An Outbreak Of Sarcocystosis In Psittacines and A Pigeon In A Zoological Collection In Brazil

Abstract

This report describes an outbreak of acute pulmonary sarcocystosis in different species of captive psittacines and in a Luzon bleeding-heart pigeon (Gallicolumba luzonica) in a zoological collection in Brazil. A majority of the birds were found dead and had exhibited no previous clinical signs. Grossly, pulmonary congestion and edema were the most-common findings. Enlarged and congested livers and spleens were also frequently observed. Microscopically, there was edema, fibrin exudation, congestion, and perivascular and interstitial lymphoplasmacytic infiltration associated with numerous sinuous schizonts of Sarcocystis sp. in the lungs. Mild to moderate myocarditis, hepatitis, splenitis, and interstitial nephritis were also observed in the birds. Immunohistochemistry confirmed Sarcocystis sp. in the capillaries of lungs, hearts, livers, and spleens of most of the birds, but also in the pancreas, kidney, intestine, proventriculus, and brain of a few birds. The probable source of Sarcocystis sp. in these birds was the wild opossum (Didelphis albiventris), a common inhabitant of a local forest that surrounds the Belo Horizonte Zoo (Fundação Zoo-Botânica). This is the first documentation of Sarcocystis infection in psittacines and a pigeon from Brazil.

