Pathology in Practice

Mackenzie E. Long1Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Shannon G. M. Kirejczyk1Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Elizabeth Howerth1Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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History

A 6-year-old 0.72-kg (1.59-lb) female squirrel monkey (Saimiri sciureus) had a peracute episode of respiratory distress characterized by discharge of blood-tinged foam from the nostrils and hypothermia (34.7°C [94.5°F]). The monkey died spontaneously soon thereafter and was submitted for necropsy. It had been housed with 2 conspecifics at a zoological institution in an exhibit enclosed by zoo-grade mesh. The 2 other squirrel monkeys were apparently healthy.

Gross Findings

On external examination, the monkey was found to be in good body condition with adequate subcutaneous and visceral adipose tissue. A mild amount of serosanguineous fluid was observed bilaterally around the nares and within the thoracic cavity. The tracheal bifurcation was filled with abundant, red-tinged fluid. The lungs were diffusely mottled pink to red and were very wet (Figure 1). The spleen was markedly enlarged and friable. The liver was pale brown with a reticular pattern and had numerous, multifocal, dark red to purple, pinpoint hemorrhages. There were no other notable gross findings.

Figure 1—
Figure 1—

Photograph of the body cavity (A) and photomicrograph of a lung tissue imprint (B) of a 6-year-old female squirrel monkey (Saimiri sciureus) that was submitted for necropsy following a peracute episode of respiratory distress. A—The lung lobes are mottled pink to dark red, and the spleen is markedly enlarged. B—In the lung tissue imprint, notice two 2− to 5-μm-long, round to elongated, intracytoplasmic organisms within a macrophage (arrow). Romanowsky-type stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 256, 6; 10.2460/javma.256.6.661

Cytologic Findings

At necropsy, tissue imprints of the lungs were prepared. Occasionally, macrophages contained 1 or 2, round to elongated, 2− to 5-μm-long organisms, each with a small, basophilic nucleus (Figure 1).

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page

Histopathologic Findings

Histologically, the alveolar septa were diffusely infiltrated by numerous macrophages, lymphocytes, plasma cells, and neutrophils and had several randomly distributed foci of lytic necrosis containing free, 2− to 5-μm-long, round to elongated protozoal tachyzoites with a small, basophilic nucleus. Alveoli contained marked amounts of edema, fibrin, and hemorrhage along with neutrophils, karyorrhectic cell debris, and macrophages. The latter often contained several intracytoplasmic tachyzoites or a single 8− to 10-μm-diameter, membrane-bound cluster of ≤ 20 tachyzoites (Figure 2). There was also moderate type II pneumocyte hyperplasia. Multifocally, perivascular tissue was expanded by edema and mild to moderate numbers of lymphocytes and plasma cells. Bronchi contained mild amounts of edema, hemorrhage, neutrophils, and macrophages.

Figure 2—
Figure 2—

Photomicrograph of a section of lung from the squirrel monkey in Figure 1. Edema, fibrin, hemorrhage, neutrophils, karyorrhectic cell debris, and macrophages are present diffusely throughout the alveoli. Occasionally, macrophages contain an 8− to 10-μm-diameter, membrane-bound group (pseudocyst) of ≤ 20 protozoal tachyzoites within a parasitophorous vacuole (arrowhead). H&E stain; bar = 20 μm. Inset—Higher-magnification image of the intracellular group of tachyzoites (arrowhead). H&E stain; bar = 5 μm.

Citation: Journal of the American Veterinary Medical Association 256, 6; 10.2460/javma.256.6.661

The splenic red pulp was diffusely engorged, and the parenchyma contained scattered, randomly distributed foci of lytic necrosis with fibrin accumulation and high numbers of the extra- and intrahistiocytic protozoal tachyzoites (Figure 3). High numbers of large macrophages with abundant phagocytosed cellular debris were present at the center of lymphoid follicles.

Figure 3—
Figure 3—

Photomicrographs of sections of the spleen from the squirrel monkey in Figure 1. A—The red pulp is diffusely congested. Numerous histiocytes contain an 8− to 10-μm-diameter, membrane-bound group of < 20 tachyzoites within a parasitophorous vacuole (arrow). H&E stain; bar = 20 μm. Inset—Higher-magnification image of intracellular groups of tachyzoites (arrows). H&E stain; bar = 5 μm. B—In a section of the spleen following immunohistochemical staining with a polyclonal antibody against Toxoplasma gondii, the tachyzoites are stained dark brown. Toxoplasma gondii –specific immunohistochemical stain and 3,3’ diaminobenzidine chromogen; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 256, 6; 10.2460/javma.256.6.661

Evaluation of several sections of liver tissue revealed numerous, randomly distributed foci of lytic necrosis with abundant hemorrhage, large numbers of macrophages, fewer lymphocytes and neutrophils, and moderate numbers of protozoal tachyzoites. Portal areas were often expanded by histiocytic and lymphocytic inflammation, which occasionally coalesced with the areas of lytic necrosis. Hepatocytes diffusely contained 1 or more discrete, round, lipid vacuoles.

