High prevalence of anticoagulant rodenticide exposure in New England Fishers (Pekania pennanti).

Anticoagulant rodenticides (ARs) are increasingly recognized as a threat to non-target species including native wildlife. Fishers (Pekania pennanti) are generally considered deep forest inhabitants that are not expected to have high exposure to ARs. To evaluate the distribution and levels of ARs in fishers, we analyzed liver samples from fisher carcasses (N = 45) opportunistically trapped across Vermont and New Hampshire between 2018 and 2019. Liquid chromatography-mass spectrometry was used to detect and quantify 11 different ARs in the liver tissue of each fisher at the time of trapping. All but one sample analyzed were positive for exposure to ARs, and 84% were positive for more than one type of AR. The most prevalent ARs detected were diphacinone (96%) and brodifacoum (80%). No samples had detectable levels of coumachlor, coumafuryl, difenacoum, pindone, or warfarin. These results are mostly consistent with findings for fishers in California as well as with a variety of rodent specializing avifauna throughout the Northeast USA but, show a higher prevalence of exposure and a different distribution of AR types than other studies. These results help establish current baseline exposure to ARs in fishers in the Northeast USA and suggest that ARs could pose a threat to wild mesocarnivore species in this region.

Avian coxiellosis in nine psittacine birds, one black-browed barbet, and one paradise tanager.

Infection with a novel species of the genus Coxiella was first described in three Swainson's blue mountain rainbow lorikeets from a zoological collection, and days later in a group of seven other psittacine birds and a toucan. We provide an update on coxiellosis in nine additional psittacines, and two non-psittacines. Psittacines originated in New England, the mid Atlantic, the Midwest, the South, and the Northwest. Psittacines most commonly had lesions in the brain, spleen, liver, and lungs, consisting of meningoencephalitis, hepatosplenomegaly, and interstitial pneumonia. Lesions contained histiocytic infiltrate, with intracytoplasmic, Gimenez-positive coccobacilli. Transmission electron microscopy revealed bacteria with trilaminar cell walls, electron dense cores, and spore-like forms. PCR revealed homology to the organism in index cases. In addition, one black-browed barbet and one paradise tanager were found with systemic coxiellosis; sequencing identified the same pathogen. These are the second piciforme and the first passerine affected by this pathogen, indicating expanded infectivity and pathogenicity.RESEARCH HIGHLIGHTS Report of coxiellosis in nine psittacines; lesions often in brain, spleen, liver, lung.Second piciforme with coxiellosis, a black-browed barbet.First case of avian coxiellosis described in a passerine, a paradise tanager.

GENETIC CHARACTERISTICS OF CANINE DISTEMPER VIRUSES CIRCULATING IN WILDLIFE IN THE UNITED STATES.

Canine distemper virus (CDV) is a highly contagious disease of wild and domestic mammals. Maintenance of CDV among wildlife plays an important role in the disease epidemiology. Wild animals, including raccoons (Procyon lotor) and gray foxes (Urocyon cinereoargenteus), serve as reservoirs of CDV and hamper the control of the disease. Recently, we discovered that at least three different CDV lineages (America-3 [Edomex], America-4, and America-5] that are genetically different from the available vaccine strains are circulating in domestic dogs in the United States. Because wildlife serve as a reservoir for the virus, it is important to determine if wildlife play a role in the maintenance and spread of these lineages. To determine the genetic characteristics of circulating strains of CDV in wildlife in various geographic regions in the United States, we studied the nucleotide sequences of the hemagglutinin (H) gene of 25 CDV strains detected in nondomestic species. The species included were free-ranging wildlife: three fishers (Martes pennanti), six foxes, one skunk (Mephitis mephitis), 10 raccoons, two wolves (Canis lupus), and one mink (Neovison vison). Strains from two species in managed care, one sloth (Choloepus didactylus) and one red panda (Ailurus fulgens), were also evaluated. Phylogenetic analysis of the H genes indicated that in addition to America-3, America-4, and America-5 lineages, there are at least two other lineages circulating in US wildlife. One of these includes CDV nucleotide sequences that grouped with that of a single CDV isolate previously detected in a raccoon from Rhode Island in 2012. The other lineage is independent and genetically distinct from other CDV strains included in the analysis. Additional genetically variable strains were detected, mainly in raccoons, suggesting that this species may be the host responsible for the genetic variability of newly detected strains in the domestic dog population.

