Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022.

We describe the pathology of natural infection with highly pathogenic avian influenza A(H5N1) virus of Eurasian lineage Goose/Guangdong clade 2.3.4.4b in 67 wild terrestrial mammals throughout the United States during April 1‒July 21, 2022. Affected mammals include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals showed primarily neurologic signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

Infection and tissue distribution of highly pathogenic avian influenza A type H5N1 (clade 2.3.4.4b) in red fox kits (Vulpes vulpes).

Avian influenza H5N1 is a highly pathogenic virus that primarily affects birds. However, it can also infect other animal species, including mammals. We report the infection of nine juvenile red foxes (Vulpes vulpes) with Highly Pathogenic Avian Influenza A type H5N1 (Clade 2.3.4.4b) in the spring of 2022 in the central, western, and northern regions of New York, USA. The foxes displayed neurologic signs, and examination of brain and lung tissue revealed lesions, with brain lesions ranging from moderate to severe meningoencephalitis. Analysis of tissue tropism using RT-PCR methods showed a comparatively lower Ct value in the brain, which was confirmed by in situ hybridization targeting Influenza A RNA. The viral RNA labelling was highly clustered and overlapped the brain lesions, observed in neurons, and grey matter. Whole viral genome sequences obtained from the affected foxes were subjected to phylogenetic and mutation analysis to determine influenza A clade, host specificity, and potential occurrence of viral reassortment. Infections in red foxes likely occurred due to preying on infected wild birds and are unlikely due to transmission between foxes or other mammals.

Pharmacokinetics of zonisamide after oral single dosing and multiple-dose escalation administration in domestic chickens (Gallus gallus).

BACKGROUND: There are few effective drugs for treatment of seizures in avian species. OBJECTIVES: To investigate the pharmacokinetics and safety of zonisamide in chickens. METHODS: Phase 1: chickens (n = 4) received a single oral dose of zonisamide at 20 mg/kg. Blood samples were collected intermittently for 36 hr after dosing. Phase 2: chickens (n = 8) received zonisamide in a dose escalation protocol (20, 30, 60 and 80 mg/kg orally every 12 hr). The dose was increased weekly, and peak and trough blood samples were collected on Days 1, 3, and 7 each week. Two birds were randomly euthanized at the end of each week. Plasma zonisamide concentrations were analysed using a commercial immunoassay. Drug concentration vs. time data were subjected to non-compartmental pharmacokinetic analysis. RESULTS: For Phase 1, peak plasma zonisamide (Cmax ) was 15 ± 3 µg/ml at 2 ± 1 hr (Tmax ). The disappearance half-life was 6.5 ± 1 hr. Mean plasma concentrations remained within the (human) therapeutic range (10-40 µg/ml) for 6 hr. For Phase 2 of the study, plasma concentrations of zonisamide remained within or close to the recommended mammalian therapeutic range for birds in the 20 and 30 mg/kg dose. Area under the curve (AUC) and Cmax were dose dependent. Two birds developed immune-mediated haemolytic anaemia. CONCLUSIONS: Zonisamide appears to be a viable drug for use in chickens at a dose of 20 mg/kg orally every 12 hr.

A NOVEL ORTHOREOVIRUS ASSOCIATED WITH EPIZOOTIC NECROTIZING ENTERITIS AND SPLENIC NECROSIS IN AMERICAN CROWS (CORVUS BRACHYRHYNCHOS).

Epizootic mortalities in American Crows (Corvus brachyrhynchos) during the winter months, referred to as winter mortality of crows, have been recorded in North America for almost two decades. The most common postmortem findings include necrotizing enteritis, colitis, and fibrinous splenic necrosis. These findings are proposed to be due to infection with a Reovirus sp. Our objectives were to characterize the pathology and seasonality of the epizootics in New York State (NYS), confirm the causative role of an Orthoreovirus sp., and determine its phylogeny. On the basis of our proposed case definition for reovirosis, we examined case data collected by the NYS Wildlife Health Program for 16 yr. A total of 558 cases of reovirosis were recorded between 2001 and 2017. Reovirosis had a clear seasonal presentation: cases occurred almost exclusively in winter months (71% in December-January). Detailed data from a 2-yr period (2016-17) demonstrated that reovirosis caused up to 70% of all recorded crow deaths during epizootic months. Crows with positive orthoreovirus isolation from the spleen or intestine were 32 times more likely to die with characteristic histologic lesions of enteritis or enterocolitis and splenic necrosis than crows with negative isolation results. An in situ hybridization probe specific to virus isolated from NYS crow reovirosis cases demonstrated a direct association between viral presence and characteristic histologic lesions. Sigma C (capsid protein) sequences of isolates from NYS crows showed high homology with Tvärminne avian virus, recently proposed as a novel Corvus orthoreovirus clade, and only distantly related to the avian orthoreovirus clade. Our study indicated that a novel orthoreovirus was the cause of winter mortality (or reovirosis) of American Crows and placed the NYS isolates in the newly proposed genus of Corvid orthoreovirus.

