Gross Findings: At necropsy, the animal was in fair to poor body condition with minimal fat stores. The caudal lung lobes were mildly overinflated, firm, and floated low in formalin. Other gross findings were related to the method of euthanasia and included penetrating thoracic gunshot trauma as well as pale tan liver and kidneys (suspected to be due to blood loss).

Histopathological Findings: Throughout the forebrain, there is widespread mild to moderate lymphoplasmacytic perivascular cuffing (Fig. 1A and 1B) and moderate to marked gliosis and neuronal necrosis (Fig. 1B). The cerebral cortex is most severely affected, the meninges and choroid are relatively mildly affected, and the white matter is rarely affected. The cerebellum is unremarkable with the exception of minimal perivascular cuffing and gliosis within cerebellar meninges and white matter. Other lesions include widespread acute pancreatic necrosis (Fig. 1C), hepatic necrosis (Fig. 1D), and bronchointerstitial pneumonia with suspect acute alveolar necrosis and moderate numbers of lungworms. The presence of lungworms, as well as artifacts within the lung associated with freezing and gunshot trauma, make it difficult to determine the cause of the inflammation and suspect necrosis in the lung.

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Morphologic Diagnoses:

1. Brain: Meningoencephalitis, lymphoplasmacytic, multifocal, moderate, with gliosis and neuronal necrosis
2. Pancreas: Necrosis, acute, multifocal, moderate
3. Liver: Hepatocellular necrosis, peracute, multifocal, mild
4. Lung: Bronchointerstitial pneumonia with suspect alveolar necrosis, multifocal, mild, with intralesional nematodes
5. Spleen and lymph node: Lymphoid depletion, mild to moderate

Ancillary diagnostic tests and results: In-house rRT-PCR testing at NWHC resulted in detection of the avian influenza matrix gene, EA/AM H5, and Eurasian lineage clade 2.3.4.4b H5 strain in oral swab, brain, lung, intestine, liver, kidney, and spleen. Highly pathogenic avian influenza (HPAI) EA/AM 2.3.4.4b H5N1 subtype was confirmed by the U.S. Department of Agriculture National Veterinary Services Laboratories (NVSL) on oral swab by sequence analysis of the hemagglutinin protease cleavage site. Avian influenza virus was not detected on rectal swab by matrix rRT-PCR at NWHC or NVSL.

Canine distemper virus, canine parvovirus, feline panleukopenia virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were not detected by PCR.

Disease: Highly pathogenic avian influenza

Etiology: HPAI EA/AM 2.3.4.4b H5N1, a reassortant of H5 goose/Guangdong HPAI and North American wild bird AI lineages

Distribution: Worldwide; HPAI H5N1 clade 2.3.4.4 was first detected in the USA in January 2022

Host range: While birds are the natural hosts of avian influenza, there have been increasing reports of mammalian infections caused by H5N1 2.3.4.4b in the USA (Fig. 2) and elsewhere. Free-ranging North American mammalian species affected include red fox (Vulpes vulpes), grey fox (Urocyon cinereoargenteus), striped skunk (Mephitis mephitis), American mink (Neovison vison), raccoon (Procyon lotor), bobcat (Lynx rufus), Virginia opossum (Didelphis virginiana), coyote (Canis latrans), fisher (Pekania pennanti), black bear (Ursus americanus), grizzly bear (Ursus arctos horribilis), mountain lion (Puma concolor), harbor seal (Phoca vitulina), gray seal (Halichoerus grypus), American river otter (Lontra canadensis), and bottlenose dolphin (Tursiops truncatus). For more information on the susceptibility of avian species, see https://www.usgs.gov/centers/nwhc/news/pathology-case-month-bald-eagle-1.

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Transmission: Evidence suggests that free-ranging mammals are infected through direct contact with infected birds or their feces (e.g., by consuming infected carcasses). Mammal-to-mammal transmission has not been documented in free-ranging populations; however, mink-to-mink transmission was suspected in an outbreak in farmed American mink.

Clinical signs: Clinical signs in mammals are similar to those in birds and typically include neurological signs such as seizures, ataxia, or tremors. Subclinical infection has also been documented.