Necrotizing Ventriculitis in Fledgling Chimney Swifts (Chaetura Pelagica) Associated With a Novel Adenovirus, Chimney Swift Adenovirus-1 (CsAdV-1).

Five chimney swift fledglings died following a progressive loss of appetite and condition while being cared for by an experienced wildlife rehabilitator. All animals had severe necrotizing and heterophilic ventriculitis, with myriad epithelial cells characterized by karyomegaly with intranuclear inclusion bodies. Transmission electron microscopy showed distention of epithelial cell nuclei and chromatin peripheralization by nonenveloped, icosahedral, 75- to 85-nm-diameter virions. Degenerate nested PCR for a highly conserved region of the adenovirus DNA polymerase gene was positive. BLAST analysis of the amplicon sequence indicated the presence of a novel adenovirus, with 74% homology to Antarctic penguin adenoviruses and 72% homology to a bat adenovirus, at low query coverages of only 65% and 63%, respectively. BLAST analysis of the predicted amino acid sequence generated the highest scores for squamate adenoviruses at 100% query coverage. Based on phylogenetic analysis of the partial amino acid sequence of the DNA polymerase, the chimney swift virus was a novel adenovirus most closely related to the Atadenovirus genus. Using a probe based on the novel viral sequence, DNA in situ hybridization identified viral nucleic acid in the nucleus. While the tentatively named chimney swift adenovirus-1 (CsAdV-1) is so far classified with the Atadenoviruses, it is relatively divergent from other members of that genus and may represent the first identified member of a new genus of Adenoviruses.

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4.

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as 'Generally Recognized as Safe' (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

Apparent fatal winter tick (Dermacentor albipictus) infestation in captive reindeer (Rangifertarandus).

The winter tick, Dermacentor albipictus (Ixodidae), commonly infests a wide variety of wild and domestic ungulates throughout North America. This one-host-tick infests animals from October to April, with moose (Alces alces) particularly affected. Animals highly infested may present with anemia, tick-induced alopecia, and alterations in thermoregulation, often resulting in death. Mortality from winter tick infestation has been reported in free-ranging woodland caribou (Rangifer tarandus caribou) and captive reindeer in Alberta, Canada. This historic report raises concern about mortality due to D. albipictus in a wider host range, specifically on translocated caribou. The aim of this report was to describe three cases of winter tick infestation in captive reindeer resulting in severe anemia and mortality likely due to the infestation in New Hampshire, northeastern United States (US). Additionally, ticks were screened molecularly for the detection of tick-borne pathogens. At time of necropsy, all three reindeer showed decreased nutritional status, marked submandibular edema, and had heavy D. albipictus infestation. None of the reindeer exhibited alopecia, which is a common clinical manifestation in moose that die from winter tick infestation. No pathogens were detected via qPCR screening. This report highlights the risk that captive cervids face in areas where winter tick is endemic; therefore, the adoption of preventive control measures should be encouraged to reduce the risk of tick infestation and potential death of these animals.

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.

Chromosome-level assembly of the gray fox (Urocyon cinereoargenteus) confirms the basal loss of PRDM9 in Canidae.

Reference genome assemblies have been created from multiple lineages within the Canidae family; however, despite its phylogenetic relevance as a basal genus within the clade, there is currently no reference genome for the gray fox (Urocyon cinereoargenteus). Here, we present a chromosome-level assembly for the gray fox (U. cinereoargenteus), which represents the most contiguous, non-domestic canid reference genome available to date, with 90% of the genome contained in just 34 scaffolds and a contig N50 and scaffold N50 of 59.4 and 72.9 Megabases, respectively. Repeat analyses identified an increased number of simple repeats relative to other canids. Based on mitochondrial DNA, our Vermont sample clusters with other gray fox samples from the northeastern United States and contains slightly lower levels of heterozygosity than gray foxes on the west coast of California. This new assembly lays the groundwork for future studies to describe past and present population dynamics, including the delineation of evolutionarily significant units of management relevance. Importantly, the phylogenetic position of Urocyon allows us to verify the loss of PRDM9 functionality in the basal canid lineage, confirming that pseudogenization occurred at least 10 million years ago.

