JPC SYSTEMIC PATHOLOGY
HEMOLYMPHATICS SYSTEMS
April 2021
H-V12 (NP)

Signalment (JPC #2415492): Adult male African green monkey (Cercopithecus aethiops)

HISTORY: One of six African green monkeys inoculated intraperitoneally with 1000 plaque-forming units of Ebola (Zaire) virus.

HISTOPATHOLOGIC DESCRIPTION:

Spleen: Diffusely the white pulp, to include both follicles and periarteriolar lymphoid sheaths, is characterized a marked paucity of mature small lymphocytes and scattered lymphocytolysis with increased numbers of tingible body macrophages (lymphoid depletion), and there is prominent perifollicular hemorrhage. Diffusely the red pulp is expanded by abundant fibrin admixed with karyorrhectic debris (necrosis) and few scattered macrophages and neutrophils.  Multifocally, the cytoplasm of macrophages contains golden-brown pigment (hemosiderin) and erythrocyte remnants (erythrophagocytosis). Splenic veins are lined by hypertrophic reactive endothelium and vessels occasionally contain fibrin thrombi.

Liver: Affecting approximately 40% of the liver are multifocal, random areas in which hepatocytes are swollen and pale with vacuolated cytoplasm (degeneration) or shrunken and angular with pyknotic or lytic nuclei (necrosis). Surrounding these areas, sinusoids are mildly expanded by fibrin and few neutrophils, monocytes, and lymphocytes. Occasionally, hepatocytes and monocytes contain round, up to 10 um diameter, eosinophilic, intracytoplasmic viral inclusions. Multifocally, large vessels occasionally contain fibrin thrombi.

MORPHOLOGIC DIAGNOSES:

1. Spleen: Lymphoid depletion, diffuse, severe, with lymphocytolysis, perifollicular hemorrhage, extensive red pulp fibrin deposition, erythrophagocytosis, and necrosis, African green monkey (Cercopithecus aethiops), primate.

2. Liver: Hepatocellular degeneration and necrosis, multifocal and random, with mild subacute inflammation, fibrin thrombi, and hepatocellular and monocytic intracytoplasmic eosinophilic viral inclusion bodies.

ETIOLOGIC DIAGNOSIS: Filoviral splenitis and hepatitis

CAUSE: Ebola virus, Zaire subtype

GENERAL DISCUSSION:

• Ebola virus (EBOV) is a filovirus, closely related to Marburg virus, that causes a highly infectious, often-fatal hemorrhagic fever in primates and humans; there are four species of Ebola virus (Zaire, Sudan, Ivory Coast, Reston) that have varying degrees of pathogenicity
• OIE listed reportable and zoonotic
• Filoviruses are enveloped, non-segmented, negative-stranded RNA viruses (order - Mononegavirales, family Filoviridae, genus - Filovirus)
  • Pleomorphic virions (filamentous, branched, "U"-shaped, "6"-shaped, circular) are 80nm in diameter and 800-1000nm in length
  • Virions replicate by budding, which causes nucleocapsids to accumulate in the cytoplasm and form closely packed arrays of bundled filaments (visible microscopically as viral inclusions)
• Ebola virus is a biosafety level 4 (BSL-4) agent but is sensitive to most common solvents, detergents, and UV light

PATHOGENESIS:

• In natural settings, the implicated reservoir host are three species of fruit bats:
  • Hypsignathus monstrosus (hammer-headed fruit bats)
  • Epomops franqueti (singing fruit bats)
  • Myonycteris torquata (little collared fruit bats)
• The mode of transmission involves direct contact with body fluids from either infected animals (e.g. gorillas and chimpanzees) or contact with a natural host
• In laboratory colonies, transmission of Ebola (Reston) has occurred through direct contact, aerosol, and fomites; experimentally, transmission has occurred via oral, parenteral, conjunctival, and aerosol inhalation routes
• The incubation period is 2-14 days; survivors clear the virus in about three weeks
• The virus targets cells of the mononuclear phagocyte system (MPS), formerly known as reticuloendothelial system; it interacts with proteins of the immune system like dendritic cells, macrophages, lymphocytes, and neutrophils, resulting in deregulation of their specific functions
• Virus-laden MPS cells rupture and release numerous virus particles into various tissues causing widespread necrosis, especially in the liver
• Endotoxemia causes release of inflammatory mediators which results in activation of complement, thrombosis, hemorrhage, tissue injury, and disseminated intravascular coagulation (DIC); DIC is a common terminal event
• Virus is able to evade the immune system by:
  • The speed of infection- animals often die before an effective immune response can be initiated
  • Its tropism for the MPS impairs any immune response that could be elicited

