JPC SYSTEMIC PATHOLOGY
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.
SLIDE A: Peripheral blood smear: Few erythrocytes (up to five per 40X high power field) contain intracellular parasites that vary in size from 1-2 um 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 um 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, etiology consistent with Plasmodium sp., 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).
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.
- Spleen, white pulp: Lymphoid hyperplasia, diffuse, moderate.
- Spleen, red pulp: Histiocytosis, multifocal, moderate, with abundant intrahistiocytic hemazoin pigment.
- 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
- Intracellular protozoan parasite of the family Plasmodiidae, genus Plasmodium that affects a wide range of vertebrates including mammals, birds, and reptiles (primates, birds, and rodents are important animal models);
- Natural infection is universal among nonhuman primates except tamarins, marmosets, and owl monkeys although they are susceptible to experimental infection
- There are numerous pathogenic Plasmodium sp.; many are species specific and usually do not cause significant disease in the normal host
- Malaria (Plasmodium ) 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)
- Research model: Plasmodium coatneyi has several biological features in common with falciparum (the species with highest morbidity and mortality in humans) (see reference 5 below):
- Considered one of the more pathogenic plasmodia in nonadapted hosts
- 48-hour cycles (tertian) of intraerythrocytic development, followed by rupture (schizogony), reinvasion of uninfected erythrocytes, and a resultant fever spike in the host
- Demonstrate microvascular cytoadherence, form rosettes of infected red blood cells with uninfected erythrocytes, and develop electron-dense knobs on the cell surface, which are theorized to play a role in cytoadhesion
- Undergo deep vascular sequestration of trophozoite- and schizont-infected red blood cells
- Demonstrate a consistent predilection for specific tissues (e.g. brain microvasculature with resultant vascular occlusion)
- knowlesi, recently recognized as a cause of human malaria, causes rapidly fatal disease in rhesus macaques and is naturally found in long-tailed and pig-tailed macaques and banded leaf monkeys; it is the only known 24 hour cycle (quotidian) malarial parasite
- Drug (artemisinin)-resistant malarial infections are becoming increasingly common especially in areas with high levels of endemic malaria maintained within populations of NHPs (e.g. Southeast Asian rainforests)
- Life cycles of various Plasmodium species are similar, two hosts required:
- Vertebrate host: (asexual reproduction): Female mosquito penetrates skin of vertebrate host (in research settings, rodents are inoculated intraperitoneally and NHPs are inoculated intravenously) à introduces sporozoites into peripheral circulation à exoerythrocytic/hepatic stage with intrahepatocyte development from sporozoite to schizont that develop thousands of merozoites à merozoites released from schizonts à merozoites enter erythrocytes (erythrocytic stage) and reside in parasitophorous vacuoles (some species appear as ring forms; the variability of location within erythrocytes and different morphologies assist in speciation) à enlarge into trophozoites à transform into schizonts that undergo asexual division to form merozoites à relatively synchronous RBC lysis that releases merozoites to infect other RBCs
- Invertebrate host (sexual reproduction), commonly Anopheles mosquitoes: Within the vertebrate host, some merozoites develop into gametocytes and remain in the blood as micro- and macrogametocytes à ingested by a female mosquito à in the mosquito’s stomach, male and female gametocytes develop into gametes, which fuse to form a motile zygote (ookinete), which enters the gastric mucosa and further develops to the oocyst stage in the gastric stroma à repeated nuclear division à rupture of oocyst à sporozoites released into hemolymph à migration to salivary gland à transferred to vertebrate host during blood meal
- Disease severity depends on host and plasmodial species depending on the degree of adaptation/naivety, with significant disease heterogeneity
- Anemia: Parasite can cause direct lysis of erythrocytes or indirect hemolysis (bystander effect, more significant than direct lysis, with splenic removal of more nonparasitized erythrocytes than parasitized erythrocytes)
- Hypersplenism: Splenic phagocytes increase in number, avidity, and efficiency during infection à removal of erythrocytes
- 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: Seen with falciparum (human); P. coatneyi, P. fragile, P. knowlesi (NHP), and P. berghei (mice); cytoadherence occurs via parasite-encoded adhesins (e.g. PfEMP-1 in P. falciparum) that bind to host cell receptors (e.g. CD36, TSP, and ICAM-1 on endothelium, leukocytes, platelets, and uninfected erythrocytes); sequestration protects the parasite from phagocytosis and destruction in the spleen and is responsible for much of the morbidity and mortality of severe malaria (particularly cerebral malaria) due to severe tissue hypoxia and subsequent cellular degeneration and necrosis
- Hemozoin pigment is insoluble ferriprotoporphyrin created from malarial digestion of hemoglobin within the host
TYPICAL CLINICAL FINDINGS:
- Minimal disease in the natural host; typically afebrile, possibly mild anemia with low-grade parasitemia; typically more severe in younger animals
- Signs are related to the level of parasitemia and appear 5-14 days post infection: irritability, cyclic fever, depression, listlessness, anorexia, weight loss, diarrhea, weakness, possibly shock-like syndrome
- CBC: thrombocytopenia, leukopenia, progressive anemia (regenerative), reticulocytosis
- Chemistry panel: suggests kidney and liver disease (acute renal tubular necrosis has been reported in severe disease, and immune mediated glomerulonephritis can be a sequela) including marked azotemia, hypertriglyceridemia, hypoalbuminemia, and increased creatinine phosphokinase