Glaucoma associated with uveal cysts and goniodysgenesis in American Bulldogs: a case series.

We present a series of three American Bulldogs with clinical signs of glaucoma and intraocular inflammation accompanied by bilateral uveal cysts and abnormal gonioscopic findings. All dogs proved refractory to medical management and were enucleated. Histopathologic findings were similar in all three and included significant preiridal fibrovascular membranes and mononuclear inflammatory infiltrates in the anterior uvea. On microscopic evaluation, cysts appeared to arise primarily from the ciliary body and iridociliary sulcus, with smaller cysts also budding from the posterior iris. Pigment dispersion was variable but consistent, involving deposition of a small number of pigment-laden cells in the dependent trabecular meshwork. Cataract formation was not noted. Glaucoma associated with uveal cysts has been described previously in Golden Retrievers and Great Danes, although clinical and histopathologic findings in those breeds are not identical to those described here. American Bulldogs with uveal cysts should have gonioscopy performed and should be monitored carefully for signs of increased intraocular pressure and intraocular inflammation. Furthermore, documentation of cyst-associated glaucoma in a third breed suggests clinicians should exercise caution in dismissing uveal cysts in dogs as incidental findings.

Accessory Genome Dynamics of Local and Global Staphylococcus pseudintermedius Populations.

Staphylococcus pseudintermedius is a major bacterial colonizer and opportunistic pathogen in dogs. Methicillin-resistant S. pseudintermedius (MRSP) continues to emerge as a significant challenge to maintaining canine health. We sought to determine the phylogenetic relationships of S. pseudintermedius across five states in the New England region of the United States and place them in a global context. The New England dataset consisted of 125 previously published S. pseudintermedius genomes supplemented with 45 newly sequenced isolates. The core genome phylogenetic tree revealed many deep branching lineages consisting of 142 multi-locus sequence types (STs). In silico detection of the mecA gene revealed 40 MRSP and 130 methicillin-susceptible S. pseudintermedius (MSSP) isolates. MRSP were derived from five structural types of SCCmec, the mobile genetic element that carries the mecA gene conferring methicillin resistance. Although many genomes were MSSP, they nevertheless harbored genes conferring resistance to many other antibiotic classes, including aminoglycosides, macrolides, tetracyclines and penams. We compared the New England genomes to 297 previously published genomes sampled from five other states in the United States and 13 other countries. Despite the prevalence of the clonally expanding ST71 found worldwide and in other parts of the United States, we did not detect it in New England. We next sought to interrogate the combined New England and global datasets for the presence of coincident gene pairs linked to antibiotic resistance. Analysis revealed a large co-circulating accessory gene cluster, which included mecA as well as eight other resistance genes [aac (6')-Ie-aph (2″)-Ia, aad (6), aph (3')-IIIa, sat4, ermB, cat, blaZ, and tetM]. Furthermore, MRSP isolates carried significantly more accessory genes than their MSSP counterparts. Our results provide important insights to the evolution and geographic spread of high-risk clones that can threaten the health of our canine companions.

Shared antibiotic resistance and virulence genes in Staphylococcus aureus from diverse animal hosts.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) poses an important threat in human and animal health. In this study, we ask whether resistance and virulence genes in S. aureus are homogeneously distributed or constrained by different animal hosts. We carried out whole genome sequencing of 114 S. aureus isolates from ten species of animals sampled from four New England states (USA) in 2017-2019. The majority of the isolates came from cats, cows and dogs. The maximum likelihood phylogenetic tree based on the alignment of 89,143 single nucleotide polymorphisms of 1173 core genes reveal 31 sequence types (STs). The most common STs were ST5, ST8, ST30, ST133 and ST2187. Every genome carried at least eight acquired resistance genes. Genes related to resistance found in all genomes included norA (fluoroquinolone), arlRS (fluoroquinolone), lmrS (multidrug), tet(38) (tetracycline) and mepAR (multidrug and tigecycline resistance). The most common superantigen genes were tsst-1, sea and sec. Acquired antibiotic resistance (n = 10) and superantigen (n = 9) genes of S. aureus were widely shared between S. aureus lineages and between strains from different animal hosts. These analyses provide insights for considering bacterial gene sharing when developing strategies to combat the emergence of high-risk clones in animals.