Pathology: Gross lesions in mammals can include pulmonary edema, congestion, or hemorrhage; less often, there may be hemorrhage or malacia in the brain. In some cases, no gross lesions may be present. Microscopically, lesions are most common in the brain and include lymphoplasmacytic (less often histiocytic, neutrophilic, or eosinophilic) perivascular cuffing, gliosis, and neuronal necrosis. Necrotizing interstitial pneumonia and myocardial necrosis with mineralization are also commonly reported. Less common findings include random acute hepatic necrosis, lymphoid depletion or necrosis, and necrosis in other tissues such as kidney, pancreas, and gastrointestinal tract. Species variations in lesion distribution have been reported.

Diagnosis: Screening tests include rRT-PCR for avian influenza virus, the H5 gene, and the H5 2.3.4.4b clade. The NVSL can confirm the identification of HPAI H5N1 2.3.4.4b by sequencing. Gross and microscopic lesions, as well as immunohistochemistry, can provide supportive evidence of disease.

Public health concerns: HPAI H5N1 2.3.4.4b is zoonotic and human infections have been reported. Infection can occur from direct contact or inhalation. Personnel working with any animal suspected to be infected with HPAI should use appropriate personal protective equipment (PPE) and follow biosafety protocols. The public should exercise caution in handling sick or dead wild birds or mammals.

Wildlife population impacts: Recent studies have pointed to possible local or regional impacts of HPAI H5N1 2.3.4.4b on wild bird populations. Bald eagle (Haliaeetus leucocephalus) nest failures up to 62% greater than historical levels have been documented in the southeastern USA in association with HPAI H5N1 2.3.4.4b infection in breeding adults and nestlings. Unprecedented mass mortality among breeding adults of colony-nesting seabirds, including sandwich terns (Thalasseus sandvicensis), common terns (Sterna hirundo), and Northern gannets (Morus bassanu), has been reported in Europe with potential implications for long-term colony success. The potential effects of HPAI H5N1 2.3.4.4b on free-ranging mammalian populations remains unknown.

Management: While population-scale management of a disease circulating in wildlife is difficult, it may be possible to reduce environmental contamination, exposure of other wildlife and domestic animals, and human-assisted spread through management responses such as carcass removal and decontamination by trained personnel.

References:

• Aguero M, Monne I, Azucena S, et al. 2022. Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022. Euro Surveill. 28(3):pii=2300001. https://doi.org/10.2807/1560-7917.ES.2023.28.3.2300001
• Alkie TN, Cox S, Embury-Hyatt C, et al. 2023. Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada. Emerg Microbes Infect. 12(1):2186608. https://doi.org/10.1080/22221751.2023.2186608.
• Elsmo EJ, Wünschmann A, Beckmen KB, et al. 2022. Pathology of natural infection with highly pathogenic avian influenza virus (H5N1) clade 2.3.4.4b in wild terrestrial mammals in the United States in 2022. bioRxiv 2023.03.10.532068; https://doi.org/10.1101/2023.03.10.532068
• Nemeth NM, Ruder MG, Poulson RL, et al. 2023. Bald eagle mortality and nest failure due to clade 2.3.4.4 highly pathogenic H5N1 influenza a virus. Sci Rep. 13(1):191. https://doi.org/10.1038/s41598-023-27446-1.
• Pohlmann A, Stejskal O, King J, et al. 2023. Mass mortality among colony-breeding seabirds in the German Wadden Sea in 2022 due to distinct genotypes of HPAIV H5N1 clade 2.3.4.4b. J Gen Virol. 104(4). https://doi.org/10.1099/jgv.0.001834.
• Puryear W, Sawatzki K, Hill N, et al. 2023. Highly pathogenic avian influenza A(H5N1) virus outbreak in New England seals, United States. Emerg Infect Dis. 29(4):786-791. https://doi.org/10.3201/eid2904.221538
• USDA Animal and Plant Health Inspection Service. Last modified April 20, 2023. 2022-2023 Detections of highly pathogenic avian influenza in mammals. https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022/2022-hpai-mammals. Accessed April 2023.