PMC6795683_01
PMC6795683_02
Gross, cytological, histopathologic, and immunohistochemical features of Sarcocystis falcatula infection in the lung of Southern rockhopper (Eudyptes chrysocome) and African (Spheniscus demersus) penguins. (A) Gross appearance of lung tissue from case 2 at necropsy. The lungs are dark red and edematous, with red-tinged fluid oozing along the periphery of the tissue. The lungs sank in formalin. (B) Cytology of tissue imprint from the lung of case 3 obtained at necropsy. The vast majority of cells are erythrocytes. A single, 2 x 8 micron, elongate to crescent-shaped tachyzoite with a small, round, slightly off-center nucleus is present at the center of the image (arrowhead). Bar = 10 mum. (C) Lung, from a 5-year-old, female, Southern rockhopper penguin (case 2), with S. falcatula pneumonia. Parabronchi and atria are frequently flooded by edema, hemorrhage and fibrin. H&E. Bar = 200 mum. (D) Lung, case 2. Air capillaries are multifocally obscured by foci of necrosis (*) containing protozoal schizonts (arrowheads), fibrin, hemorrhage, and low numbers of heterophils. The interstitium is hypercellular due to low numbers of lymphocytes and macrophages. H&E. Bar = 20 mum. Inset: High magnification image highlights the elongate to serpentine appearance of an intracellular schizont (case 3). (E) Lung, case 1. Pneumocytes lining the parabronchus in the top left of the image are hypertrophied (arrowhead) and the air space is filled with edema, fibrin, macrophages, and scant hemorrhage. The interstitium is expanded by inflammatory cell infiltrates, and multifocally obscured by necrosis, hemorrhage, and an accumulation of fibrin and edema. H&E. Bar = 20 mum. Inset: High magnification image with a schizont (arrowhead), which exhibits a "sunburst" pattern, with merozoites radiating around a clear zone. (F) Immunohistochemistry for S. neurona polyclonal antibody on lung tissue (case 2) demonstrates strongly immunoreactive, elongate to serpentine schizonts (arrowheads), free merozoites, and macrophages containing phagocytosed debris. DAB chromogen with hematoxylin counterstain. Bar = 10 mum.
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F1
Cytologic examination of lung tissue imprints from case 3 showed crescent-shaped, 2 x 4-8 mum protozoal zoites (Figure 1B), toxic heterophils and multinucleated giant cells. Parabronchi were flooded with hemorrhage, edema, and fibrin (Figure 1C). Air capillaries were obscured by foci of necrosis with fibrin exudation, and accumulations of heterophils, hemorrhage, and protozoal schizonts (Figure 1D). Air spaces were multifocally lined by hypertrophied epithelial cells (Figure 1E). In cases 2 and 3, schizonts were numerous and elongate, often conforming to the shape of capillaries (Figure 1D, inset), while schizonts were fewer and this classic serpentine morphology of S. falcatula was not observed in case 1. In all cases, schizonts occasionally exhibited a "sunburst" arrangement, in which merozoites radiated around a centralized clearing (Figure 1E, inset). On immunohistochemistry, protozoa exhibited strong immunoreactivity (Figure 1F) for polyclonal S. neurona antibodies and variable immunoreactivity for N. caninum and T. gondii antibodies (Table 1).
PMC6795683_03
Gross, cytological, histopathologic, and immunohistochemical features of Sarcocystis falcatula infection in the lung of Southern rockhopper (Eudyptes chrysocome) and African (Spheniscus demersus) penguins. (A) Gross appearance of lung tissue from case 2 at necropsy. The lungs are dark red and edematous, with red-tinged fluid oozing along the periphery of the tissue. The lungs sank in formalin. (B) Cytology of tissue imprint from the lung of case 3 obtained at necropsy. The vast majority of cells are erythrocytes. A single, 2 x 8 micron, elongate to crescent-shaped tachyzoite with a small, round, slightly off-center nucleus is present at the center of the image (arrowhead). Bar = 10 mum. (C) Lung, from a 5-year-old, female, Southern rockhopper penguin (case 2), with S. falcatula pneumonia. Parabronchi and atria are frequently flooded by edema, hemorrhage and fibrin. H&E. Bar = 200 mum. (D) Lung, case 2. Air capillaries are multifocally obscured by foci of necrosis (*) containing protozoal schizonts (arrowheads), fibrin, hemorrhage, and low numbers of heterophils. The interstitium is hypercellular due to low numbers of lymphocytes and macrophages. H&E. Bar = 20 mum. Inset: High magnification image highlights the elongate to serpentine appearance of an intracellular schizont (case 3). (E) Lung, case 1. Pneumocytes lining the parabronchus in the top left of the image are hypertrophied (arrowhead) and the air space is filled with edema, fibrin, macrophages, and scant hemorrhage. The interstitium is expanded by inflammatory cell infiltrates, and multifocally obscured by necrosis, hemorrhage, and an accumulation of fibrin and edema. H&E. Bar = 20 mum. Inset: High magnification image with a schizont (arrowhead), which exhibits a "sunburst" pattern, with merozoites radiating around a clear zone. (F) Immunohistochemistry for S. neurona polyclonal antibody on lung tissue (case 2) demonstrates strongly immunoreactive, elongate to serpentine schizonts (arrowheads), free merozoites, and macrophages containing phagocytosed debris. DAB chromogen with hematoxylin counterstain. Bar = 10 mum.
