JPC SYSTEMIC PATHOLOGY

HEMOLYMPHATIC SYSTEM

February 2024

H-P03

 

 

SIGNALMENT: Juvenile male cynomolgus macaque

 

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

 

HISTOPATHOLOGICAL DESCRIPTION:  

SLIDE A: Peripheral blood smear: Few erythrocytes (up to five per 40X high power field) contain intracellular parasites that vary from 1-2 µm ring forms to larger trophozoites that occupy up to 50% of the erythrocyte. The cytoplasm of the parasites stains light blue and often contains small, 1 µm or less, black-brown, anisotropic pigment granules (hemozoin or malaria pigment). There is mild anisocytosis and polychromasia of erythrocytes.  

 

MORPHOLOGIC DIAGNOSIS: Cytologic specimen, peripheral blood smear:  Trophozoites, intraerythrocytic, few, with hemozoin pigment, cynomolgus macaque (Macaca fascicularis), nonhuman primate.

 

SLIDE B: Spleen: Multifocally, primarily within the red pulp, there are increased numbers of macrophages that often contain abundant intracytoplasmic, faintly birefringent, golden-brown, globular pigment (hemozoin).  Diffusely, periarteriolar lymphoid sheaths are moderately expanded by increased numbers of lymphocytes (lymphoid hyperplasia).

 

Lymph node: Similar pigment is seen within macrophages of the adjacent splenic lymph node cortical and medullary sinuses. 

 

Liver: Kupffer cells are mildly increased in number and often contain abundant intracytoplasmic hemozoin as previously described. Multifocally, there are moderate numbers of lymphocytes and plasma cells surrounding portal areas and to a lesser extent surrounding centrilobular veins. Hepatocytes are diffusely swollen with lacy cytoplasm (glycogenosis).

 

MORPHOLOGIC DIAGNOSIS: 

1. Spleen, white pulp: Lymphoid hyperplasia, diffuse, moderate, cynomolgus macaque (Macaca fascicularis), nonhuman primate.
2. Spleen and lymph node: Histiocytosis, multifocal, moderate, with abundant intrahistiocytic hemazoin pigment.
3. Liver: Kupffer cell hyperplasia, multifocal, moderate with abundant intrahistiocytic hemazoin pigment, and mild, multifocal, periportal lymphoplasmacytic hepatitis.

 

CAUSE: Plasmodium sp. (P. knowlesi) 

 

ETIOLOGIC DIAGNOSIS: Plasmodial erythroparasitemia 

 

CONDITION: Malaria 

 

GENERAL DISCUSSION:  

• Order Hemosporidia is composed of blood-borne, intracellular apicomplexan protozoa that include parasites of three main genera: Plasmodium (the cause of malaria and avian malaria), Hemoproteus sp., and Leucocytozoon sp.
• There are several species of Plasmodium that can affect a wide range of vertebrates, including mammals, birds, and reptiles and are an important cause of disease in primates (“malaria”) and birds (“avian malaria”)
  1. There are numerous pathogenic Plasmodium sp.; many are species specific and usually do not cause significant disease in the normal/adapted host
  2. Plasmodium coatneyi infection in macaques is an important research model as it has several biological features in common with P. falciparum (the species with highest morbidity and mortality in humans)
  3. Natural infection is universal among NHPs except tamarins, marmosets, and owl monkeys (susceptible to experimental infection)
• Malaria (Plasmodium spp.) parasites can be classified on the basis of host infected (human, monkey, or anthropoid ape), parasite morphology, or type of cyclic fever produced (quotidian has a 24-hour cycle, tertian has a 48-hour cycle, and quartan has a 72-hour cycle)

 

LIFE CYCLE:

• Life cycles of various Plasmodium species are similar with two hosts required (indirect lifecycle): 

1. Vertebrate host: (asexual or schizogonic phase): Female mosquito with infected saliva bite -> Sporozoites injected into peripheral circulation:
   1. Exoerythrocytic or liver phase: Invade macrophages and endothelial cells (especially in lungs, heart, liver, and spleen) -> Develop into first generation exoerythrocytic schizont/meront -> Asexual reproduction to form merozoites -> Cell rupture, merozoite release -> Repeat or invade RBCs
   2. Erythrocytic or blood phase: Merozoites invade erythrocytes -> Develop into trophozoites -> Form erythrocytic meronts (additional asexual stage -> RBC lysis, additional RBCs infected) or gametocytes (macrogametocytes or microgametocytes) -> Gametocytes remain inside RBC until ingested by insect host 
2. Invertebrate host (sexual or sporogonic phase), an arthropod vector, usually an Anopheles (or Culex) mosquito: Female mosquito ingests RBCs with gametocytes -> Sporogony occurs
   1. Male and female gametocytes develop into gametes -> Fuse to form a motile zygote (ookinete), which enters the gastric epithelium and develops into oocyst -> Oocyst ruptures and sporozoites released into hemolymph -> Migration to salivary gland -> Infectious sporozoites transferred to vertebrate host 

