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Behavioral, clinical, and pathological characterization of acid metalliferous water toxicity in mallards

From September to November 2000, United States Fish and Wildlife Service biologists investigated incidents involving 221 bird deaths at 3 mine sites located in New Mexico and Arizona. These bird deaths primarily involved passerine and waterfowl species and were assumed to be linked to consumption of acid metalliferous water (AMW). Because all of the carcasses were found in or near pregnant leach solution ponds, tailings ponds, and associated lakes or storm water retention basins, an acute-toxicity study was undertaken using a synthetic AMW (SAMW) formulation based on the contaminant profile of a representative pond believed to be responsible for avian mortalities. An acute oral-toxicity trial was performed with a mixed-sex group of mallards ( Anas platyrhynchos ). After a 24-h pretreatment food and water fast, gorge drinking was evident in both SAMW treatment and control groups, with water consumption rates greatest during the initial drinking periods. Seven of nine treated mallards were killed in extremis within 12 h after the initiation of dose. Total lethal doses of SAMW ranged from 69.8 to 270.1 mL/kg (mean ± SE 127.9 ± 27.1). Lethal doses of SAMW were consumed in as few as 20 to 40 min after first exposure. Clinical signs of SAMW toxicity included increased serum uric acid, aspartate aminotransferase, creatine kinase, potassium, and P levels. PCV values of SAMW-treated birds were also increased compared with control mallards. Histopathological lesions were observed in the esophagus, proventriculus, ventriculus, and duodenum of SAMW-treated mallards, with the most distinctive being erosion and ulceration of the kaolin of the ventriculus, ventricular hemorrhage and/or congestion, and duodenal hemorrhage. Clinical, pathological, and tissue-residue results from this study are consistent with literature documenting acute metal toxicosis, especially copper (Cu), in avian species and provide useful diagnostic profiles for AMW toxicity or mortality events. Blood and kidney Cu concentrations were 23- and 6-fold greater, respectively, in SAMW mortalities compared with controls, whereas Cu concentrations in liver were not nearly as increased, suggesting that blood and kidney concentrations may be more useful than liver concentrations for diagnosing Cu toxicosis in wild birds. Based on these findings and other reports of AMW toxicity events in wild birds, we conclude that AMW bodies pose a significant hazard to wildlife that come in contact with them.

New Mexico, Arizona

Beak deformities in Buteo jamaicensis (Red-tailed Hawk) signal possible emergence of avian keratin disorder among raptors

Beginning in the late 1990s, an unusual cluster of beak deformities was observed among Buteo jamaicensis (Red-tailed Hawk) in the Pacific coastal region of North America. However, information was not available to determine the scope of this problem nor to assess a potential link between beak deformities in hawks and avian keratin disorder (AKD), an emerging disease primarily among passerines that causes overgrowth of the keratinized layer of the beak. To help address these knowledge gaps, we compiled capture records, citizen science reports, and other available data from across North America. As part of a long-term raptor monitoring program in Washington, we detected anomalously high rates of beak deformities in 29% and other keratin abnormalities in an additional 19% of B. jamaicensis between 2014 and 2021. We also identified more than 100 other AKD-like cases in B. jamaicensis , largely clustered in the Pacific coastal region, with most reports from 2000 to present. Pathologic examination of affected hawks revealed gross and microscopic features consistent with AKD in passerines, while shared epidemiological patterns, including timing of emergence, provided further evidence that this disease may impact an ecologically diverse suite of species. We detected poecivirus, a novel avian virus implicated as a likely cause of AKD in Poecile atricapillus (Black-capped Chickadee), in 1 out of 10 swabs collected from live B. jamaicensis , with gross beak deformities, suggesting the possibility of a shared viral etiology across species. However, additional work would be needed to determine the underlying cause of beak deformities in and their potential impacts on B. jamaicensis . By documenting this cluster of beak deformities, we hope to raise awareness among the ornithological community, including raptor researchers, to better track current and future outbreaks of beak deformities and related conditions.

