JPC SYSTEMIC PATHOLOGY

HEMOLYMPHATIC SYSTEM

April 2024

H-V05

Signalment: (JPC #1834129): Group of 40–60-pound pigs

HISTORY: Found comatose and hypothermic. Necropsy findings included cachexia, severe congestion and edema of the lungs, and mild discoloration of the liver.

HISTORY: Tissues from one of a group of 40–60-pound pigs, which were inoculated with an infectious agent. This pig was euthanized 7 days post-inoculation.

HISTOPATHOLOGICAL DESCRIPTION: Spleen: Diffusely, there is lymphoid depletion within white pulp characterized by lymphocyte necrosis with loss of architecture and replacement by moderate amounts of eosinophilic cellular and karyorrhectic debris (lytic necrosis). Multifocally red pulp contains mild to moderate amounts of necrotic debris, fibrin, and increased numbers of neutrophils and erythrophagocytic macrophages. Occasionally, arteriolar walls are disrupted, expanded, and replaced by eosinophilic fibrillar to homogenous fibrin admixed with degenerate inflammatory cells and non-degenerate macrophages, lymphocytes, and neutrophils, and small amounts of necrotic cellular debris (fibrinonecrotizing vasculitis). Diffusely, vessels are lined by hypertrophied endothelium. There is diffuse, moderate congestion.

MORPHOLOGIC DIAGNOSIS: Spleen: Lymphoid necrosis, diffuse, subacute, severe, with multifocal necrotizing vasculitis, breed unspecified, porcine.

ETIOLOGIC DIAGNOSIS: Asfarviral splenic necrosis

CAUSE: African swine fever virus (ASFV)

CONDITION: African pig disease, wart hog disease

GENERAL DISCUSSION:

African swine fever (ASF) is a highly contagious, highly fatal febrile hemorrhagic disease of suids that is endemic in sub-Saharan Africa and Sardinia, and is spreading throughout Europe and Asia
1. International spread of ASF is primarily through infected pork products in garbage fed to pigs
ASF virus (ASFV) is an enveloped dsDNA virus and is the only member of family Asfarviridae; there are multiple genotypes from different geographic regions with high, moderate, and low virulence strains
The pathogenesis, mechanisms of injury, and clinical outcomes of ASF are very similar to those of classical swine fever (H-V06), although ASF is generally much more severe
1. Unlike CSF, ASF does NOT infect epithelium, but the degeneration of vascular endothelium and the fibrin arterial changes are identical
ASF is typically seen as a peracute or acute disease (high morbidity and mortality); virulence diminishes with time, thus subacute, chronic, and inapparent forms become increasingly evident; survivors remain persistently infected

PATHOGENESIS:

ASFV natural hosts include warthogs and bushpigs and it exists in a natural cycle between warthogs and the soft tick vector Ornithodoros moubata
1. ASFV is infective throughout various nymph stages, and there is trans-stadial, transovarial, and sexual transmission
2. Other arthropods, including other species of Ornithodoros ticks as well as stable flies (Stomoxys calcitrans) can harbor the virus
3. Transmission to domestic pigs occurs by tick bites or ingestion of warthog tissues or secretions
ASFV is transmitted through a variety of routes, including direct contact with infected animals, contaminated fomites, feces, ingestion of contaminated feed (e.g., with pig meat products), inhalation, iatrogenic, and Ornithodorus ticks
1. Transplacental infection with viral strains of low virulence may result in persistently infected piglets
2. In clinical domestic pigs, all body fluids/tissues contain large amounts of virus
3. ASFV is very stable and can survive for months in refrigerated meat and for years in frozen meat
Following inhalation or ingestion, ASFV replicates in the tonsils (epithelial cells of tonsil crypts) and head/neck lymph nodes -> Marked viremia
Macrophages are the primary target cells, especially mature (CD163 and CD107a+) macrophages; viral glycoproteins p12, p30, and p54 play a major role in attaching to and entering target cells
Proinflammatory cytokines (IL-1, IL-6, tumor necrosis factor-α) released from macrophages cause endothelial cell lysis in all organ systems -> Vasculitis, systemic petechial and ecchymotic hemorrhages, coagulation cascade activation, DIC, circulatory collapse, shock, and death
1. Hemorrhages attributed to endothelial damage and marked thrombocytopenia
2. There is also widespread cell death via cell lysis and apoptosis -> Severe loss of monocytes, macrophages, and lymphocytes in all organ systems
3. Activation of pulmonary intravascular macrophages, cytokine release -> Pulmonary edema, neutrophil sequestration, microthrombi in septal capillaries
4. ASFV also modulates macrophages signaling pathways, interfering with expression of immunomodulatory genes -> Allows for persistent infections
5. Increased vascular permeability in the initial stage of acute infection is dependent on viremia and elevated concentration of serum nitric oxide (NO) (Tatoyan, J Comp Pathol. 2019)
Later in disease, other cells, including megakaryocytes, tonsillar epithelial cells, hepatocytes, renal epithelial cells, and endothelial cells, become infected
Chronic infections produce immune complexes -> Vascular damage (e.g., fibrinoid vasculitis) and glomerulonephritis

