JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
May 2019
C-V04

SIGNLAMENT (JPC # 2741020):  Chilean flamingo

HISTORY:  This animal was weak and ataxic.  Sections of the heart were positive via immunohistochemistry and in situ hybridization for West Nile virus.

HISTOPATHOLOGIC DESCRIPTION:  Heart:  Multifocally affecting approximately 50% of the myocardium, separating and surrounding cardiomyocytes and Purkinje fibers, and multifocally infiltrating the epicardium, are numerous lymphocytes, plasma cells and macrophages.  Adjacent cardiomyoctes are often shrunken, hypereosinophilic, or fragmented with loss of cross striations, have pyknotic or karyolytic nuclei, and are surrounded by eosinophilic cellular and karyorrhectic debris (necrosis).  Rare cardiomyocytes are pale, swollen and vacuolated (degenerate). Frequently, similar inflammatory cells infiltrate the epicardium. Previously mentioned inflammatory cells extend into adjacent epicardial adipose tissue, which is diffusely composed of shrunken and variably sized adipocytes (fat atrophy).

MORPHOLOGIC DIAGNOSIS: 

1. Heart:  Pancarditis, necrotizing, lymphoplasmacytic and histiocytic, multifocal to coalescing, moderate, subacute, Chilean flamingo (Phoenicopterus chilensis), avian.
2. Heart, epicardium: Fat atrophy, diffuse, moderate. 

ETIOLOGIC DIAGNOSIS:  Flaviviral myocarditis

CAUSE:  West Nile virus

GENERAL DISCUSSION:

• West Nile virus (WNV), an arbovirus of the family Flaviviridae, genus Flavivirus (enveloped RNA virus), was first identified in Uganda in 1937 and introduced into the northeastern US in 1999
• Zoonotic disease that can cause fatal disease in humans, horses, birds, and other mammalian and reptilian species
  • Horses are the most susceptible domestic animals; pathogenicity depends on virus strain
• Birds are the reservoir for sylvatic transmission, with humans and horses becoming infected during the urban transmission cycle and considered incidental dead-end hosts
  • Brain and heart are defined features of disease in birds
  • Corvids (crows) and jays are major amplifying hosts- high titer viremia and high mortality
  • Finches/sparrows (low titer viremia and variable mortality) may be primary maintenance hosts in North America
• Divided into two genetic lineages:
  • Lineage 1 WNV in North America (made up of different clades, some of which are virulent); Kunjin virus is variant of lineage 1 in Australia
  • Lineage 2 WNV (Africa) strains are nonpathogenic or cause mild disease in humans/horses
• Mosquitos (especially Culex and Aedes) are considered the primary vectors in the US; ticks (Argas and Amplyomma) are capable of harboring the virus; frequency of disease related to seasonal mosquito activity (i.e. summer and fall)

PATHOGENESIS:

• Transmitted via mosquito bites and (rarely) by direct contact, fomites or ingestion
  • Signs of WNV in naïve areas: increased wild bird mortality and increased number of cases of equine encephalitis
• Susceptibility (in avians) varies by species; most susceptible avian species: crows, ravens, jays, magpies, owls, some raptors
  • Psittacines also susceptible (lesions seen in multiple organ systems, especially myocardium and mononuclear-phagocyte system; CNS involvement not as prominent)
• In susceptible species: injection of virus-containing saliva from mosquito> replication in local tissues and lymph nodes> high-titer viremia> viral invasion of multiple organ systems with widespread necrosis, hemorrhage and inflammation
  • Dissemination occurs before development of a full adaptive immune response, so the innate immune system (especially macrophages) are important in resistance to infection
  • Cellular targets include CNS (cerebellum) and PNS; cardiomyocytes; macrophages and monocytes; multiple epithelial cell types; oocytes; fibrous connective tissues
  • Mechanism for crossing blood-brain-barrier in CNS infection is unclear
• Virus targets cells of the mononuclear phagocyte system; inhibits production of pro-inflammatory and antiviral cytokines such as IL1 and type 1 interferon; targets molecules important in signaling pathways necessary for macrophage activation
• The disassociation of virion density from lesion severity suggests an immunopathologic mechanism in the pathogenesis of WNV disease in horses

TYPICAL CLINICAL FINDINGS:

• Weakness and recumbency; ataxia, tremors, abnormal head posture, circling
• Sudden death without premonitory signs, especially in crows