Ornithology

Novel picornavirus associated with avian keratin disorder in Alaskan birds

Avian keratin disorder (AKD), characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees ( Poecile atricapillus ) in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100%) AKD-affected individuals were positive, while only 2/9 (22%) control individuals were infected with poecivirus. Two northwestern crows ( Corvus caurinus ) and two red-breasted nuthatches ( Sitta canadensis ) with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD.

mBio

Necrotizing enteritis as a cause of mortality in Laysan albatross, Diomedea immutabilis, chicks on Midway Atoll, Hawaii

A necropsy survey of Laysan albatross, Diomedea immutabilis , chicks on Midway Atoll in June 1993, 1994, and 1995 revealed 54% (21/39), 67% (49/71), and 93% (15/16), respectively, to have enteritis as the most severe pathologic finding. The lesion was limited to the ileum, ceca, and large intestine. We were unable to attribute a single infectious etiology to this lesion. Many birds with enteritis also exhibited renal lesions similar to those encountered in chickens experimentally deprived of water. We propose that enteritis is a significant cause of mortality in Laysan albatross chicks on Midway and that it may be a sequela to dehydration. It is likely that the pathology of dehydration in Laysan albatross differs from that in chickens largely because of diet.

Midway Atoll

Multi-decade mortality and a novel homolog of hepatitis C virus in bald eagles ( Haliaeetus leucocephalus ), the national bird of the USA

The bald eagle (Haliaeetus leucocephalus) once experienced near-extinction but has since rebounded. For decades, bald eagles near the Wisconsin River, USA, have experienced a lethal syndrome with characteristic clinical and pathological features but unknown etiology. Here, we describe a novel hepacivirus-like virus (Flaviviridae: Hepacivirus) identified during an investigation of Wisconsin River eagle syndrome (WRES). Bald eagle hepacivirus (BeHV) belongs to a divergent clade of avian viruses that share features with members of the genera Hepacivirus and Pegivirus. BeHV infected 31.9% of eagles spanning 4,254 km of the coterminous USA, with negative strand viral RNA demonstrating active replication in liver tissues. Eagles from Wisconsin were approximately 10-fold more likely to be infected than eagles from elsewhere. Eagle mitochondrial DNA sequences were homogeneous and geographically unstructured, likely reflecting a recent population bottleneck, whereas BeHV envelope gene sequences showed strong population genetic substructure and isolation by distance, suggesting localized transmission. Cophylogenetic analyses showed no congruity between eagles and their viruses, supporting horizontal rather than vertical transmission. These results expand our knowledge of the Flaviviridae, reveal a striking pattern of decoupled host/virus coevolution on a continental scale, and highlight knowledge gaps about health and conservation in even the most iconic of wildlife species.

Scientific Reports

Macroscopic, histologic, and ultrastructural lesions associated with avian keratin disorder in Black-capped Chickadees (Poecile atricapillus)

An epizootic of beak abnormalities (avian keratin disorder) was recently detected among wild birds in Alaska. Here we describe the gross, histologic, and ultrastructural features of the disease in 30 affected adult black-capped chickadees (Poecile atricapillus). Grossly, there was elongation of the rhamphotheca, with varying degrees of lateral deviation, crossing, and gapping between the upper and lower beak. Not uncommonly, the claws were overgrown, and there was alopecia, scaling, and crusting of the skin. The most prominent histopathologic features in the beak included epidermal hyperplasia, hyperkeratosis, and core-like intrusions of necrotic debris. In affected birds, particularly those with moderate to severe beak overgrowth, there was remodeling of premaxillary and mandibular bones and various dermal lesions. Lesions analogous to those found in beaks were present in affected claws, indicating that this disorder may target both of these similar tissues. Mild to moderate hyperkeratosis occurred in other keratinized tissues, including skin, feather follicles, and, occasionally, sinus epithelium, but typically only in the presence of microbes. We did not find consistent evidence of a bacterial, fungal, or viral etiology for the beak lesions. The changes observed in affected birds did not correspond with any known avian diseases, suggesting a potentially novel hyperkeratotic disorder in wild birds.

Alaska

High costs of infection: Alphavirus infection reduces digestive function and bone and feather growth in nestling house sparrows (Passer domesticus)

Increasingly, ecoimmunology studies aim to use relevant pathogen exposure to examine the impacts of infection on physiological processes in wild animals. Alphaviruses are arthropod-borne, single-stranded RNA (ssRNA) viruses (“arboviruses”) responsible for millions of cases of human illnesses each year. Buggy Creek virus (BCRV) is a unique alphavirus that is transmitted by a cimicid insect, the swallow bug, and is amplified in two avian species: the house sparrow ( Passer domesticus ) and the cliff swallow ( Petrochelidon pyrrhonota) . BCRV, like many alphaviruses, exhibits age-dependent susceptibility where the young are most susceptible to developing disease and exhibit a high mortality rate. However, alphavirus disease etiology in nestling birds is unknown. In this study, we infected nestling house sparrows with Buggy Creek virus and measured virological, pathological, growth, and digestive parameters following infection. Buggy Creek virus caused severe encephalitis in all infected nestlings, and the peak viral concentration in brain tissue was over 34 times greater than any other tissue. Growth, tissue development, and digestive function were all significantly impaired during BCRV infection. However, based on histopathological analysis performed, this impairment does not appear to be the result of direct tissue damage by the virus, but likely caused by encephalitis and neuronal invasion and impairment of the central nervous system. This is the first study to examine the course of alphavirus diseases in nestling birds and these results will improve our understanding of age-dependent infections of alphaviruses in vertebrate hosts.