Full text

Case Report—
An Outbreak of Sarcocystosis in Psittacines and a Pigeon in a Zoological Collection
in Brazil
R. Ecco,
A
M. M. Luppi,
B
M. C. C. Malta,
B
M. R. Arau
´jo,
A
R. M. C. Guedes,
A
and H. L. Shivaprasad
CD
A
Veterinary School, Universidade Federal de Minas Gerais. Belo Horizonte, Av. Anto
ˆnio Carlos, 6627, Belo Horizonte,
MG 31270-901 Minas Gerais, Brazil
B
Fundac¸a
˜o Zoo-Bota
ˆnica de Belo Horizonte, Av. Otacı
´lio Negra
˜o de Lima, 8000, Pampulha, Belo Horizonte, 31365-450, Minas Gerais, Brazil
C
California Animal Health and Food Safety Laboratory System, Fresno Branch, 2789 S. Orange Avenue, University of California, Davis,
Fresno, CA 93725, U.S.A.
Received 5 April 2008; Accepted and published ahead of print 17 June 2008
SUMMARY. This report describes an outbreak of acute pulmonary sarcocystosis in different species of captive psittacines and in
a Luzon bleeding-heart pigeon (Gallicolumba luzonica) in a zoological collection in Brazil. A majority of the birds were found dead
and had exhibited no previous clinical signs. Grossly, pulmonary congestion and edema were the most-common findings. Enlarged
and congested livers and spleens were also frequently observed. Microscopically, there was edema, fibrin exudation, congestion, and
perivascular and interstitial lymphoplasmacytic infiltration associated with numerous sinuous schizonts of Sarcocystis sp. in the
lungs. Mild to moderate myocarditis, hepatitis, splenitis, and interstitial nephritis were also observed in the birds.
Immunohistochemistry confirmed Sarcocystis sp. in the capillaries of lungs, hearts, livers, and spleens of most of the birds, but
also in the pancreas, kidney, intestine, proventriculus, and brain of a few birds. The probable source of Sarcocystis sp. in these birds
was the wild opossum (Didelphis albiventris), a common inhabitant of a local forest that surrounds the Belo Horizonte Zoo
(Fundac¸a
˜o Zoo-Bota
ˆnica). This is the first documentation of Sarcocystis infection in psittacines and a pigeon from Brazil.
RESUMEN. Reporte de Caso—Un brote de Sarcocistosis en psita
´cidas y una paloma en una coleccio
´n de un zoolo
´gico en Brasil.
Este reporte describe un brote de sarcocistosis pulmonar aguda en diferentes especies cautivas de psita
´cidas y una paloma
Corazo
´n Sangrante de Luzo
´n(Gallicolumba luzonica) de una coleccio
´n de un zoolo
´gico en Brasil. La mayorı
´a de las aves fueron
encontradas muertas sin que hubieran mostrado ninguna sintomatologı
´a clı
´nica. En las lesiones macrosco
´picas los hallazgos ma
´s
comunes fueron la congestio
´n y el edema pulmonar. Tambie
´n se observo
´frecuentemente hepatomegalia y esplenomegalia.
Microsco
´picamente habı
´a edema, exudacio
´n fibrinosa e infiltracio
´n linfoplasmocı
´tica perivascular e intersticial asociada con la
presencia de numerosos esquizontes sinuosos de Sarcocystis sp en los pulmones. Tambie
´n se observo
´en estas aves de moderada a
severa miocarditis, hepatitis, esplenitis y nefritis intersticial. Por medio de pruebas de inmunohistoquı
´mica se confirmo
´la presencia
de Sarcocystis sp en los capilares del pulmo
´n, corazo
´n, hı
´gado y bazo de la mayorı
´a de las aves afectadas, pero tambie
´nenpa
´ncreas,
rin
˜o
´n, intestino, proventrı
´culo y cerebro de algunas aves. La posible fuente de Sarcocystis sp en estas aves fue la zarigu
¨eya silvestre
(Didelphis albiventris), un habitante comu
´n del bosque local que rodea el Zoolo
´gico de Belo Horizonte (Fundacio
´n Zoo-Bota
´nica).
Esta es la primera documentacio
´n de una infeccio
´n con Sarcocystis en psita
´cidas y una paloma en Brasil.
Key words: Sarcocystis falcatula, schizogony, schizonts, merogony, merozoites, psittacine, pigeon, pulmonary edema,
immunohistochemistry
Abbreviations: H&E 5hematoxylin and eosin; IHC 5immunohistochemistry; PAS 5periodic acid-Schiff; TTBS 5Tween-
Tris buffered saline
Sarcocystis spp. are ubiquitous protozoan parasites that require two
hosts to complete their life cycle (23). The sexual phase takes place in
the definitive host, a carnivore, and results in the production of
infective sporocysts that are shed in the feces (27). Asexual
reproduction occurs in the intermediate host and is characterized
by schizogony (merogony) and formation of sarcocysts in skeletal
muscle. In general, Sarcocystis spp. have specific intermediate hosts.
In contrast, Sarcocystis falcatula can use a large variety of bird species
as intermediate hosts, including passeriformes, psittaciformes, and
columbiformes (1,2,10,18,25,28,30). In addition, S. falcatula has a
prolonged schizogony (5 mo or more) and is highly pathogenic to
intermediate hosts, especially to psittacines, mainly due to the fatal
pulmonary presentation (2,5,10,14). The merogony phase in the
intermediate hosts takes place primarily in the endothelial cells of
arteries, capillaries, veins, and venules of lungs, liver, kidney, brain,
heart and skeletal muscle (25,26). Mature sarcocysts can be observed
in cardiac and skeletal muscles (1,21).
Sarcocystis falcatula infections have been reported in North
America and in the North American opossum, Didelphis virginiana,
which has been considered the definitive host for S. falcatula
(10,11,29). It is known that North American opossum (D.
virginiana) is the intermediate host for at least 3 species of
Sarcocystis:S. falcatula,S. neurona, and S. speeri; however, S. neurona
and S. speeri are not pathogenic to birds (4,6,10).
The opossum D. albiventris, in Argentina, and the South
American opossum D. marsupialis,inSa
˜o Paulo, Brazil, have also
been identified as definitive hosts of S. falcatula and S. falcatula-like
protozoa (4,6,10). The S. falcatula-like protozoon identified from
one of the opossums (D. albiventris) was recognized as a new species
based on structural difference and molecular characterization. It was
named S. lindsayi, but its importance still has to be determined (5,8).
Outbreaks of acute fatal Sarcocystis infection have been reported in
various species of birds, especially in psittacines in the United States
D
Corresponding author. California Animal Health and Food Safety
Laboratory System, Fresno Branch, 2789 South Orange Avenue, Fresno, CA
93725. E-mail: hlshivaprasad@ucdavis.edu
AVIAN DISEASES 52:706–710, 2008
706