fvets-06-00340-g0001.jpg
PMC6795683_03_fvets-06-00340-g0001.jpg
F1
Cytologic examination of lung tissue imprints from case 3 showed crescent-shaped, 2 x 4-8 mum protozoal zoites (Figure 1B), toxic heterophils and multinucleated giant cells. Parabronchi were flooded with hemorrhage, edema, and fibrin (Figure 1C). Air capillaries were obscured by foci of necrosis with fibrin exudation, and accumulations of heterophils, hemorrhage, and protozoal schizonts (Figure 1D). Air spaces were multifocally lined by hypertrophied epithelial cells (Figure 1E). In cases 2 and 3, schizonts were numerous and elongate, often conforming to the shape of capillaries (Figure 1D, inset), while schizonts were fewer and this classic serpentine morphology of S. falcatula was not observed in case 1. In all cases, schizonts occasionally exhibited a "sunburst" arrangement, in which merozoites radiated around a centralized clearing (Figure 1E, inset). On immunohistochemistry, protozoa exhibited strong immunoreactivity (Figure 1F) for polyclonal S. neurona antibodies and variable immunoreactivity for N. caninum and T. gondii antibodies (Table 1).
Hepatic and splenic pathology in Southern rockhopper penguins with fatal Sarcocystis falcatula infection. (A) Spleen, case 2. The splenic parenchyma is markedly hypercellular, with numerous hematopoietic cell precursors, lymphocytes, histiocytes, and plasma cells. There are multifocal to coalescing zones of coagulative to lytic necrosis (*) surrounded by a rim of hemorrhage. H&E. Bar = 200 mum. Inset: High magnification image of a S. falcatula schizont (arrowhead) within an area of necrosis in the spleen of case 3. Bar = 10 mum. (B) Liver, case 2. Portal regions are hypercellular, with moderate numbers of lymphocytes, plasma cells, macrophages and granulocytic precursor cells, which compress hepatocytes multifocally. Bar = 100 mum. Left insert: Sinusoids are congested, with macrophages containing phagocytosed cell debris, erythrocyte fragments and hemosiderin (arrowhead). H&E. Bar = 10 mum. Right inset: Immunohistochemistry for S. neurona highlights a schizont with radially arranged merozoites. Bar = 10 mum.
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F2
There were also coalescing foci of coagulative to lytic necrosis, and case 3 had scattered, intraendothelial schizonts (Figure 2A). All cases had multifocal lymphohistiocytic portal hepatitis and extramedullary granulopoiesis (Figure 2B); cases 2 and 3 had low numbers of schizonts within sinusoids (Figure 2B, right inset). All cases also had moderate numbers of sinusoidal macrophages containing fragments of erythrocytes and intracytoplasmic hemosiderin (Figure 2B, left inset).
Ultrastructure of Sarcocystis falcatula asexual life stages by Transmission Electron Microscopy (TEM) in the lung of a 32-year-old, female, Southern rockhopper penguin (case 3). (A) TEM demonstrates an intraendothelial S. falcatula schizont. The schizont expands the endothelial cell (EC), is irregularly shaped and is separated from the EC cytoplasm by a thin membrane. Several nuclear spindles (arrowheads) project from the schizont nucleus (N). The endothelial cell abuts a capillary containing erythrocytes (RBC) and is supported by a basement membrane (BM). Bar = 2 mum. (B) Higher magnification TEM image of the S. falcatula schizont nucleus (N) from (A). Several conically arranged microtubules (mt) comprise the nuclear spindle (ns) of a developing merozoite. Bar = 600 nm. (C) TEM shows numerous, up to 2 x 6 mum merozoites clustered within an endothelial cell (EC). Inset: Higher magnification of a merozoite showing rhoptries (rh) and micronemes (mn). Bar = 2 mum. (D) A mature merozoite is present in a focus of necrotic lung tissue on TEM. The merozoite has a central, round nucleus (n), an anterior conoid (con), numerous micronemes (mn) at the posterior pole, and a double-membrane pellicle (pel). Bar = 800 nm.
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F3
Transmission electron microscopy revealed protozoa with ultrastructural features compatible with Sarcocystis spp. (Figure 3). One schizont had a multilobulated nucleus with nuclear spindles arranged along the periphery (Figures 3A,B). Merozoites had a round, central nucleus, an anterior conoid and several tear-shaped, electron-dense micronemes at the posterior end (Figures 3C,D).