TYPICAL CLINICAL FINDINGS:

• Fever, anorexia, weight loss, lethargy, rash, lymphadenopathy, conjunctivitis, vomiting, diarrhea, petechiae, ecchymoses, hematemesis, melena, prostration, death
• Thrombocytopenia, neutrophilia, early lymphopenia followed by reactive leukocytosis with atypical lymphocytes in survivors
• Significantly elevated liver enzymes - LDH, AST, ALT
• Coagulation abnormalities with fibrin degradation products consistent with DIC

TYPICAL GROSS FINDINGS:

• Maculopapular rash, splenomegaly, widespread petechial hemorrhages, well-demarcated hemorrhage in proximal duodenum, interstitial pneumonia
• Hemothorax and/or hemoperitoneum due to massive internal bleeding (DIC)

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Lymphoid depletion and necrosis of germinal centers surrounded by hemorrhage of the mantle zone and massive fibrin deposition in the spleen
• Widespread, multifocal necrosis especially in the liver, spleen, lung, and adrenal cortex (within the zona glomerulosa, with little or no inflammation)
• Systemic lesions consistent with DIC including fibrin thrombi in capillaries of gastrointestinal tract, glomeruli, lung, and hepatic sinusoids; marked fibrin deposition in spleen
• Amphophilic to eosinophilic intracytoplasmic inclusion bodies in many tissues
• Kidney: Interstitial nephritis

ADDITIONAL DIAGNOSTIC TESTS:

• Clinical signs and histopathology are highly suggestive
• Paired serum samples using IFA (indirect fluorescent antibodies)
• Virus isolation, antigen-capture enzyme immunoassay, reverse transcriptase-polymerase chain reaction (RT-PCR)
• Immunohistochemistry: Antigenic positivity reported in cells of the MPS, hepatocytes, adrenal cortical cells, endothelial cells, fibrocytes, and fibrin thrombi

DIFFERENTIAL DIAGNOSIS:

Hemorrhagic fever viruses:

• Simian hemorrhagic fever virus (SHFV; Arterivirus, H-V04): Causes a highly infectious, fatal disease in macaques with clinical signs and gross pathology very similar to Ebola but no hepatic nor adrenal cortical necrosis
  • Reston Ebola outbreak associated with concurrent infection with SHFV
• Kyasanur Forest disease virus (Flavivirus): Lesions in Langurs and bonnet macaques include multifocal hepatocelluar necrosis and hemorrhage in adrenal glands, brain, kidney, and lung
• Marburg virus (Filovirus): causes severe and often fatal hemorrhagic fever
• Yellow fever virus (Flavivirus, D-V23): Mosquito transmitted hemorrhagic fever virus of New World monkeys; asymptomatic in African nonhuman primate species
  • Asian macaques get a rapidly fatal disease similar to humans; lesions are multifocal hepatocellular necrosis with councilman bodies and fatty degeneration of remaining hepatocytes; DIC and depletion of vitamin K-dependent clotting factors leads to bleeding diathesis; lymphoid depletion may be prominent
  • Dengue virus (Flavivirus): Sylvatic cycle involves Aedes spp. mosquitoes and nonhuman primates; nonhuman primates do not have clinical signs with experimental infection; causes hemorrhagic fever in humans
  • Machupo virus (MACV; Arenavirus): Foci of degeneration and cell death in the haired skin, liver, pancreas, adrenal glands, lymph nodes, tongue, esophagus, salivary glands, renal pelvis, small intestine, and large intestine; lymphohistiocytic interstitial pneumonia; nonsuppurative encephalitis; is the source agent of outbreaks of Bolivian hemorrhagic fever (BHF) in remote endemic regions of South America
    • Macrophages in the tracheobronchial lymph node were the first cells to demonstrate visible viral antigen after viral aerosolization
      • Guinea pigs are a viable animal model for MACV infection with a course of infection similar to humans and NHP