Oral Capillariosis Due to Eucoleus dispar in Migrating Sharp-shinned Hawks (Accipiter striatus) in New York, USA, 2016-18.

Oral lesions focused around the oral commissures were documented in several Sharp-shinned Hawks (Accipiter striatus) in the 2016-18 spring migration season at a banding station located on the southern shore of Lake Ontario, New York, US. Samples of the inflamed and caseous lesions repeatedly tested negative for Trichomonas gallinae and poxvirus; however, large numbers of capillariid eggs and embedded worms were consistently present. Morphologically, the nematodes were identified as Eucoleus dispar, which was confirmed by PCR and genetic sequencing. The affected hawks displayed no other clinical signs of illness, were in good body condition, and were released back into their migration pathway shortly after examination and testing. We report a unique clinical presentation for oral capillariosis in A. striatus.

Eastern equine encephalitis in puppies in Michigan and New York states.

Eastern equine encephalitis virus (EEEV) is classified as a select agent and is capable of causing mortality in humans and a number of veterinary species. Herein, we describe 3 cases of EEE in puppies in Michigan and New York. Two puppies were euthanized following an acute history of seizures and obtundation. A littermate of one of these puppies died 2 wk earlier following a history of anorexia and fever. All 3 puppies lacked significant gross anatomic lesions at autopsy and tested negative for rabies virus. In all 3 puppies, histologic examination revealed necrotizing, neutrophilic, lymphoplasmacytic meningoencephalitis with strong positive immunohistochemical labeling for EEEV antigen in neurons and glial cells. The diagnosis of EEE was confirmed by PCR in one puppy and by plaque reduction neutralization testing in the other 2 dogs. EEE is rare in dogs, and has only been reported in puppies. The initial clinical signs of EEE in puppies are typically nonspecific, including anorexia, fever, and diarrhea, but rapidly progress to severe neurologic disease characterized by seizures and recumbency. Although rare, EEE should be considered as a differential diagnosis for neurologic disease in puppies, especially after more common diseases, such as canine distemper, rabies, and toxoplasmosis have been ruled out.

Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response.

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.

Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius).

BACKGROUND: Respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified. FINDINGS: Pneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae. CONCLUSIONS: This report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses.

B-cell multicentric lymphoma as a probable cause of abortion in a Quarter horse broodmare.

A 5-year-old Quarter horse broodmare was evaluated for inappetence, depression, and diarrhea 13 days after aborting a 9-month gestation fetus. Clinical and laboratory examination ruled out uterine rupture and peritonitis. Ultrasonography of the uterus combined with cytological analysis of peritoneal fluid suggested the existence of diffuse lymphoma. A multicentric B-cell lymphoma involving the uterus and ovary was confirmed at necropsy and histopathological examination.

Lymhome multicentrique à cellules B comme cause possible d'avortement chez une jument poulinière Quarter Horse. Une jument Quarter horse de 5 ans a été présentée pour anorexie, baisse d'état général et diarrhée, trente jours après un avortement à 9 mois de gestation. Lors de l'examen clinique initial, rupture utérine et péritonite ont pu être éliminées. L'analyse cytologique des liquides péritonéaux et pleuraux aspirés suggéra un lymphome diffus confirmé en nécropsie lors de l'examen histopathologique.(Traduit par les auteurs).

Metastatic pancreatic carcinoma and bronchioloalveolar adenomas in an Egyptian fruit bat (Rousettus aegyptiacus).

An adult female, intact Egyptian fruit bat (Rousettus aegyptiacus) was presented for lethargy, anorexia, and markedly reduced flying activity. Physical and ultrasound examinations were suggestive of an abdominal mass with free fluid within the abdomen. Based on the poor and deteriorating clinical condition of the animal, euthanasia was elected. Gross necropsy revealed an irregular thickening at the root of the mesentery and a diffusely, dark-red liver with rounded hepatic margins. Histologic examination revealed extensive neoplastic effacement of the pancreas with invasion into the surrounding mesentery and mesenteric lymph nodes and metastatic spread to the liver. Based on the morphology of the neoplastic cells, the involvement of the pancreas, and immunohistochemistry, a diagnosis of metastatic pancreatic carcinoma was made. Additionally, two small neoplasms were identified in the lungs. These masses were distinct from the carcinoma, and their morphology was consistent with bronchioloalveolar adenomas. This is the first known report of either benign pulmonary lesions or pancreatic carcinomas in the order Chiroptera.