 

PATHOGENESIS:  

• Disease severity depends on host and Plasmodium species, degree of adaptation/naivety, with significant disease heterogeneity 
  1. In the natural host, organisms produce little disease, usually without fever, possible mild anemia associated with a low-grade parasitemia
  2. In aberrant/nonadapted hosts, infection often produces severe disease or death
     • In nonadapted hosts, exoerythrocytic schizogony is more prominent and RBC merozoites can go back to infect macrophages and endothelial cells, restarting the exoerythrocytic or liver phase
  3. Malaria in most NHPs is generally not fatal, however it may cause debilitation and disease and can be precipitated by stress, concurrent disease, splenectomy, or immunosuppression; these NHPs can be malaria risk to human handlers (blood-borne route)
• Cyclic fever coincides with the relatively synchronous rupture of parasitized erythrocytes with release of toxic metabolites into the bloodstream
• Sequestration of erythrocytes in the microvasculature; in brain capillaries, leads to sludging of blood and cerebral malaria in humans and simian primates (not apes)
  1. Vascular occlusion can be worsened due to swollen, meront-filled endothelial cells -> Tissue hypoxia and necrosis
• Death may be due to circulatory shock, respiratory insufficiency, cardiac tamponade due to pericardial effusion, and/or massive release of inflammatory mediators (cytokine storms)

 

TYPICAL CLINICAL FINDINGS:

• Signs are related to the level of parasitemia (worse in aberrant hosts): Irritability or depression/malaise, cyclic fever; pale mucus membranes (anemia)
  1. Other signs may include vomiting and/or diarrhea or splenic rupture
  2. There may be neurological signs including motor incoordination, seizures, and paralysis
• CBC: Thrombocytopenia, leukopenia, progressive anemia (regenerative), reticulocytosis
• Splenectomy increases susceptibility to a number of blood-borne infections, including malaria
• Disease typically more severe in younger animals

 

TYPICAL GROSS FINDINGS:

• Marked splenomegaly, hepatomegaly
• Diffuse brown discoloration of multiple organs especially spleen and liver due to hemazoin pigment within phagocytic cells,
• Cardiopulmonary: Pulmonary pallor and edema; hydropericardium, myocarditis
• Bone marrow: Dark red due to erythroid regeneration (and hemazoin)
• Also reported: 
  1. Cerebral hemorrhage, splenic rupture
  2. Malaria during pregnancy can lead to complications such as abortion, low-birth-weight infants, intrauterine growth retardation, anemia, and congenital malaria

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Liver and spleen: Lymphoid hyperplasia and expansion of the mononuclear phagocytic system cells (e.g., Kupffer cell hyperplasia (liver), lymphoid follicular hyperplasia (spleen)), phagocytic cells (as well as erythrocytes) containing black granular pigment (hemazoin or malaria pigment), and lymphocytic, histiocytic and plasmacytic inflammation (hepatitis, splenitis), extramedullary hematopoiesis and hemosiderosis, +/- hepatocellular and/or splenic cellular necrosis
  1. Hemazoin (β-hematin) is a refractile, birefringent pigment created from malarial digestion of hemoglobin (dimers of heme) within the host; it does not stain with iron stains (e.g., Perl’s) and must be differentiated from acid hematin
• Exoerythrocytic schizonts/meronts in macrophages and endothelial cells within the spleen, liver, lungs and other organs (e.g., kidney, brain) (approximately 15x20 µm)
• Bone marrow: Hypercellular due to erythroid +/- myeloid hyperplasia, hemazoin-laden macrophages
• Cardiovascular, pulmonary: Granulocytic or histiocytic interstitial pneumonia, myocarditis
• Also reported: Lower nephron (tubular) necrosis and/or glomerulopathy of the kidney

 

ULTRASTRUCTURAL FINDINGS:

 

ADDITIONAL DIAGNOSTIC TESTS:  

 

DIFFERENTIAL DIAGNOSIS:

Blood parasites in NHPs:

Blood parasites in Avians:

