16-year-old, male, bar-headed goose, (Anser indicus).Between
September 9 and September 17, 2015, three bar-headed geese housed in an open,
outdoor enclosure near Village Lagoon died unexpectedly. The first two had been
found dead on exhibit, while the third had been hospitalized shortly before
death with clinical signs of labored breathing and lethargy. The hospitalized
goose had a CBC within normal limits. At the same time, a fourth bar-headed
goose was hospitalized with diarrhea.
spleen has a few ill-defined, pale tan foci of necrosis extending from
the serosa into the parenchyma. The pancreas has 10 to 15 scattered, tan,
well-circumscribed necrotic foci measuring up to 0.2 cm in diameter. The
entire small and large intestine are filled with tan to green, fibrous, clumped
soft material that coats the mucosa in some regions and sloughs away along
other portions. Within the ceca, there is a small to moderate amount of
tan-green watery fluid and tan fibrous strands that loosely adhere to the
and pancreas: Sections are mildly autolyzed. In the duodenum, villi are
markedly blunted and frequently fragmented. The majority of crypts are dilated
with hypereosinophilic, karyorrhectic cellular debris (crypt abscesses), and
glandular epithelial cells are frequently shrunken and hypereosinophilic with
nuclear pyknosis (necrotic). The villous epithelium is sloughed and is
regionally replaced by luminal bands of necrotic sloughed cells, degenerate
heterophils, and large numbers of embedded bacteria (pseudomembrane). Moderate
to large numbers of lymphocytes with fewer plasma cells populate the lamina
propria. Throughout the exocrine pancreas, there are multiple, randomly
distributed foci of necrosis, characterized by hypereosinophilia, loss of
cellular detail and karyorrhexis, and small numbers of heterophils. Foci of
necrosis are often surrounded by a band of acinar cells with marked zymogen
depletion. There are occasional
ill-defined regions of exocrine pancreatic hyperplasia, with lobules of tightly
packed, slightly haphazardly arranged acinar cells delineated by coarse fibrous
Immunohistochemistry for West Nile Virus (performed in-house): Strong positive cytoplasmic immunoreactivity within intestinal epithelial cells or foci of crypt necrosis, pancreatic acinar cells, glial nodules in the brain, renal tubular epithelial cells, cardiomyocytes, hepatocytes, and in the spleen (macrophages).
1. Duodenum: Severe, diffuse, acute, necrotizing
enteritis with segmental pseudo-membranes and mixed bacteria
Moderate, multifocal, acute necrosis
and virus isolation were performed by the California Animal Health and Food
Safety diagnostic laboratory (CAHFS).
West Nile Virus (avian) PCR: Virus detected in brain
Virus isolation: West Nile Virus Avian Influenza Matrix Gene qRT-PCR: Not detected in lung, duodenum, or spleen. Avian Paramyxovirus-1 qRT-PCR: Not detected in lung, duodenum, or spleen. Bacterial culture, colon contents (IDEXX): 3+ Escherichia coli, 1+ Aeromonas species, 2+ Normal positive flora. No Salmonella, Shigella, Pleisiomonas, Edwardseilla, Aeromonas or Yersinia were isolated.
Intraerythrocytic protozoan trophozoites/Babesia bovis
and histopathology findings on all three bar-headed geese (Anser indicus)
were consistent with severe systemic viral infection, with acute necrosis
most severely affecting the gastrointestinal tract, but also including
pancreas, trachea, spleen, esophagus, feather follicle epithelium and brain.
The three mortalities within a short time span, in conjunction with the
spectrum of histologic lesions (widespread tissue necrosis), met internal
criteria requiring notification of government regulatory officials (California
Department of Food and Agriculture, CDFA) and testing to rule out
highly-pathogenic avian influenza (HPAI). Immediate biosecurity measures were
implemented. Suspicion of HPAI was heightened by the fact that bar-headed geese
were the migratory waterfowl species predominantly affected in H5N1 HPAI virus
epidemics in China in 2005 and 2006.6,9 Within approximately 48
hours of notifying CDFA, we received negative test results for HPAI, and CDFA
lifted the quarantine. Concurrently, West Nile virus (WNV) infection in all
three geese was confirmed by in-house immunohistochemistry. These results were
corroborated by PCR and virus isolation performed by the California Animal
Health and Food Safety diagnostic laboratory (CAHFS). The presence of such
striking, acute intestinal crypt necrosis was considered to be unusual for WNV
in an avian species, especially with only minimal brain lesions and an absence
of myocardial lesions.
