is unknown. Opportunistic sampling of sympatric species and testing of banked serum was used to investigate potential host range and spatiotemporal patterns of WFBV exposure. Antibodies were detected in Herring Gulls (Larus argentatus), Ring-billed Gulls (Larus delawarensis), a White-winged Scoter (Melanitta fusca), and a Black Scoter (Melanitta nigra). These findings demonstrate the likely occurrence of fall/winter transmission, expand our understanding of the host range of the virus, and provide further insight into the epidemiology of WFBV in the northeastern US.

Aquatic Bird Bornavirus-Associated Disease in Free-Living Canada Geese ( Branta canadensis ) in the Northeastern USA.

During the winter of 2013-14, 22 Canada geese ( Branta canadensis ) were admitted to the Wildlife Clinic at the Cummings School of Veterinary Medicine at Tufts University with nonspecific neurologic abnormalities and emaciation. Five of these geese, along with three geese that were submitted dead, were evaluated via histopathology, immunohistochemistry, and reverse transcription PCR (RT-PCR) for bornaviruses. Histopathologically, six of the eight birds had lymphoplasmacytic encephalitis. One bird, which also had encephalitis, had a dilated esophagus. Lead poisoning, West Nile virus, avian influenza, and avian paramyxovirus infection were excluded from the diagnosis. Brain tissue from all eight geese was positive for bornaviral N-antigen on immunohistochemistry. Frozen brain tissue from five birds was available for bornavirus RT-PCR. Three of the five birds were positive for the bornavirus M gene. Formalin-fixed paraffin-embedded brain tissue was evaluated on the remaining three geese via RT-PCR, with one of these geese testing positive. A bornavirus was subsequently cultured in duck embryo fibroblasts from the brain of one Canada Goose. This virus genome was sequenced, and the virus was identified as aquatic bird bornavirus 1. We were unable to identify any unusual features of this genome that would account for its apparent pathogenicity, given that subclinical infection with bornavirus in waterfowl is common in North America.

Plasmid-mediated resistance to cephalosporins and quinolones in Escherichia coli from American crows in the USA.

American crow (Corvus brachyrhynchos) faeces were tested for Escherichia coli with plasmid-mediated quinolone resistance (PMQR), extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. A total of 590 faecal samples were collected at four roosting sites in the USA and cultivated on selective media. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to assess clonality. Transferability of resistance genes was studied using conjugation and transformation bioassays. In total, 78 (13%, n = 590) cefotaxime-resistant isolates were obtained, of which 66 and 12 displayed AmpC and ESBL phenotypes, respectively. Fifty-four AmpC-producing isolates carried blaCMY-2 . Isolates producing ESBLs contained genes blaCTX-M-27 (5 isolates), blaCTX-M-15 (4), blaCTX-M-14 (2) and blaCTX-M-1 (1). Ninety isolates (15%, n = 590) with reduced susceptibility to ciprofloxacin were obtained, among which 14 harboured PMQR genes aac(6')-Ib-cr (4 isolates), qnrB19 (3), qnrS1 (2), qnrA1 (2), qnrB2 (1), qnrB6 (1) and qnrD3 (1). High genetic diversity was revealed by PFGE and MLST. Epidemiologically important E. coli clones (e.g., ST131, ST405) were identified. Plasmids carrying blaCMY-2 were assigned predominantly to IncA/C (8 plasmids), IncI1/ST23 (5) and IncI1/ST12 (3). The study demonstrates a widespread occurrence of E. coli with ESBL, AmpC and PMQR genes associated with clinically important multidrug-resistant clones and epidemic plasmids, in American crows.

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

UNLABELLED: Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Bathing birds bias beta-diversity: frequent dispersal by gulls homogenizes fauna in a rock-pool metacommunity.

Metacommunity theory generally predicts that regional dispersal of organisms among local habitat patches should influence spatial patterns of species diversity. In particular, increased dispersal rates are generally expected to increase local (alpha) diversity, yet homogenize local communities across the region (decreasing beta-diversity), resulting in no change in regional (gamma) diversity. Although the effect of dispersal on alpha-diversity has garnered much experimental attention, the influence of dispersal rates on diversity at larger spatial scales (beta and gamma) is poorly understood. Furthermore, these theoretical predictions are not well tested in the field, where other environmental factors (e.g., habitat size, resource density) likely also influence species diversity. Here, we used a system of freshwater rock pools on Appledore Island, Maine, USA, to test the effects of dispersal rate on species diversity in metacommunities. The pools exist in clusters (metacommunities) that experience different levels of dispersal imposed by gulls (Larus spp.), which we show to be frequent passive dispersers of rock-pool invertebrates. Although previous research has suggested that waterbirds may disperse aquatic invertebrates, our study is the first to quantify the rate at which such dispersal occurs and determine its effects on species diversity. In accordance with theory, we found that metacommunities experiencing higher dispersal rates had significantly more homogeneous local communities (reduced beta-diversity) and that gamma-diversity was not influenced by dispersal rate. Contrary to theoretical predictions, however, alpha-diversity in the rock pools was not significantly influenced by dispersal. Rather, local diversity was significantly positively related to local habitat size, and both alpha- and gamma-diversity were influenced by the physicochemical environment of the pools. These results provide an important field test of metacommunity theory, highlighting how local and regional factors interact to drive patterns of species diversity in metacommunities, and demonstrate that waterbirds are indeed important dispersal vectors for aquatic invertebrates.

American crows as carriers of vancomycin-resistant enterococci with vanA gene.

We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to '1', consistent with that shown in the 'Results' section and Figure 2.].

Surveillance of Wildlife Diseases: Lessons from the West Nile Virus Outbreak.

The West Nile virus outbreak of 1999 revealed many weaknesses in this country's ability to respond to disease threats that cross species lines. There were issues of poor communication among human, domestic animal, and wildlife health agencies that delayed diagnosis; a lack of diagnostic capacity of wildlife agencies at the state level; the exclusion of captive wildlife from any surveillance efforts; an inability to visualize the geospatial relationship between the human and avian outbreaks in a timely manner; and marked disparities of funding levels across agencies. Wildlife has played an important role in recent emerging infectious diseases, and it is clear that a One Health approach will be necessary to respond to future threats. The question is, are we any better prepared to recognize and respond to a wildlife-related emerging infectious disease than we were 14 years ago? Have the lessons of WNV been learned?

Pilot study of antimicrobial-resistant Escherichia coli in herring gulls (Larus argentatus) and wastewater in the northeastern United States.

Wildlife may be an important reservoir of antibiotic-resistant bacteria and resistance genes. In this pilot study, the prevalence and patterns of antimicrobial resistance in Escherichia coli cultured from wild herring gull (Larus argentatus) feces and human wastewater at Cape Cod, Massachusetts, USA, was compared. Antimicrobial susceptibility was tested using Kirby-Bauer disk diffusion with seven antimicrobial agents. A high proportion of antimicrobial agent-resistant E. coli isolates (59.2%) were detected in wastewater samples compared with a lower prevalence of 17.5% in gull feces. In addition, there was a large proportion of isolates with intermediate susceptibility (93.0%) in gull feces. Although similar resistance patterns and shared resistance genes suggest possible wastewater contamination of the local environment, the relatively low frequency of resistance and high prevalence of intermediate susceptibility detected in E. coli cultured from gull feces depict a complex model of antimicrobial resistance among E. coli strains of wildlife origin.