Importance of differential expression of Marek's disease virus gene pp38 for the pathogenesis of Marek's disease.

The role of pp38 in the pathogenesis of Marek's disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B-based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Sp1 A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated.

Expression of the C-type lectins DC-SIGN or L-SIGN alters host cell susceptibility for the avian coronavirus, infectious bronchitis virus.

Infectious bronchitis virus (IBV), an avian coronavirus, is a cause of great economic loss in the poultry industry. The virus mainly infects respiratory epithelium, but can be also detected in other organs. The functional receptor for the virus has not been found and field strains of IBV do not infect conventional cell lines. Recently, it has been shown that the C-type lectins DC-SIGN/L-SIGN can promote entry of several coronaviruses. Here we examine whether DC-SIGN/L-SIGN are entry determinants for IBV. We show that by introducing human DC-SIGN/L-SIGN into non-permissive cells, infection by the IBV is dramatically increased. DC-SIGN mediated infection was inhibited by mannan and anti-lectin antibodies, and was independent of sialic acid levels on the cell. Enhancement of IBV infection also occurred for different serotypes of IBV. Our findings demonstrated that even in the absence of avian-specific receptor, DC-SIGN-like lectins are capable of mediating efficient IBV infection.

Oral contraceptives decrease the prevalence of ovarian cancer in the hen.

Ovarian cancer is the leading cause of reproductive cancer death in U.S. women. This high mortality rate is due to the lack of early detection methods and ineffectiveness of therapy for advanced disease. Until more effective screening methods and therapies are developed, chemoprevention strategies are warranted. The hen has a high spontaneous prevalence of ovarian cancer and has been used as a model for studying ovarian cancer chemoprevention. In this study, we used the hen to determine the effect of progestin alone, estrogen alone, or progestin and estrogen in combination (as found in oral contraceptives) on ovarian cancer prevalence. We found that treatment with progestin alone and in combination with estrogen decreased the prevalence of ovarian cancer. A significant risk reduction of 91% was observed in the group treated with progestin alone (risk ratio = 0.0909; 95% CI: 0.0117-0.704) and an 81% reduction was observed in the group treated with progestin plus estrogen (risk ratio = 0.1916; 95% CI = 0.043-0.864). Egg production was also significantly reduced in these treatment groups compared with control. We found no effect of progestin, either alone or in combination with estrogen, on apoptosis or proliferation in the ovary, indicating that this is not the likely mechanism responsible for the protective effect of progestin in the hen. Our results support the use of oral contraceptives to prevent ovarian cancer and suggest that ovulation is related to the risk of ovarian cancer in hens and that other factors, such as hormones, more than likely modify this risk.

Recovery of little brown bats (Myotis lucifugus) from natural infection with Geomyces destructans, white-nose syndrome.

Geomyces destructans produces the white fungal growth on the muzzle and the tacky white discoloration on wings and ears that characterize white-nose syndrome (WNS) in cave-hibernating bats. To test the hypothesis that postemergent WNS-infected bats recover from infection with G. destructans, 30 little brown bats (Myotis lucifugus) were collected in May 2009 from a WNS-affected hibernation site in New Jersey. All bats were confirmed to be infected with G. destructans using a noninvasive fungal tape method to identify the conidia of G. destructans and polymerase chain reaction (PCR). The bats were then held in captivity and given supportive care for 70 days. Of the 26 bats that survived and were humanely killed after 70 days, 25 showed significant improvement in the external appearance of wing membranes, had no microscopic evidence of infection by G. destructans, and had wing tissue samples that were negative for G. destructans by PCR. A subset of the bats was treated topically at the beginning of the rehabilitation study with a dilute vinegar solution, but treatment with vinegar provided no added advantage to recovery. Provision of supportive care to homeothermic bats was sufficient for full recovery from WNS. One bat at day 70 still had both gross pathology and microscopic evidence of WNS in wing membranes and was PCR-positive for G. destructans. Dense aggregates of neutrophils surrounded the hyphae that remained in the wing membrane of this bat.

110 years of Avipoxvirus in the Galapagos Islands.