Sections of the intestines had scattered, dilated crypts with attenuated to eroded epithelium and intraluminal, sloughed karyorrhectic cell debris and few neutrophils. In the large intestine, these crypts occasionally contained long, slender bacilli and were frequently elongated; among the crypt cells, there were several mitotic figures. The lamina propria was diffusely infiltrated with mild to moderate numbers of lymphocytes and plasma cells. There was also a focus of crypt necrosis within the duodenum. The adjacent lymph node between the duodenum and pancreas contained a large focus of lytic necrosis with a low number of tachyzoites. The brain had a small focus of lytic necrosis within the left basal ganglion, which contained a high number of protozoal tachyzoites and abundant hemorrhage.

Immunohistochemical analysis with polyclonal goat antibody against Toxoplasma gondii was performed on formalin-fixed lung and spleen specimens. Myriad, intra- and extracellular tachyzoites throughout the splenic red pulp and fewer organisms throughout the lungs had strong immunopositivity for T gondii (Figure 3).

Morphologic Diagnoses and Case Summary

Morphologic diagnosis: severe, acute, multifocal, necrotizing pneumonitis, splenitis, hepatitis, encephalitis, and mesenteric lymphadenitis with protozoal tachyzoites, consistent with disseminated T gondii infection; and moderate, subacute, lymphoplasmacytic enterocolitis with multifocal crypt necrosis, crypt hyperplasia, and slender bacilli within crypt lumina.

Case summary: disseminated toxoplasmosis in a captive squirrel monkey.

Comments

The squirrel monkey of the present report had developed disseminated infection with T gondii. Although infection of the squirrel monkey with other cyst-forming coccidian parasites including Sarcocystis spp and Neospora spp was possible, T go nd ii infection was considered the most likely differential diagnosis because of the high susceptibility of New World monkeys to the parasite and the dissemination of infection. Following infection, signs of systemic illness including malaise and hypothermia as well as signs of respiratory distress (eg, dyspnea and serosanguineous or foamy nasal discharge) commonly develop acutely.1 In a study1 evaluating fatal toxoplasmosis in New World primates, the most common gross and histologic findings were pulmonary congestion and edema, interstitial pneumonia, splenomegaly, necrotic splenitis, multifocal necrotic hepatitis, and mesenteric lymphadenitis. Cardiovascular, CNS, and gastrointestinal tract lesions were also observed.1 Among those affected New World primates, protozoal organisms morphologically consistent with T gondii were detected in the lungs, liver, spleen, and lymph nodes. For definitive diagnosis, immunohistochemical analysis of appropriate tissue sections for T gondii antigen is recommended.2 In the case described in the present study, T gondii infection was confirmed by detection of strong, T gondii–specific, cytoplasmic immunolabeling of tachyzoites in formalin-fixed, paraffin-embedded sections of lungs and spleen.

For the squirrel monkey of the present report, it was unclear whether the intestinal changes were attributable to the T gondii infection because tachyzoites were not observed in the sections of the intestines following routine or immunohistochemical staining. In the colon, pathological changes were associated with, but not necessarily caused by, long, slender bacilli. These bacteria were most likely Brachyspira spp but could potentially have been Clostridium piliforme. Brachyspira infections occasionally result in nonlethal diarrhea, whereas infection with C piliforme (the cause of Tyzzer disease) can result in colitis, hepatitis, and myocarditis and be fatal. The presence of the bacilli might have been an incidental finding; alternatively, the bacilli may have contributed to gastrointestinal inflammation and dissemination of T gondii from the intestines.

