Conference 24 - 2016 Case: 3 20170426

Signalment:

27-year-old male African penguin, (Spheniscus demersus).This captive bird was at the end of a molting period and had been depressed and lethargic for a couple of days before being found dead.

Gross Description:

The lungs were described as congested by the submitting institution, which performed the necropsy. A complete set of formalin-fixed tissues was submitted for histopathologic examination.

Histopathologic Description:

Lung: Variably obscuring the parenchyma throughout the section are interstitial and luminal accumulations of inflammatory cells, eosinophilic fluid (edema), and sometimes necrotic cellular debris. Moderate numbers of macrophages and fewer heterophils are present in the lumens of parabronchi, atria and air capillaries mixed with amorphous to globular eosinophilic material and erythrocytes, and these air spaces are often lined by hypertrophied epithelial cells. Rarely, within disrupted air capillary walls, there are irregularly round to slightly elongated clusters of approximately 2-4 X 1 µm, fusiform, basophilic, protozoal zoites. There is prominent infiltration of septal and perivascular interstitium by moderate to large numbers of lymphocytes, plasma cells, and fewer macrophages, along with numerous nodular aggregates of macrophages containing brown anisotropic pigment (anthracosis).

Morphologic Diagnosis:

Lung: severe diffuse necrotizing and lymphohistiocytic interstitial pneumonia with epithelial hypertrophy, edema, hemorrhage and protozoal schizonts.

Lab Results:

Immunohistochemistry: In house: Toxoplasma gondii (commercial rabbit polyclonal antibody) weakly positive with some non-specific background staining CAHFS
Laboratory, Davis, CA
CAHFS Laboratory, Davis, CA
CAHFS Laboratory, Davis, CA
sp. (rabbit polyclonal antibody) strongly positive
Toxoplasmagondii (rabbit polyclonal antibody) negative
PCR: Wildlife Conservation Society, Bronx Zoo, NY
Two assays on formalin-fixed, paraffin-embedded lung: Apicomplexan 18S rRNA gene and Sarcocystis-specific ITS-1 18S rRNA gene both positive sequences of products match Sarcocystis falcatula

Condition:

Sarcocystic falcatula, African penguin

Contributor Comment:

Death of this penguin was due to a severe interstitial pneumonia caused by a protozoal infection. Occasional clusters of very small, elongated protozoal organisms present in air capillary walls throughout the lung were suggestive of apicomplexan schizonts, so differential diagnoses initially included Sarcocystis sp., Toxoplasma gondii, and Plasmodium sp. Both malaria and toxoplasmosis have previously been reported as causes of interstitial pneumonia in penguins,^6,13whereas sarcocystosis is a common cause of pneumonia in various avian species but has not been described in penguins. The only relevant immunohistochemical stain available in house was a rabbit polyclonal antibody against T. gondii (BioGenex, San Ramon, CA). Organisms exhibited weak but positive reactivity with this immunostain. Because cross-reactivity between different cyst-forming apicomplexans has been reported with polyclonal antibodies, such as between T. gondii and Neospora caninum^1,11 and between N. caninum and Sarcocystis sp.,¹² more specific diagnostic methods were pursued. No fresh/frozen lung tissue was available, so formalin-fixed paraffin-embedded lung tissue was submitted to the Wildlife Conservation Society for apicomplexan and Sarcocystis-specific PCR assays. The DNA sequences obtained from both of these positive assays were 100% matches to Sarcocystis falcatula. The next closest match was S. neurona with a 98-99% identity. The pneumonia in this penguin is therefore attributed to S. falcatula. Subsequent additional immunohistochemical staining performed at the CAHFS laboratory in Davis, CA, confirmed strongly positive labelling of the protozoa with a Sarcocystis antibody. The Toxoplasma gondii immunohistochemical stain performed by this laboratory was negative.