West Nile Virus is in the genus Flavivirus, family Flaviviridae, and is serologically classified within the Japanese encephalitis antigenic group. The virus is distributed worldwide and has a wide potential host range, but is maintained primarily in a bird-mosquito cycle. Wild birds (especially corvids) act as amplifying hosts. Culex spp mosquitoes are the primary vector, although the virus is found in other vectors (other mosquito species, ticks) of undetermined significance in transmission.3,5 Horizontal transmission2 and transmission through prey or contaminated water have also been reported.5 There are seven genetic lineages of WNV strains, with two major lineages, lineage 1 and lineage 2. WNV genetic lineage 1 is widespread geographically, including in North America. Lineage 2 WNV strains correspond primarily to enzootic areas in Africa, with recent detection in Europe. Lineage 1 strains have been considered more virulent, but both have been implicated in significant disease outbreaks in birds.3,5 Mortality due to WNV has been documented in 24 orders of birds from North America, including anseriforms.5
In mammals, after a bite by an infected mosquito, the virus replicates in keratinocytes, cutaneous dendritic cells, endothelial cells, and fibroblasts, followed by viremia and hematogenous spread.3 In avian species, the mechanism and sites of replication are not completely understood. Experimentally in birds, virus has been detected in blood within 45 minutes of mosquito biting, suggesting that primary viremia can occur without local replication.5 Clinical disease develops upon viral invasion of major organs and/or the CNS, usually by 5-6 days post-infection.7 In most infected birds, virus is detectable first in the spleen, followed by spread to other visceral organs, and then to the CNS.5 Lesions of WNV in birds are often more extensive in less susceptible species, such as chickens, with multi-organ failure, peracute death and few to no lesions in highly susceptible species like corvids. Chronic, persistent WNV infections have also been documented in some species of birds, including house finches (Haemorhous mexicanus) and Western scrub-jays (Aphelocoma californica).7
In most orders of birds, histologic lesions of WNV are primarily found in the CNS, heart, liver, kidney, and spleen. Typical lesions in the CNS include mononuclear meningo-encephalitis with perivascular cuffing, gliosis, and glial nodules. In other organs, lesions are characterized by lympho-plasmacytic and histiocytic inflammation, accompanied by cellular degeneration or necrosis. While the intestinal lesions seen in the present case, with striking enterocyte and crypt necrosis, have been described in WNV-infected corvids and other passerines, they have not been described in Anseriformes.1,2, 4,5, 8 Reports of naturally and experimentally infected geese of various species have emphasized pronounced myocardial lesions and moderate to severe mononuclear meningoencephalitis.1,2, 4, 8
1. Small intestine: Enteritis, necrotizing, diffuse, severe
with crypt abscesses, bar-headed goose, Anser indicus.
Pancreas: Pancreatitis, necrotizing, random, multifocal, mild.
This excellent case
demonstrates the widespread tissue tropism that West Nile virus (WNV) has in
avian species. Most wild birds infected with WNV have a prolonged viremia
allowing dissemination of the virus throughout the body, affecting nearly every
organ. Typically, microscopic lesions associated with WNV viremia are
lymphoplasmacytic and histiocytic inflammation with degeneration and necrosis
within the central nervous system (CNS), heart, spleen, kidney, and liver. The
virus is distributed throughout the world and outbreaks can cause acute death
in a variety of different avian species. Highly susceptible birds include
crows, jays, and magpies may die so acutely that there are little to no
macroscopic lesions. Chronic disease, characterized by severe inflammatory
dehydration, hemorrhage, and congestion, occurs in avian species with
lower susceptibility to WNV infection such as owls, hawks, and psittacine
birds. Choroiditis, iridocyclitis, and retinal necrosis leading to progressive
visual impairment and blindness are reported in naturally WNV infected
red-tailed hawks and experimentally infected partridges and pheasants.
As mentioned by the contributor, the virus is transmitted predominantly by Culex sp. mosquitoes. Corvid birds, such as the American crow, are the main amplifying host and the virus is maintained in a bird-mosquito-bird lifecycle. Despite being dead end hosts, a variety of mammalian species can be infected, with humans and horses particularly susceptible to developing clinical disease. Transmission occurs during the late spring to early fall during favorable weather conditions for the mosquito vector. In contrast to avian species, lesions in horses are confined to the central nervous system, primarily within the grey matter of the brainstem and thoracolumbar spinal cord and less commonly in the cerebrum and cervical spinal cord. Histologic lesions are typically lymphoplasmacytic meningo-encephalomyelitis with glial nodules, lymphohistiocytic perivascular cuffing, ring hemorrhages, neuronal degeneration, and necrosis. The preferred modality for postmortem diagnosis of WNV infection in mammals includes histologic identification of the previously mentioned lesions, in addition to polymerase chain reaction (PCR) testing of the brainstem for WNV antigens. Even in severe equine cases, viral antigen is typically scant within the central nervous system making immunohistochemistry (IHC) in-situ hybridization (ISH) less useful in horses.
Ruminants, dogs, cats, and pigs are susceptible to infection, but usually only have transient and subclinical disease; however, a recent report in Veterinary Pathology describes severe lympho-plasmacytic meningoencephalitis in six WNV infected sheep with neurological signs in California. In contrast to horses, a large amount of viral antigens accumulated in the CNS of sheep in this study. As a result, both PCR and IHC were useful testing modalities in these sheep. Viral antigens in avian species are widespread and the contributor provides excellent quality images of strong positive cytoplasmic immunoreactivity for WNV antigen within intestinal epithelial cells and crypts and pancreatic acinar cells.
After injection of the virus by the mosquito vector, the virus is deposited in the extracellular matrix where it can propagate in keratinocytes and infect Langerhans dendritic cells and tissue macrophages. The virus then spreads hematogenously via the leukocyte tracking system. Viral envelope proteins E2 and E1 are responsible for organ attachment and endocytosis respectively. Although not completely understood, entry of the virus into the CNS likely involves a combination of breakdown of the blood-brain barrier by proinflammatory cytokines and retrograde axonal transport from the peripheral nervous system.
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