- Coagulation assays: severe coagulopathy with increase in fibrin degradation products and decrease in proteins C and S (suggestive of a consumptive process)
TYPICAL GROSS FINDINGS:
- Diffuse brown discoloration of multiple organs especially kidneys and liver, also lungs, heart, and brain; due to vascular congestion and hemozoin pigment within erythrocytes and monocytes
- Marked splenomegaly, hepatomegaly, lymphadenomegaly
- Bone marrow: dark red due to erythroid regeneration
- Kidneys: often congested and slightly enlarged (glomerulonephritis and/or acute tubular necrosis)
- Brain: Diffuse pink color
- Skin: Rare reports of cutaneous necrosis secondary to disseminated intravascular coagulopathy causing thrombosis of superficial vasculature (reported with coatneyi and P. falciparum)
- Eye: retinal hemorrhages are common (and diagnostically significant) in humans but not currently recorded in NHP models
- Petechiae and ecchymoses of multiple tissues especially the epicardium
TYPICAL LIGHT MICROSCOPIC FINDINGS:
- Lymphoid and macrophage hyperplasia (lymphocytosis and histiocytosis) in the spleen, liver, and bone marrow; erythrophagocytosis in spleen, liver, lymph nodes
- Erythrocyte sequestration in microvasculature of multiple tissues (most common in the myocardium with coatneyi), often forming rosettes of uninfected cells bound to a parasitized cell (more readily visible with electron microscopy)
- Spleen: lymphoid follicular hyperplasia, activated macrophages, and red pulp sequestration with parasitized erythrocytes and birefringent hemozoin pigment
- Liver: Hyperplasia of Kupffer cells with intracytoplasmic hemozoin, parasites, and cellular debris; occasionally diffuse centrilobular necrosis (rare in humans)
- Bone marrow: Erythropoiesis +/- myeloid hyperplasia
- Blood: monocytosis, parasitized erythrocytes, and occasionally large hemozoin-laden monocytes
- Kidneys: Mesangioproliferative glomerulopathy (IgG, IgM, and C3 deposition on thickened basement membranes, not noted with coatneyi); acute tubular necrosis has been reported with hyaline degeneration (possible protein resorption), hemoglobin casts, and cellular casts
- Lungs: Hemozoin-laden macrophages
- Brain: Cerebral malaria; parasitized red cells partially or completely occlude blood vessels evidenced by perivascular edema, hemorrhage, and peripheral necrosis in conjunction with increased cytoadhesion of parasitized cells (sequestration); frequent intraerythrocytic and intramonocytic hemozoin pigment within the neuropil
- Placenta: In severe cases, infarction, fibrinoid necrosis, fibrin deposition, and mononuclear and polymorphonuclear fibrinonecrotizing placentitis
- Rosettes: Erythrocytes form rosettes of uninfected cells bound to a central parasitized cell ( coatneyi, P. falciparum, P. fragile)
- Conical electron-dense “knobs”: Function in cytoadhesion (including formation of rosettes); not present in infections with vivax, P. cynomolgi, P. knowlesi, or the ring stages of P. falciparum and P. coatneyi
- Erythrocyte morphology: distortion and prolongation of cytoplasm that interdigitates with endothelial invaginations and projections, hemozoin crystals, intraerythrocytic parasites (schizonts predominate)
ADDITIONAL DIAGNOSTIC TESTS:
- PCR, serology (IFA preferred)
- Detection of trophozoites in erythrocytes is the gold standard for laboratory diagnosis; examination is conducted using blood prepared as thin films (routine blood smear, used for speciation) and thick films (involves erythrocyte lysis, more sensitive), stained with Giemsa or Wright-Giemsa;
- Highly visible and diagnostic hemozoin pigment may be detected in erythrocytes that contain more mature trophozoites or schizonts, as well as intraerythrocytic tinctorial stippling (Maurer’s spots, require Giemsa or modified Leishman stain for visualization)
Blood Parasites in NHPs:
- Plasmodium spp.
- Hepatocystis spp: See Comparative Pathology section
- Genus Plasmodium: >250 species, with new species and novel host-parasite interactions regularly reported
- Human: Four species of Plasmodium commonly infect man: 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
- NHP: coatneyi (several biological features in common with P. falciparum, see reference #5), P. knowlesi (zoonotic potential), P. cynomolgi, P. brasilianum, P. inui, P. schwetzi, P. simium (zoonotic potential), many others
- Avian: Rarely reported in domestic pigeons, canaries, turkeys, penguins (the most significant parasitic disease in captive penguins housed outdoors), falcons, bald eagles, and cliff swallows; often not host-specific; disease is similar to malaria in humans; 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
- Rodents: A separate group of malarial parasites infects rodents and are commonly used as a research model; however, cerebral malaria in rodents is due to nonsequestration-mediated immune-mediated encephalitis (whereas NHP and human cerebral malaria is sequestration-mediated)
- Reptiles: Lizards and snakes; floridense and P. mexicanum
- Genus Haemoproteus:
- Avian: Host-specific; gametocytes are only seen within erythrocytes; appears similar to Plasmodium 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
- Poultry: meleagridis
- Pigeons, doves: columbae, H. Saccharovi
- Waterfowl: nettionis
- Genus Leucocytozoon:
- 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
- Chickens: andrewsi
- Ducks: simondi
- Turkeys: smithi
- Guinea fowl: neavei
- Genus Hepatocystis: 25 recognized species, infects nonhuman primates; lifecycle is similar to malaria except schizogony occurs in hepatocytes rather than erythrocytes (forming grossly visible hepatic cysts) and therefore there is no cyclical fever, transmitted by Culicoides ; incidence can exceed malaria
- Old world primates: Hepatocystis kochi, H. semnopitheci
- African Green Monkeys: bouillezi, H. cercopitheci, H. kochi and H. simiae
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
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