In North America, the definitive host for S. falcatula and the closely related S. neurona is the Virginia opossum, Didelphis virginiana, which sheds infective sporocysts in feces.⁹ Opossums had been seen on the premises of the aquarium institution where this penguin was housed. Infection of various bird species, most naturally grackles and cowbirds, as intermediate hosts is typically through ingestion of food contaminated with opossum feces, but insects such as cockroaches can serve as mechanical vectors.² In the intermediate host, asexual reproduction (mero-gony/schizogony) occurs in endothelial cells in various tissues but especially in the lung.^9,10 The merozoites produced eventually infect skeletal and cardiac muscle cells and form sarcocysts filled with bradyzoites, which are infective to the definitive host upon ingestion of the intermediate host tissue.⁹ Replication in pulmonary endothelial cells can result in severe interstitial pneumonia, the most common form of fatal infection, although two other clinical forms of disease have been characterized: a neurologic form and a muscular form.¹⁴ The neurologic form has been reported in psittacines and raptors.^14,15

Fatal pulmonary infection with S. falcatula has been reported most often in psittacines,^2-5,14 and this is thought to be the first report in a penguin. The microscopic features of the pneumonia in this penguin were similar to what has been described in other avian species infected with S. falcatula, namely edema, fibrin deposition, congestion, hemorrhage, mononuclear inflammatory infiltrates, endothelial cell lysis, and pneumocyte hyperplasia.^7,10,14 The schizonts were rare in this case, occasionally exhibiting a sunburst or somewhat elongated arrangement, but characteristic serpentine forms that conform to pulmonary capillary lumens^2-5,7-10,14 were not seen in this penguin. Identification of the infected cells as endothelial cells was not possible in this case from microscopic examination alone. A complete set of tissues was examined histologically from this penguin, including brain, skeletal muscle and heart, but no extrapulmonary schizonts or sarco-cysts were seen. There was, however, a mild lymphohistiocytic portal hepatitis in this bird, which can be seen with S. falcatula infection,^7,8 and for which no other etiology was identified.

JPC Diagnosis:

Lung: Pneumonia, interstitial, necrotizing and lympho-histocytic, multifocal, moderate with intracellular apicomplexan zoites, African penguin, Spheniscus demersus.

Conference Comment:

The contributor provides an outstanding summary of avian sarcocystosis. The genus Sarcocystis is a large group of cyst-forming apicomplexan protozoal coccidian parasites that affect mammals, birds, and reptiles. They have also been rarely reported in amphibians and fish.^3,4,11,15 Sarcocystis falcatula, similar to all other members of this genus, utilizes a two-host life cycle based on the predator and prey dynamic. Carnivores and omnivores are the definitive hosts and become infected by preying upon intermediate hosts (usually herbivores) which contain mature sarcocysts in muscular or neural tissue. After digestion of the sarcocyst wall, bradyzoites are released and invade the intestinal epithelium of the carnivorous definitive host. These bradyzoites undergo sexual gametogony and develop into micro-(male), macro-(female) gamonts. Fertilization leads to the formation of infective oocysts, which sporulate in the intestinal lumen and are shed in the feces. Similar to other common apicomplexan coccidian parasites (Besnoitia, Frenkelia, Isospora, Toxoplasma), sporulated oocysts contain two sporocysts with four sporozoites each.^3,9

Sporulated oocysts are then ingested by susceptible intermediate hosts, such as herbivorous mammals or birds. After ingestion by the intermediate host, sporozoites excyst in the intestine and undergo two generations of asexual merogony (schizogony). Sporozoites are released and first migrate to endothelial cells where they undergo the first two generation of asexual reproduction, resulting in the development of meronts.^3,9,11,15 In birds, merogony is most pronounced in vascular endothelial cells within the lungs. Pulmonary capillary endothelial cells can become markedly swollen and inflamed, leading to lymphohistiocytic vasculitis, airway edema, interstitial pneumonia, and even vascular obstruction in birds with a heavy protozoan burden.^11,15 Merogony has also been reported in vessels of the liver, pancreas, spleen, adrenal glands and heart.¹¹In animals that survive the maturation of the second generation of meronts, merozoites are liberated from meronts within capillaries and enter circulating mononuclear cells. Merozoites then undergo endodyogeny and enter muscle fibers to eventually form sarcocysts containing bradyzoites. They will remain encysted and viable in the muscular or nervous tissue until they are ingested by the definitive host to complete the lifecycle. There are over 90 Sarcocystis sp. known to infect mammals, birds, and reptiles. The vast majority of species are considered non-pathogenic; however, some species, such as S. falcatula in birds, are associated with severe clinical disease in the intermediate host.^3,4,11,15