TYPICAL GROSS FINDINGS:

• Nonspecific and inconsistent; often none
• Hemorrhage of the brain and meninges, especially cerebellum
• Splenomegaly; splenitis
• Heart: enlargement, flaccidity, pale streaks within the myocardium
  • Myocardial hemorrhage; mottled, pale foci
• Intraosseous calvarial hemorrhage
• Intestinal mucosal hemorrhage
• Renal and pulmonary congestion or hemorrhage
• Hemorrhagic tracheitis has also been reproted

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Heart: multifocal, lymphoplasmacytic, myocarditis with necrosis
• Necrotizing splenitis; pancreatitis; adrenalitis; nephritis; hepatitis
• Spleen: Lymphoid depletion
• Central nervous system
  • Hemorrhage in cerebellar folia, cerebrum, brain stem, cervical spinal cord
  • Degeneration and necrosis of cerebellar molecular layer and Purkinje cells; cerebral neurons only rarely affected
  • Lymphoplasmacytic meningoencephalitis; perivascular cuffing; gliosis
• Enterocolitis, lymphoplasmacytic, chronic

ULTRASTRUCTURAL FINDINGS:

• Virions 35-45 nm in diameter, dense core surrounded by a thin, diffuse outer layer (typical flaviviral morphology)
• Virions in cytoplasmic vacuoles, less frequently in rough endoplasmic reticulum
• 100 nm smooth membrane vesicles (SMV) in dilated rough endoplasmic reticulum and vacuoles (characteristic of flaviviruses)
• Marked vesiculation and vacuolation of the cytoplasm, with disorganization of the rough endoplasmic reticulum and Golgi apparatus

ADDITIONAL DIAGNOSTIC TESTS:

• Immunohistochemistry (lacks specificity)
• In situ hybridization
• Reverse transcriptase-polymerase chain reaction (RT-PCR) assays
• Virus isolation (requires Biosafety level 3 lab)
• Hemagglutination inhibition; serology (rising titer)

DIFFERENTIAL DIAGNOSIS:

• Exotic Newcastle’s disease (Paramyxoviridae; rubulavirus) or highly pathogenic avian influenza (Orthomyxoviridae; influenzavirus A): Both cause mortality in chickens and turkeys; however, WNV is seldom a fatal infection in these species
  • CNS lesion, congestion, edema, and hemorrhage can be present in both and in various tissues
• Eastern equine encephalitis (Togaviridae; alphavirus): Arbovirus known to be pathogenic for birds

COMPARATIVE PATHOLOGY:

• Others birds:
  • Bald eagle / Golden eagle: WNV induced lesions noted in the brain, eyes, heart
    • Most noteworthy histologic lesion included lymphoplasmacytic encephalitis, predominantly in the cerebrum
  • Ducks: WNV causes weakness, inability to stand
  • Red tailed hawks: recent reports of ocular lesions to include pectenitis, retinal and choroidal inflammation, and retinal necrosis
• Vertebrates (other than birds) generally have low-titer viremia
• Horses: Pronounced, if not exclusive tropism for the brain and spinal cord, leading to neuronal necrosis, predominantly lymphocytic polioencephalomyelitis extending from basal nuclei, brain stem to sacral spinal cord (worst lesions in ventral horns of thoracolumar segments), gliosis/glial nodules
  • Antigen scarce in lesions; no antigen in the PNS
  • IgM-capture enzyme-linked immunosorbent assay is more sensitive than RT-PCR in horses
• Sheep: reported cases of WNV induced lymphoplasmacytic meningoencephalitis in California
• Dogs: Usually no clinical disease (rare reports of CNS and/or cardiac disease)
• Alligators: Multiorgan involvement (similar to wild birds) described in juveniles
• Reported in squirrels: Encephalitis, myelitis and myocarditis
• Humans with WNV: Non-specific flu-type symptoms; fatalities in older people and children; predominately brainstem lymphohistiocytic glial nodules

Arboviruses:

• Alphavirus (Togaviridae): EEE, WEE, VEE
• Flavivirus (Flaviviridae): WNV, Japanese encephalitis virus, St. Louis encephalitis virus, Dengue, yellow fever, tick-borne encephalitis virus
• Bunyavirus (Bunyaviridae): California encephalitis virus, LaCrosse virus
• Phlebovirus (Bunyaviridae): Rift Valley fever virus, sandfly fever virus