TYPICAL CLINICAL FINDINGS:

Peracute: Severe acute viral hemorrhagic fever with high morbidity/mortality; death in 1-3 d
1. Pigs typically die before development of anti-ASF immunoglobulin, probably due to the demise of antigen-presenting cells in lymph nodes, spleen, and liver
Acute: High fever, anorexia, lethargy, weakness, recumbency, respiratory distress, diarrhea, neurological signs, and erythema, as well as cyanosis of ears, tails, lower legs, and abdomen
1. Death usually within 7-9 days of infection
Subacute and chronic: Dyspnea, vomiting, and nasal and conjunctival discharge
1. Fever is usually transient and affected pigs either die or recover within 3-4 weeks
Pregnant sows may abort at any time during pregnancy once clinical signs are noted
Severe thrombocytopenia and leukopenia

TYPICAL GROSS FINDINGS:

ASFV induces severe vascular changes that cause pulmonary edema, disseminated intravascular coagulation, and hemorrhages in different organs with melena, epistaxis, erythema, renal petechiae, and diffuse hemorrhages in lymph nodes
1. Lymph nodes are severely swollen and appear grossly like blood clots due to severe hemorrhage and necrosis
2. Hemorrhages on serosal surfaces
3. Renal cortical, medullary, and pelvic hemorrhage (turkey egg kidney)
4. Marked splenomegaly (congestion); the spleen is friable and dark black
5. Meninges may be congested, edematous, or hemorrhagic
6. Hydropericardium and hydrothorax
7. Pulmonary and gall bladder edema (not typically present in CSF pigs)
Aborted fetuses: Anasarca and mottled lungs/liver; petechia of placenta, skin, and myocardium
Chronic: Small hemorrhagic infarcts in skin (especially in areas of trauma), erythema and cyanosis; arthritis, lobular pulmonary consolidation with chronic interstitial pneumonia; fibrinous pericarditis and pleuritis; and glomerulonephritis

TYPICAL LIGHT MICROSCOPIC FINDINGS:

Extensive necrosis of lymphocytes and monocyte/macrophage system cells
Degeneration and necrosis of vascular endothelium with fibrinoid change as well as hemorrhage, and/or thrombosis
1. There may be necrosis of other cells, including hepatocytes
Hematopoietic: Depletion of lymphoid tissue with widespread necrosis of macrophages and lymphocytes; hemorrhage within lymph nodes
Kidney: Renal tubular degeneration with amorphous casts in the medulla, acute glomerular capillary thrombosis, or subacute to chronic focal segmental glomerulonephritis

ADDITIONAL DIAGNOSTIC TESTS:

PCR for the p72 gene and immunofluorescent antigen detection in tissue smears offer rapid diagnostic options.
1. Sections of tonsil, lymph node, kidney, and/or spleen as well as blood samples are the most useful for diagnosis
2. Bone marrow was found to provide the highest viral load and a high probability of virus detection (especially useful in a decomposed carcass); ear biopsies may also be useful for surveillance programs (Flannery, J Vet Diagn Invest. 2020)
3. Meat juice samples (obtained via a freeze-thaw cycle at the slaughterhouse) was shown to have decreased, but comparable, sensitivity to splenic swabs with rtPCR for surveillance (Okwasiimire, J Vet Diagn Invest. 2023)
Quantitative PCR (qPCR) technique developed to detect B646L gene (which codes for the p72 protein) using FFPET (formalin-fixed, paraffin-embedded tissue), enabling testing at a BSL-2 diagnostic laboratory (ASFV is classified as a BSL-3 level pathogen) (Urbaniak, J Vet Diagn Invest. 2023)

DIFFERENTIAL DIAGNOSIS:

Differentials for “turkey egg kidney” include: ASF, CSF, salmonellosis, porcine dermatitis nephropathy syndrome (circovirus-associated disease), and Erysipelothrix rhusiopathiae
Other common causes of skin discoloration include Erysipelothrix rhusiopathiae (diamond-shaped skin lesion), Salmonella choleraesuis (most common on the abdomen and ears), and porcine dermatitis nephropathy syndrome
Classical swine fever (Porcine pestivirus): Lesions of ASF are usually indistinguishable from CSF
1. Hemorrhage and edema are usually more frequent and severe in ASF
2. Splenomegaly and hematoma-like visceral lymph nodes are more characteristic of ASF
3. Infarction may be more characteristic of CSF
4. There is no cross-protection conferred between classical swine fever virus (CSFV) and ASFV
Other differentials include pasteurellosis and other septicemias