Relationship between Uveal Inflammation and Viral Detection in 30 Cats with Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) virus is the most common infectious cause of uveitis in cats. Confirmatory diagnosis is usually only reached at postmortem examination. The relationship between the histologic inflammatory pattern, which depends on the stage of the disease, and the likelihood of detection of the viral antigen and/or RNA has not been investigated. We hypothesized that viral detection rate by either immunohistochemistry, in situ hybridization or RT-qPCR is dependent upon the predominant type of uveal inflammatory response (i.e., pyogranulomatous vs. plasmacytic). Thus, the aims of this study were to evaluate cases of FIP-induced uveitis, localize the viral antigen and RNA, and assess the relationship between the inflammatory pattern (macrophage- vs. plasma cell-rich) and the likelihood of detecting the FIP antigen and/or RNA. We evaluated 30 cats with FIP-induced uveitis. The viral antigen and/or RNA were detected within uveal macrophages in 11/30 cases, of which 8 tested positive by RT-qPCR. Correlation analysis determined a weak to moderate but significant negative correlation between the degree of plasmacytic uveal inflammation and the likelihood of detecting the FIP antigen and RNA. This study suggests that predominance of plasmacytic inflammation in cases of FIP uveitis reduces the odds of a confirmatory diagnosis through the viral detection methods available.

Genome Sequencing of Methicillin-Resistant and Methicillin-Susceptible Mammaliicoccus sciuri from Diseased Animals.

Mammaliicoccus sciuri (previously Staphylococcus sciuri) is a frequent colonizer of mammals. We report the draft genomes of a methicillin-resistant strain (2254A) isolated from an armadillo and a methicillin-susceptible strain (6942A) from a cow. Genomes were sequenced using long-read Nanopore sequencing.

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.

Fatal bronchopneumonia caused by skunk adenovirus 1 in an African pygmy hedgehog.

Eleven adult African pygmy hedgehogs ( Atelerix albiventris) were added to a group of 35 animals, and within 10 d, respiratory distress affected 8 of 35 resident animals in the group, but none of the introduced animals. Three animals died following onset of clinical signs. Tissues from one animal were collected and submitted for histopathology, which revealed acute necrotizing bronchopneumonia and tracheitis with intraepithelial intranuclear inclusion bodies. Electron microscopy identified 75-90 nm diameter encapsulated icosahedral virions. Degenerate nested PCR analysis identified adenovirus within the affected lung tissue. Deep sequencing showed 100% homology to skunk adenovirus 1 (SkAdV-1). Adenoviruses are usually species-adapted and -specific, but our case supports the single previous report of non-skunk infection with SkAdV-1, indicating that this virus can infect other species, and further shows that it can cause fatal disease.

Identification of a Novel Gammaherpesvirus in Canada lynx (Lynx canadensis).

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22-53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8-31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study.

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.

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.

Biphasic malignant melanoma adenocarcinoma in the digit of a dog.

A 7-y-old Black Russian Terrier dog was evaluated for chronic lameness of the right forelimb and concurrent weight loss. Clinical examination revealed a pigmented mass arising from digit 4 of the right forelimb; the digit was amputated and submitted for histologic evaluation. Histologically, the neoplasm was composed of ill-defined streams, sheets, and clusters of melanocytes admixed with a distinct population of malignant epithelial cells forming glands and nests. The diagnosis was a biphasic malignant melanoma adenocarcinoma, a rarely reported neoplasm in human medicine that has not been described in veterinary medicine, to our knowledge.