REFERENCES:

1. Bowen R, Greene C, Rickettsial, chlamydial and mycoplasmal diseases. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed. St. Louis, MO: Saunders Elsevier; 2012:214-217.
2. Eckstrand CD,et al. Diagnostic exercise: High mortality in a flock of chukar partridge chicks (Alectoris chukar) in California. Vet Pathol. 2015 ;52(1):189-92.
3. Fulton RM, Boulianne M. Bacterial diseases. In: Boulianne M, ed.  Avian Disease Manual. 7th ed. Jacksonville, FL: American Association of Avian Pathologists, Inc; 2013:16-24.
4. Gamino V, Escribano-Romero E, Blázquez AB, Gutiérrez-Guzmán AV, Martín-Acebes MÁ, Saiz JC, Höfle U. Experimental north american west nile virus infection in the red-legged partridge (Alectoris rufa). Vet Pathol. 2016 May;53(3):585-593
5. Gamino V, Escribano-Romero E, Gutiérrez-Guzmán AV, Blázquez AB, Saiz JC, Höfle U. Oculopathologic findings in flavivirus-infected gallinaceous birds. Vet Pathol. 2014 Nov;51(6):1113-1116.
6. Grant Maxie M, Youssef S. Nervous system. In: Grant Maxie M, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. Philadelphia, PA: Elsevier; 2016:374-375.
7. Hofmeister EK, Lund M, Shearn Bochsler V. West Nile Virus Infection in American Singer Canaries: An Experimental Model in a Highly Susceptible Avian Species. Vet Pathol. 2018 Jul;55(4):531-538.
8. MacLachlan NJ, Dubovi EJ. Fenner’s Veterinary Virology. 4th ed. London, UK: Academic Press; 2017: 533-535.
9. Nemeth NM, Bosco-Lauth AM, Williams LM, Bowen RA, Brown JD. West Nile Virus Infection in Ruffed Grouse ( Bonasa umbellus): Experimental infection and protective effects of vaccination. Vet Pathol. 2017 Nov;54(6):901-911.
10. Nemeth NM, Thomsen BV, Spraker TR, et al. Clinical and pathologic responses of american crows (Corvus brachyrhynchos) and fish crows (C ossifragus) to experimental West Nile virus infection. Vet Pathol. 2011; 48(6):1061-1074.
11. Palmieri C, Franca M, Uzal F, et al. Pathology and immunohistochemical findings of West Nile virus infection in psittaciformes. Vet Pathol. 2011; 48(5):975-984.
12. Porter MB, Long MT, Getman LM, Giguere S, et al. West Nile virus encephalomyelitis in horses: 46 cases (2001). J Am Vet Med Assoc. 2003; 222(9):1241-1247.
13. Rimoldi G, Mete A, Adaska JM, Anderson ML, Symmes KP, Diab S. West nile virus infection in sheep. Vet Pathol. 2017 Jan;54(1):155-158.
14. Schmidt RE, Reavill DR, Phalen DN. In: Schmidt RE, Reavill DR, Phalen DN. eds. Pathology of Pet and Aviary Birds, Second Edition. Iowa State Press. Ames, Iowa. 2015: 224-225.
15. Toplu N, Oquzoglu T, et al. West Nile virus infection in horses: Detection by immunohistochemistry, in-situ hybridization, and ELISA. Vet Pathol. 2015; 52(6):1073-1076.
16. Williams J, Mentoor J, et al. Comparative pathology of neurovirulent lineage 1 (NY99/385) and lineage 2 (SPU93/01) West Nile virus infections in BALBc mice. Vet Pathol. 2015; 52(1): 140-151.
17. Wünschmann A, Armién AG, Khatri M, Martinez LC, Willette M, Glaser A, Alvarez J, Redig P. Ocular lesions in red-tailed hawks (Buteo jamaicensis) with naturally acquired west nile disease. Vet Pathol. 2017 Mar;54(2):277-287.
18. Wünschmann A, Timurkaan N, Armien AG, Bueno Padilla I, Glaser A, Redig PT. Clinical, pathological, and immunohistochemical findings in bald eagles (Haliaeetus leucocephalus) and golden eagles (Aquila chrysaetos) naturally infected with West Nile virus. J Vet Diagn Invest. 2014 Sep;26(5):599-609.

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