Classical swine fever African swine fever virus

Splenic infarcts Splenomegaly, NO infarcts

No pulmonary edema Pulmonary edema, especially septa

Patchy interstitial pneumonia

Button ulcers in colon Massive hemorrhagic necrosis in colon

Rare frank lymphoid necrosis Diffuse severe lymphoid necrosis

(there is a lack of mature

lymphoid tissue)

No particular liver lesions Necrosis of periportal hepatocytes

Lymphoid infiltrates

Rare renal tubular degeneration Common renal tubular degeneration

Generally no brain lesions Perivascular cuffing with necrotic

debris in brain

Meningoencephalomyelitis

Infects epithelium No epithelial infection

	ASF (acute)	ASF (subacute)	CSF	PRRS	Swine erysipelas	Septicemic salmonellosis	PDNS
Skin	Erythema	Erythema	Erythema	Cyanosis	Diamond skin lesions	Cyanosis	Macules and papules
Lymph nodes	Marbled aspect	Hemorrhagic	Marbled aspect	Swelling or marbled	Marbled aspect	Swelling	Marbled aspect
Spleen	Hyperemic splenomegaly	Partial hyperemic splenomegaly or focal infarct	Multiple infarcts at the margin	Scattered infarcts or white spots on the surface	Hyperemic splenomegaly	Splenomegaly	-
Kidney	Petechial hemorrhages	Petechial hemorrhages, perirenal edema	Petechial hemorrhages	Petechial hemorrhages	Petechial hemorrhages	Petechial hemorrhages	Glomerulonephritis
Gall bladder	Petechial hemorrhages	Wall edema	Petechial hemorrhages	-	-	-	-
Tonsils	-	-	Necrotic areas	Swelling or with hemorrhages	-	-	-
Intestine	Petechial hemorrhages	Petechial hemorrhages	Button ulcers	-	-	Necrotic enterocolitis	-
Thrombocytopenia	Absence or slight (late)	Transient	Intense (early)	Absent	Absent	Absent	Absent
Other	-	-	Nervous signs; congenital malformation (congenital form)	Thymic atrophy; interstitial pneumonia	Arthritis and vegetative endocarditis (chronic form)	Nervous signs; congestion of gastric mucosa; necrotic foci in the liver; bronchopneumonia	-

Adapted from: Table 3 Differential diagnosis of African swine fever on the basis of gross lesions. In: Sanchez-Vincaino JM, Mur L, Gomez-Villamandos JC, Carrasco L. An update on the epidemiology and pathology of African swine fever. J Comp Pathol. 2015;152(1):9-21.

COMPARATIVE PATHOLOGY:

Only infects members of the Suidae family; the bush pig and warthog act as reservoirs
Warthogs are susceptible to infection but do not develop clinical signs, with most being infected during the first few weeks of life and develop a generalized viremia, sufficient to infect ticks that feed on them

REFERENCES:

Cantile C, Youssef S. Nervous System. In: Maxie MG, ed. Jubb, Kennedy & Palmer's Pathology of Domestic Animals. Vol 1. 6th ed. St. Louis, MO: Elsevier; 2016:398, 452.
Flannery J, Ashby M, Moore R, et al. Identification of novel testing matrices for African swine fever surveillance. J Vet Diagn Invest. 2020; 32(6):961-963.
Gal A, Castillo-Alcala F. Cardiovascular System, Pericardial Cavity, and Lymphatic Vessels. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:569, 691.
Lopez A, Martinson SA. Respiratory System, Thoracic Cavities, Mediastinum, and Pleurae. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:569, 627.
Martinez MAJ, Gasper DJ, Mucino MCC, Terio KA. Suidae and Tayassuidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. London, UK: Academic Press; 2018:209-212.
Okwasiimire R, Nassali A, Ndoboli D, et al. Comparison of diaphragm meat juice and muscle swab samples to spleen and spleen swab samples for the detection of African swine fever viral nucleic acid. J Vet Diagn Invest. 2023;35(2):145-152.
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Tatoyan MR, Ter-Pogossyan ZR, Semerjyan AB, et al. Serum concentrations of vascular endothelial growth factor, stromal cell-derived factor, nitric oxide and endothelial DNA proliferation in development of microvascular pathology in acute African Swine Fever. J Comp Pathol. 2019;167:50-59.
Urbaniak K, Meekins DA, Davis AS, Richt JA, Trujillo JD. Development of a sensitive, high-throughput extraction protocol for qPCR detection of African swine fever virus in formalin-fixed, paraffin-embedded tissues. J Vet Diagn Invest. 2023;35(3):284-288.
Valli VEOT, Kiupel M, Bienzle D, Wood RD. Hematopoietic System. In: Maxie MG, ed. Jubb, Kennedy & Palmer's Pathology of Domestic Animals. Vol 3. 6th ed. St. Louis, MO: Elsevier; 2016:168, 181-182.
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