PLoS ONE

Experimental infection of a North American raptor, American kestrel (Falco sparverius), with highly pathogenic avian influenza virus (H5N1)

Several species of wild raptors have been found in Eurasia infected with highly pathogenic avian influenza virus (HPAIV) subtype H5N1. Should HPAIV (H5N1) reach North America in migratory birds, species of raptors are at risk not only from environmental exposure, but also from consuming infected birds and carcasses. In this study we used American kestrels as a representative species of a North American raptor to examine the effects of HPAIV (H5N1) infection in terms of dose response, viral shedding, pathology, and survival. Our data showed that kestrels are highly susceptible to HPAIV (H5N1). All birds typically died or were euthanized due to severe neurologic disease within 4-5 days of inoculation and shed significant amounts of virus both orally and cloacally, regardless of dose administered. The most consistent microscopic lesions were necrosis in the brain and pancreas. This is the first experimental study of HPAIV infection in a North American raptor and highlights the potential risks to birds of prey if HPAIV (H5N1) is introduced into North America.

PLoS ONE

Lethal infection of wild raptors with highly pathogenic avian influenza H5N8 and H5N2 viruses in the USA, 2014–15

An outbreak of highly pathogenic avian influenza (HPAI) led to heavy losses of poultry in commercial farms in North America in 2014–15. Enhanced surveillance by virologists and pathologists at the US Geological Survey National Wildlife Health Center and its partners resulted in the identification of lethal infections with clade 2.3.4.4 subgroup icA2 H5N8 and novel reassortant H5N2 viruses in diverse wild raptor species that died concomitant with the poultry epizootic in the US. A range of pathologic abnormalities were present in dead raptors, including necrotizing encephalitis and myocarditis, pancreatic necrosis, and pulmonary congestion and edema. Raptors are highly susceptible to disease caused by infection with HPAI clade 2.3.4.4 viruses.

Journal of Wildlife Diseases

Clinical pathology results from cranes with experimental West Nile Virus infection

Sandhill cranes ( Grus canadensis ) were vaccinated for and then challenged with West Nile virus. Resulting titers demonstrated protection in the vaccinated-challenged cranes as compared to the unvaccinated-challenged cranes. Clinical pathology results showed challenged cranes, whether vaccinated or not, had a decrease in their hematocrits and an elevation of 2.5-fold in their white blood cell counts as compared to unchallenged control sandhill cranes. No differences were apparent in the differential counts of heterophils and lymphocytes.

Proceedings of the Association of Avian Veterinari

Suspected fusariomycotoxicosis in sandhill cranes (Grus canadensis): clinical and pathological findings.

In 1985 and 1986, large-scale natural die-offs of sandhill cranes in Texas were attributed to fusariomycotoxicosis. These birds demonstrated a progressive loss of motor control to the neck, wings, and legs. Based on necropsy and/or histopathology of 31 cranes, the most common lesions involved skeletal muscle and included hemorrhages, granulomatous myositis, thrombosis, and vascular degeneration. Serum chemistry results revealed that levels of creatinine kinase, aspartate aminotransferase, and alanine aminotransferase were above published normals. However, only alanine aminotransferase was higher in clinically affected cranes than in normal cranes collected from the same area.

Texas

Aquatic bird disease and mortality as an indicator of changing ecosystem health

We analyzed data from pathologic investigations in the United States, collected by the USGS National Wildlife Health Center between 1971 and 2005, into aquatic bird mortality events. A total of 3619 mortality events was documented for aquatic birds, involving at least 633 708 dead birds from 158 species belonging to 23 families. Environmental causes accounted for the largest proportion of mortality events (1737 or 48%) and dead birds (437 258 or 69%); these numbers increased between 1971 and 2000, with biotoxin mortalities due to botulinum intoxication (Types C and E) being the leading cause of death. Infectious diseases were the second leading cause of mortality events (20%) and dead birds (20%), with both viral diseases, including duck plague ( Herpes virus ), paramyxovirus of cormorants ( Paramyxovirus PMV1) and West Nile virus ( Flavivirus ), and bacterial diseases, including avian cholera ( Pasteurella multocida ), chlamydiosis ( Chalmydia psittici ), and salmonellosis ( Salmonella sp.), contributing. Pelagic, coastal marine birds and species that use marine and freshwater habitats were impacted most frequently by environmental causes of death, with biotoxin exposure, primarily botulinum toxin, resulting in mortalities of both coastal and freshwater species. Pelagic birds were impacted most severely by emaciation and starvation, which may reflect increased anthropogenic pressure on the marine habitat from over-fishing, pollution, and other factors. Our study provides important information on broad trends in aquatic bird mortality and highlights how long-term wildlife disease studies can be used to identify anthropogenic threats to wildlife conservation and ecosystem health. In particular, mortality data for the past 30 yr suggest that biotoxins, viral, and bacterial diseases could have impacted >5 million aquatic birds.