Toxoplasma gondii is an obligate intracellular, apicomplexan protozoan. The parasite infects most warmblooded species including humans, with an estimated prevalence of 30% to 50% in the human population worldwide.3 Species of the family Felidae are the only definitive host in which the parasite undergoes sexual replication in the intestinal epithelial cells to form oocysts. These oocysts are then shed in the feces and can contaminate water or food stores, thereby facilitating their ingestion by intermediate hosts. It is unclear how the squirrel monkey of the present report became infected with T gondii. It may have ingested water or food items contaminated with T gondii oocysts, given that felids were unlikely to have had access to the enmeshed exhibit area. Felids and intermediate hosts can also become infected via ingestion of raw or undercooked meat from infected animals wherein the parasite has encysted in tissues.4 Upon ingestion of a sporulated oocyst, sporozoites emerge and disseminate from the intestinal tract to other tissues by a hematogenous route and via the lymphatics. The sporozoites can invade any cell type either by direct penetration or phagocytosis, where they become surrounded by a parasitophorous vacuole and will then multiply to form a pseudocyst. At this point, the sporozoites are termed tachyzoites, which continue to replicate by endodyogeny until the host cell can no longer support the parasites and the cell ruptures.2 Released tachyzoites can then invade additional cells, wherein they may differentiate into bradyzoites as early as 3 days after infection and where they may persist indefinitely.2 In the definitive host, these asexual stages can occur or the bradyzoites may enter intestinal tissues and undergo the sexual stage of development to form zygotes that develop into oocysts.2 There is no specific treatment for T gondii infection in exposed animals; control measures include prevention of environmental contamination and exclusion of vector species from animal enclosures.2

References

  • 1. Epiphanio S, Sinhorini IL, Catao-Dias JL. Pathology of toxoplasmosis in captive new world primates. J Comp Pathol 2003;129:196204.

  • 2. Dubey JP. The life cycle of Toxoplasma gondii. In: Ajioka JW, Soldati D, eds. Toxoplasma: molecular and cellular biology. Norfolk, England: Horizontal Bioscience, 2007;313.

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  • 3. Flegr J, Prandota J, Sovičková M, et al. Toxoplasmosis—a global threat. Correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. PLoS One 2014;9:e90203.

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  • 4. Webster JP, Kaushik M, Bristow GC, et al. Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol 2013;216:99112.

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Contributor Notes

Dr. Long's present address is Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

Dr. Kirejczyk's present address is Division of Pathology, Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329.

Address correspondence to Dr. Howerth (howerth@uga.edu).
  • View in gallery
    Figure 1—

    Photograph of the body cavity (A) and photomicrograph of a lung tissue imprint (B) of a 6-year-old female squirrel monkey (Saimiri sciureus) that was submitted for necropsy following a peracute episode of respiratory distress. A—The lung lobes are mottled pink to dark red, and the spleen is markedly enlarged. B—In the lung tissue imprint, notice two 2− to 5-μm-long, round to elongated, intracytoplasmic organisms within a macrophage (arrow). Romanowsky-type stain; bar = 10 μm.

  • View in gallery
    Figure 2—

    Photomicrograph of a section of lung from the squirrel monkey in Figure 1. Edema, fibrin, hemorrhage, neutrophils, karyorrhectic cell debris, and macrophages are present diffusely throughout the alveoli. Occasionally, macrophages contain an 8− to 10-μm-diameter, membrane-bound group (pseudocyst) of ≤ 20 protozoal tachyzoites within a parasitophorous vacuole (arrowhead). H&E stain; bar = 20 μm. Inset—Higher-magnification image of the intracellular group of tachyzoites (arrowhead). H&E stain; bar = 5 μm.

  • View in gallery
    Figure 3—

    Photomicrographs of sections of the spleen from the squirrel monkey in Figure 1. A—The red pulp is diffusely congested. Numerous histiocytes contain an 8− to 10-μm-diameter, membrane-bound group of < 20 tachyzoites within a parasitophorous vacuole (arrow). H&E stain; bar = 20 μm. Inset—Higher-magnification image of intracellular groups of tachyzoites (arrows). H&E stain; bar = 5 μm. B—In a section of the spleen following immunohistochemical staining with a polyclonal antibody against Toxoplasma gondii, the tachyzoites are stained dark brown. Toxoplasma gondii –specific immunohistochemical stain and 3,3’ diaminobenzidine chromogen; bar = 20 μm.

  • 1. Epiphanio S, Sinhorini IL, Catao-Dias JL. Pathology of toxoplasmosis in captive new world primates. J Comp Pathol 2003;129:196204.

  • 2. Dubey JP. The life cycle of Toxoplasma gondii. In: Ajioka JW, Soldati D, eds. Toxoplasma: molecular and cellular biology. Norfolk, England: Horizontal Bioscience, 2007;313.

    • Search Google Scholar
    • Export Citation
  • 3. Flegr J, Prandota J, Sovičková M, et al. Toxoplasmosis—a global threat. Correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. PLoS One 2014;9:e90203.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Webster JP, Kaushik M, Bristow GC, et al. Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol 2013;216:99112.

    • Crossref
    • Search Google Scholar
    • Export Citation

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