As mentioned by the contributor, the only known definitive host for this protozoan in North America is the opossum (Didelphis virginiana). Most Sarcocystis sp. infect a specific intermediate host; however, Sarcocystis falcatula is unique in that it can infect a heterogeneous population of avian intermediate hosts from numerous avian species (Passeriformes, Psittaciformes, and Columbiformes).^4,9,11,15 Birds become infected by eating feed contaminated with opossum feces containing infective oocysts or sporocysts or ingestion of cockroaches, which is the main mechanical vector for the parasite.²

References:

1. Barr BC, Conrad PA, Dubey JP, Anderson ML. Neospora-like encephalomyelitis in a calf: Pathology, ultrastructure and immunoreactivity. J Vet Diagn Invest. 1991;3:39-46.

2. Clubb SL, Frenkel JK. Sarcocystis falcatula of opossums: Transmission by cockroaches with fatal pulmonary disease in psittacine birds. J Parasitol. 1992; 78(1):116-124.

3. Gardiner CH, Fayer R, Dubey JP. Apicomplexa. In: An Atlas of Protozoan Parasites in Animal Tissues. 2nd ed. Armed Forces Institute of Pathology, American Registry of Pathology, Washington DC; 1998:20-42.

4. Godoy SN, De Paula CD, Cubas ZS, Matushima ER, Catão-Dias JL. Occurrence of Sarcocystis falcatula in captive psittacine birds in Brazil. J Avian Med Surg. 2009; 23(1):18-23.

5. Hillyer EV, Anderson MP, Greiner EC, Atkinson CT, Frenkel JK. An outbreak of Sarcocystis in a collection of psittacines. J Zoo Wildlife Med. 1991;22(4):434-445.

6. Ploeg M, Ultee T, Kik M. Disseminated toxoplasmosis in black-footed penguins (Spheniscus demersus). Avian Dis. 2011; 55:701-703.

7. Smith JH, Neill PJG, Dillard III EA, Box ED. Pathology of experimental Sarcocystis falcatula infections of canaries (Serinus canarius) and pigeons (Columba livia). J Parasitol. 1990; 76(1):59-68.

8. Smith JH, Neill PJG, Box ED. Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the budgerigar (Melopsittacus undulates). III. Pathologic and quantitative parasitologic analysis of extrapulmonary disease. J Parasitol. 1989; 75(2):270-287.

9. Smith JH, Meier JL, Neill PJG, Box ED. Pathogenesis of Sarcocystis falcatula in the budgerigar. I. Early pulmonary schizogony. Lab Invest. 1987; 56(1): 60-71.

10. Smith JH, Meier JL, Neill PJG, Box ED. Pathogenesis of Sarcocystis falcatula in the budgerigar. II. Pulmonary pathology. Lab Invest. 1987; 56(1):72-84.

11. Sundermann CA, Estridge BH, Branton MS, Bridgman CR, Lindsay DS. Immunohistochemical diagnosis of Toxoplasma gondii: potential for cross-reactivity with Neospora caninum. J Parasitol. 1997; 83(3):440-443.

12. Uzêda RS, Schares G, Ortega-Mora LM, et al. Combination of monoclonal antibodies improves immuno-histochemical diagnosis of Neospora caninum. Vet Parasitol. 2013; 197:477-486.

13. Vanstreels RET, da Silva-Filho RP, Kolesnikovas CKM, et al. Epidemiology and pathology of avian malaria in penguins undergoing rehabilitation in Brazil. Vet Res. 2015; 46:30.

14. Villar D, Kramer M, Howard L, Hammond E, Cray C, Latimer K. Clinical presentation and pathology of sarcocystosis in psittaciform birds: 11 cases. Avian Dis. 2008; 52:187-194.

15. Wünschmann A, Rejmanek D, Conrad PA, et al. Natural fatal Sarcocystis falcatula infections in free-ranging eagles in North America. J Vet Diagn Invest. 2010; 22:282-289.