Marine Ecology Progress Series

The pathogenesis of a North American H5N2 clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata)

Background Aquatic waterfowl, particularly those in the order Anseriformes and Charadriiformes , are the ecological reservoir of avian influenza viruses (AIVs). Dabbling ducks play a recognized role in the maintenance and transmission of AIVs. Furthermore, the pathogenesis of highly pathogenic AIV (HPAIV) in dabbling ducks is well characterized. In contrast, the role of diving ducks in HPAIV maintenance and transmission remains unclear. In this study, the pathogenesis of a North American A/Goose/1/Guangdong/96-lineage clade 2.3.4.4 group A H5N2 HPAIV, A/Northern pintail/Washington/40964/2014, in diving sea ducks (surf scoters, Melanitta perspicillata ) was characterized. Results Intrachoanal inoculation of surf scoters with A/Northern pintail/Washington/40964/2014 (H5N2) HPAIV induced mild transient clinical disease whilst concomitantly shedding high virus titers for up to 10 days post-inoculation (dpi), particularly from the oropharyngeal route. Virus shedding, albeit at low levels, continued to be detected up to 14 dpi. Two aged ducks that succumbed to HPAIV infection had pathological evidence for co-infection with duck enteritis virus, which was confirmed by molecular approaches. Abundant HPAIV antigen was observed in visceral and central nervous system organs and was associated with histopathological lesions. Conclusions Collectively, surf scoters, are susceptible to HPAIV infection and excrete high titers of HPAIV from the respiratory and cloacal tracts whilst being asymptomatic. The susceptibility of diving sea ducks to H5 HPAIV highlights the need for additional research and surveillance to further understand the contribution of diving ducks to HPAIV ecology.

BMC Veterinary Research

A lesser scaup (Aythya affinis ) naturally infected with Eurasian 2.3.4.4 highly pathogenic H5N1 avian influenza virus – Movement ecology and host factors

Despite the recognized role of wild waterfowl in the potential dispersal and transmission of highly pathogenic avian influenza (HPAI) virus, little is known about how infection affects these birds. This lack of information limits our ability to estimate viral spread in the event of an HPAI outbreak, thereby limiting our abilities to estimate and communicate risk. Here we present telemetry data from a wild Lesser Scaup ( Aythya affinis ), captured during a separate ecology study in the Chesapeake Bay, Maryland. This bird tested positive for infection with clade 2.3.4.4 HPAI virus of the A/goose/Guangdong/1/1996 (Gs/GD) H5N1 lineage (results received post-release) during the 2021–22 ongoing outbreaks in North America. While the infected bird was somewhat lighter than other adult males surgically implanted with transmitters (790g, ߂ = 868g, n = 11), it showed no clinical signs of infection at capture, during surgery, nor upon release. The bird died 3d later, pathology undetermined as the specimen was not able to be recovered. Analysis of movement data within the 3d window showed that the infected individual's maximum and average hourly movements (3894.3m, 428.8m respectively) were noticeably lower than noninfected conspecifics tagged and released the same day (߂ = 21594.5m, ߂ = 1097.9m, respectively; n = 4). We identified four instances where the infected bird had close contact (fixes located within 25m and 15 min) with another marked bird during this time. Collectively, these data suggest that the HPAI positive bird observed in this study may have been shedding virus for some period prior to death, with opportunities for direct bird to bird or environmental transmission. Although limited by low sample size and proximity to the time of tagging, we hope that these data will provide useful information as managers continue to respond to this ongoing outbreak event.

