27-year-old male African penguin, (Spheniscus
bird was at the end of a molting period and had been depressed and lethargic
for a couple of days before being found dead.
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.
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).
Lung: severe diffuse necrotizing and
lymphohistiocytic interstitial pneumonia with epithelial hypertrophy, edema,
hemorrhage and protozoal schizonts.
Immunohistochemistry: In house: Toxoplasma gondii (commercial rabbit
polyclonal antibody) weakly positive with some non-specific background
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
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
Sarcocystic falcatula, African penguin
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,6,13 whereas 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 caninum1,11
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 lumens2-5,7-10,14 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.
interstitial, necrotizing and lympho-histocytic, multifocal, moderate with
intracellular apicomplexan zoites, African penguin, Spheniscus
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
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
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