The role of disease in regulating populations is controversial, partly owing to the absence of good disease records in historic wildlife populations. We examined birds collected in the Galapagos Islands between 1891 and 1906 that are currently held at the California Academy of Sciences and the Zoologisches Staatssammlung Muenchen, including 3973 specimens representing species from two well-studied families of endemic passerine birds: finches and mockingbirds. Beginning with samples collected in 1899, we observed cutaneous lesions consistent with Avipoxvirus on 226 (6.3%) specimens. Histopathology and viral genotyping of 59 candidate tissue samples from six islands showed that 21 (35.6%) were positive for Avipoxvirus, while alternative diagnoses for some of those testing negative by both methods were feather follicle cysts, non-specific dermatitis, or post mortem fungal colonization. Positive specimens were significantly nonrandomly distributed among islands both for mockingbirds (San Cristobal vs. Espanola, Santa Fe and Santa Cruz) and for finches (San Cristobal and Isabela vs. Santa Cruz and Floreana), and overall highly significantly distributed toward islands that were inhabited by humans (San Cristobal, Isabela, Floreana) vs. uninhabited at the time of collection (Santa Cruz, Santa Fe, Espanola), with only one positive individual on an uninhabited island. Eleven of the positive specimens sequenced successfully were identical at four diagnostic sites to the two canarypox variants previously described in contemporary Galapagos passerines. We conclude that this virus was introduced late in 1890's and was dispersed among islands by a variety of mechanisms, including regular human movements among colonized islands. At present, this disease represents an ongoing threat to the birds on the Galapagos Islands.

Condition, innate immunity and disease mortality of inbred crows.

Cooperatively breeding American crows (Corvus brachyrhynchos) suffer a severe disease-mediated survival cost from inbreeding, but the proximate mechanisms linking inbreeding to disease are unknown. Here, we examine indices of nestling body condition and innate immunocompetence in relationship to inbreeding and disease mortality. Using an estimate of microsatellite heterozygosity that predicts inbreeding in this population, we show that inbred crows were in relatively poor condition as nestlings, and that body condition index measured in the first 2-33 days after hatching, in addition to inbreeding index, predicted disease probability in the first 34 months of life. Inbred nestlings also mounted a weaker response along one axis of innate immunity: the proportion of bacteria killed in a microbiocidal assay increased as heterozygosity index increased. Relatively poor body condition and low innate immunocompetence are two mechanisms that might predispose inbred crows to ultimate disease mortality. A better understanding of condition-mediated inbreeding depression can guide efforts to minimize disease costs of inbreeding in small populations.

Non-West Nile virus-associated mortality in a population of American crows (Corvus brachyrhynchos): a gross and histopathologic study.

The American crow (Corvus brachyrhynchos) is a common urban and rural inhabitant of the Northeast and Midwest United States that is commonly infected with West Nile virus (WNV). The current study was initiated to determine non-WNV-associated causes of mortality in the American crow. All animals (40/40) tested negative for WNV infection via polymerase chain reaction and had no evidence of infection based on immunohistochemistry. Common gross necropsy findings included external trauma (6/40), hepatosplenomegaly (6/40), poxviral dermatitis (5/40), and pneumonia (3/40). Common histologic findings included endoparasitism (32/40), multifocal hepatic and splenic necrosis (7/40), pigment accumulation in the spleen (5/40), and disseminated bacterial infection (3/40). The most significant and debilitating diseases included fungal pneumonia and poxvirus-associated lesions. The present report increases the knowledge of diseases present in the American crow population.

Delivery of foreign antigens by engineered outer membrane vesicle vaccines.

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.

Histopathologic criteria to confirm white-nose syndrome in bats.

White-nose syndrome (WNS) is a cutaneous fungal disease of hibernating bats associated with a novel Geomyces sp. fungus. Currently, confirmation of WNS requires histopathologic examination. Invasion of living tissue distinguishes this fungal infection from those caused by conventional transmissible dermatophytes. Although fungal hyphae penetrate the connective tissue of glabrous skin and muzzle, there is typically no cellular inflammatory response in hibernating bats. Preferred tissue samples to diagnose this fungal infection are rostral muzzle with nose and wing membrane fixed in 10% neutral buffered formalin. To optimize detection, the muzzle is trimmed longitudinally, the wing membrane is rolled, and multiple cross-sections are embedded to increase the surface area examined. Periodic acid-Schiff stain is essential to discriminate the nonpigmented fungal hyphae and conidia. Fungal hyphae form cup-like epidermal erosions and ulcers in the wing membrane and pinna with involvement of underlying connective tissue. In addition, fungal hyphae are present in hair follicles and in sebaceous and apocrine glands of the muzzle with invasion of tissue surrounding adnexa. Fungal hyphae in tissues are branching and septate, but the diameter and shape of the hyphae may vary from parallel walls measuring 2 microm in diameter to irregular walls measuring 3-5 microm in diameter. When present on short aerial hyphae, curved conidia are approximately 2.5 microm wide and 7.5 microm in curved length. Conidia have a more deeply basophilic center, and one or both ends are usually blunt. Although WNS is a disease of hibernating bats, severe wing damage due to fungal hyphae may be seen in bats that have recently emerged from hibernation. These recently emerged bats also have a robust suppurative inflammatory response.