COMPARATIVE PATHOLOGY:

Animal models include guinea pigs, hamsters, mice, and rabbits:

• Guinea pigs were infected via aerosolization with guinea pig–adapted Zaire Ebolavirus (variant: Mayinga) and developed lethal interstitial pneumonia that was distinct from lesions described in guinea pigs challenged subcutaneously, nonhuman primates challenged by the aerosol route, and natural infection in humans
  • Intracytoplasmic inclusion bodies were observed in many alveolar macrophages
  • Perivasculitis was noted within the lungs
  • Rhesus macaques were infected via aerosolization with Zaire strain:
    • Important and unique findings include early infection of the respiratory lymphoid tissues, early fibrin deposition in the splenic white pulp, and perivasculitis and vasculitis in superficial dermal blood vessels of haired skin with rash
    • Initial infection occurred in the respiratory lymphoid tissues, fibroblastic reticular cells, dendritic cells, alveolar macrophages, and blood monocytes
    • Virus spread to regional lymph nodes, where significant viral replication occurred
    • Virus secondarily infected many additional blood monocytes and spread from the respiratory tissues to multiple organs, including the liver and spleen
    • Viremia, increased temperature, lymphocytopenia, neutrophilia, thrombocytopenia, and increased alanine aminotransferase, aspartate aminotransferase, g-glutamyl transpeptidase, total bilirubin, serum urea nitrogen, creatinine, and hypoalbuminemia were measurable mid to late infection
    • Infection progressed rapidly with whole-body destruction of lymphoid tissues, hepatic necrosis, vasculitis, hemorrhage, and extravascular fibrin accumulation
    • Hypothermia and thrombocytopenia were noted in late stages with the development of disseminated intravascular coagulation and shock
  • Dogs in endemic areas of Africa have developed titers to the virus
  • Pigs – Reston EBOV found to be in coinfection with PRRSV from domestic pigs

REFERENCES:

1. Bell TM, Bunton TE, Shaia CI, et al. Pathogenesis of Bolivian hemorrhagic fever in Guinea pigs. Vet Pathol. 2016; 53(1):190-199.
2. Bell TM, Shaia CI, Bunton TE, Robinson CG. Pathology of experimental Machupo Virus infection, Chicava strain, in Cynomolgus Macaques (Macaca fascicularis) by intramuscular and aerosol exposure. Vet Pathol. 2015; 52(1):26-37.
3. Lowenstine LJ, McManamon R, Terio KA. Apes. In: Terio KA, McAloose D, St. Leger J, Eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 390-391.
4. MacLachlan NJ, Dubovi EJ, eds. Fenner’s Veterinary Virology. 5th ed. London, UK: Elsevier Inc; 2017: 373-380.
5. Martinez MAJ, Mucino MCC, Terio KA. Suidae and Tayassuidae. In: Terio KA, McAloose D, St. Leger J, Eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 214.
6. Twenhafel NA, Shaia CI, Bunton TE, et al. Experimental aerosolized guinea pig adapted Zaire Ebolavirus (Variant: Mayinga) causes lethal pneumonia in Guinea pigs. Vet Pathol. 2015; 52(1):21-25.
7. Twenhafel NA, Mattix MD, Johnson JC, et al. Pathology of experimental aerosol Zaire Ebolavirus infection in Rhesus Macaques. Vet Pathol. 2013; 50(3): 514-529.
8. Wachtman L, Mansfield K. Viral diseases of nonhuman primates. In: Abee CH, Mansfield K, Tardiff S, Morris T, eds. Nonhuman Primates in Biomedical Research: Diseases. 2^nd San Diego, CA: Academic Press; 2012:38-41.
9. Zumbrum EE. Mission critical: Mobilization of essential animal models for Ebola, Nipah, and Machupo virus Vet Pathol. 2015; 52(1):18-20.

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