• Plasmodium sp.
• Genus Haemoproteus:
  1. Avian: Host-specific; gametocytes are only seen within erythrocytes; appears similar to Plasmodium sp. gametocytes; transmitted by biting flies and midges in which sporogony occurs à schizonts/meronts commonly infect pulmonary vascular endothelium or other visceral endothelium à merozoites invade erythrocytes and mature, or there may be a second cycle of schizogony in cardiac and skeletal muscle (turkeys reported with skeletal muscle fusiform cysts); rarely symptomatic, anemia and hepatomegaly have been reported
  2. Poultry: H. meleagridis
  3. Pigeons, doves: H. columbae, H. Saccharovi
  4. Waterfowl:  H. nettionis
• Genus Leucocytozoon (H-P06)
  1. Avian: Target erythrocytes and cardiac myocytes; transmitted by culicoid midges and simuliid flies (black flies); generally clinically inapparent, with acute outbreaks in young poultry and chronic disease in older birds; surviving birds are carriers; the most pronounced lesion is splenomegaly; Leucocytozoon speciation may be inaccurate
  2. Chickens: L. andrewsi
  3. Ducks: L. simondi
  4. Turkeys: L. smithi
  5. Guinea fowl: L. neavei
• Histological identification and differentiation of tissue stages and intraerythrocytic stages of Hemoproteus sp., Plasmodium sp., and Leucocytozoon sp. is frequently challenging
  1. Hemozoin is seen with Plasmodium and Hemoproteus but not Leucocytozoon 

 

COMPARATIVE PATHOLOGY:

• NHP: Numerous species are described
  1. P. knowlesi: Natural host is cynomolgus macaque (among others) and can produce infections in rhesus macaques that are nearly always fatal; zoonotic
  2. P. brazilianum is sometimes a markedly pathogenic species and is the most common malarial parasite of New World monkeys
  3. P. coatneyi: Considered to be homologous to P. malariae in humans; zoonotic; possesses several biological features in common with P. falciparum); P. knowlesi (zoonotic potential); P. Cynomolgi; P.  inui; P. schwetzi; P. simium (zoonotic potential); many others
• Avian: 
  1. Rarely reported in domestic pigeons, canaries, turkeys, penguins (the most significant parasitic disease in captive penguins housed outdoors), falcons, bald eagles, and cliff swallows
  2. Often not host-specific; disease is similar to malaria in humans
  3. P. relictum is one of the most widespread species and has played a significant role in the decline of many endangered Hawaiian bird species; spread primarily by Culex sp. of mosquito
• Reptiles: Infections generally considered minimally pathogenic
  1. Black and white tegus: Plasmodium infection identified on blood smear, successfully treated using antimicrobials (Pereira, J Comp Pathol. 2021)
• Human: Four species of Plasmodium commonly infect humans: P. falciparum (highest morbidity and mortality), P. vivax, P. ovale, and P. malariae; recently, P. knowlesi has been discovered as a significant cause of human malaria

Other intraerythrocytic parasites:

• Cats: Cytauxzoon felis, Babesia cati
• Cattle: Anaplasma marginale, Anaplasma centrale, Babesia bovis, Babesia bigemina, Theileria mutans, Theileria annulata
• Deer: Theileria cervi
• Dogs: Babesia canis, Babesia gibsoni
• Horses: Babesia equi, Babesia caballi
• Sheep: Babesia ovis, Babesia motasi

 

REFERENCES:

1. Crespo R, Franca MS, Fenton H, Shivaprasad HL. Galliformes and Colubriformes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. London, UK: Academic Press; 2018:767-768. 
2. Fitz-Coy SH. Parasitic Diseases. In: Boulianne M ed. Avian Disease Manual. 7^th ed. Madison, WI: Omnipress; 2019:132-133.
3. Lombardini ED, Turner GDH, Brown AE, Inamnuay L, Kaewamatawong T, Sunyakumthorn P, Ferguson DJP. A systematic analysis of ultrastructural lesions in the Plasmodium coatneyi splenectomized rhesus macaque model of severe malaria. Vet Pathol. 2022 Sep;59(5):873-882.
4. Lowenstine LJ, McManamon R, Terio KA. Apes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. London, UK: Academic Press; 2018:400, 402. 
5. Lowenstein LJ, Osborn KG. Strait K, Else JG, Eberhard ML. Respiratory System Diseases of Nonhuman Primates. In: Abee CR, Mansfield K, Tardif S, Morris T. Nonhuman Primates in Biomedical Research: Volume 2: Diseases. 2^nd ed. San Diego, CA: Elsevier; 2012: 491, 527.
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