Transboundary and Emerging Diseases

Acute oral toxicity of sodium cyanide in birds

Sensitivities of six avian species, black vulture ( Coragyps atratus ), American kestrel ( Falco sparverius ), Japanese quail ( Coturnix japonica ), domestic chicken ( Gallus domesticus ), eastern screech-owl ( Otus asio ), and European starling ( Sturnus vulgaris ), to acute poisoning by sodium cyanide (NaCN) were compared by single dose LD50's. Three species, domestic chickens, black vultures, and turkey vultures ( Cathartes aura ), were dosed with NaCN to determine cyanide residues in those that died and also in survivors, in addition to postmortem fate. Three flesh-eating species (black vulture, American kestrel, and eastern screech-owl; LD50's 4.0–8.6 mg/kg) were more sensitive to NaCN than three species (Japanese quail, domestic chicken, and European starling; LD50's 9.4–21 mg/kg) that fed predominantly on plant material. Elevated concentrations of cyanide were found in the blood of birds that died of cyanide poisoning; however, concentrations in birds that died overlapped those in survivors. Blood was superior to liver as the tissue of choice for detecting cyanide exposure. No gross pathological changes related to dosing were observed at necropsy.

Journal of Wildlife Diseases

Climate change and coastal wetland salinization: Physiological and ecological consequences for Arctic waterfowl

Coastal wetland salinization related to warming climate has the potential to impact ecological systems globally. In Alaska, the Yukon-Kuskokwim Delta (YKD) supports large concentrations of breeding water birds and is an ecologically important area for conservation of migratory bird biodiversity. On the YKD, the majority of waterfowl nest in low elevation coastal tundra where storm surges drive saline water into terrestrial wetland habitats. Because newly hatched water birds lack functional salt glands to process saline water, salinization may negatively impact their growth and survival. We investigated potential demographic impacts of wetland salinization by conducting controlled physiological experiments to determine consequences of saline water exposure to spectacled Somateria fischeri and Steller's eider Polysticta stelleri ducklings, and analysed habitat suitability based on experimentally defined tolerance thresholds and sampled wetland salinity levels. We found physiological and behavioural pathologies in <1 week old ducklings at exposure levels of 6 parts per thousand (ppt), and found some evidence of reduced growth in ducklings first exposed after 1 week of age. We conclude that the acute toxicity threshold for ducklings <1 week of age is ≤6 ppt. We found that the area of coastal tundra where wetland salinity exceeded 6 ppt varied considerably among years, suggesting that some portion of the breeding range is unsuitable for rearing ducklings in some years. However, we also found that most eiders in most years nested in areas with salinity at or exceeding the tolerance threshold for ducklings, suggesting that nesting eiders do not appear to avoid saline habitats. We suggest that localized fresh water refugia currently may allow resilience to salinization during the critical period. Understanding how species and habitats respond to climate driven changes is essential for predicting future patterns of distribution and abundance, and is necessary for making informed decisions about conservation priorities. Our study provides insights into the extent of wetland salinization in Alaska, mechanism of impact and current ecological consequences on avian communities depending on these habitats. With ongoing climate change, the probability of species crossing physiological tolerance thresholds of wetland salinity may change in the future.

Functional Ecology

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria

Background Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. Methods The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria . Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. Results Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. Conclusion Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

Malaria Journal

West Nile virus infection in American singer canaries: An experimental model in a highly susceptible avian species

This study investigated the susceptibility of American singer canaries ( Serinus canaria ) to West Nile virus (WNV) infection. Adult canaries were inoculated with 10 5 , 10 2 , and 10 1 plaque forming units (PFU) of WNV. All birds became infected and mortality occurred by 5 days postinoculation. The load of viral RNA as determined by RT-qPCR was dose dependent, and was higher at all doses than the level of viral RNA detected in American crows ( Corvus brachyrhynchos ) challenged with 10 5 PFU of WNV. In a subset of birds, viremia was detected by virus isolation; canaries inoculated with 10 1 PFU of WNV developed viremia exceeding 10 10 PFU/mL serum, a log higher than American crows inoculated with 10 5 PFU of virus. In canaries euthanized at 3 days postinoculation, WNV was isolated at >10 7 PFU of virus/100 mg of lung, liver, heart, spleen, and kidney tissues. Pallor of the liver and splenomegaly were the most common macroscopic observations and histologic lesions were most severe in liver, spleen, and kidney, particularly in canaries challenged with 10 2 and 10 1 PFU. Immunoreactivity to WNV was pronounced in the liver and spleen. IgG antibodies to WNV were detected in serum by enzyme immunoassay in 11 of 21 (52%) challenged canaries and, in 4 of 5 (20%) of these sera, neutralization antibodies were detected at a titer ≥ 1:20. American singer canaries provide a useful model as this bird species is highly susceptible to WNV infection.

Veterinary Pathology