(2,16,24). Among psittacines, Old World psittacines such as
budgerigars (Melopsittacus undulatus), cockatiels (Nymphicus hollan-
dicus), African grey parrots (Psittacus erithacus erithacus), and various
species of cockatoos (Cocatua spp.) are highly susceptible to S.
falcatula (2). Psittacines from the New World are relatively resistant
to S. falcatula, but nestlings of certain psittacine species are
susceptible (2). Contaminated feed and water from the feces of
opossum is one of the ways birds acquire S. falcatula. In one of the
studies, it was shown that cockroaches can also act as mechanical
vectors of infectious forms of S. falcatula (2). Flies and fomites can
also be mechanical carriers for S. falcatula (1).
Sarcocystis associated with encephalitis has been reported in other
species of birds in the United States such as chickens, wild turkeys,
golden eagles, bald eagles, northern goshawks, northern gannets, and
straw-necked ibis, but the species of Sarcocystis which causes
encephalitis in birds is not known (7,20,22).
Sarcocystosis outbreaks in various species of birds, including
psittacines, passerines, and other birds from zoological collections
and aviaries, have been reported in the United States. There are no
such reports of S. falcatula infection in birds from outside the United
States (6); however, there are reports of Sarcocystis in capercailles
(Tetrao urogallus) from Sweden and Finland, although the species of
Sarcocystis could not be determined (9,13). There is one report from
Germany in which S. kirmsei was identified in the brain of a hill
mynah (Gracula religiosa), but no details were provided regarding its
life cycle and definitive host (15). Also, the species of Sarcocystis from
the South American opossum, D. albiventris, which is infectious to
birds in Brazil, is not known. Therefore, the purpose of this paper is
to document, for the first time, that in Brazil, fatal pulmonary
sarcocystosis was found in eight psittacines and one pigeon; and to
discuss the role of the opossum, D. albiventris, as a potential source
of an unidentified Sarcocystis sp. in psittacines and a pigeon.
MATERIALS AND METHODS
Birds. Birds in the study diagnosed with sarcocystosis included five
ring-necked parakeets (Psittacula krameri), two bare-eyed cockatoos
(Cacatua sanguinea), one Congo African grey parrot (Psittacus erithacus
erithacus), and a male Luzon bleeding-heart pigeon (Gallicolumba
luzonica). The Philippines is the natural habitat of the Luzon bleeding-
heart pigeon. The pigeon was a mature male bird of unknown age that
had been housed in the zoological collection of Belo Horizonte, Brazil
for many years. Among the ring-necked parakeets, a species originally
found in Africa and Asia, there was an adult pair (male and female)
introduced into the aviary of the zoo six months before the episode of
sarcocystosis began. Three other young ring-necked parakeets (one
female and two males) were hatched in the zoo. Two adult cockatoos,
species originally found in Australia, had been in the zoo for many years.
The African grey parrot from Africa, an adult male, was hatched in
captivity in a different institution and had been housed in the aviary at
the zoo for more than 10 years.
Housing. The total number of birds present in the aviary that died of
Sarcocystis included 15 ring-necked parakeets, three bare-eyed cockatoos,
two African grey parrots, and one Luzon bleeding-heart pigeon. Each
species of birds was kept in a separate enclosure, except the pigeon,
which shared the aviary with two violet turacos (Musophaga violacea). All
the birds were kept in similar aviaries in an area of 3 3233 m each.
The aviaries were enclosed by wire mesh on all sides, including the roof,
had a brick wall in the back, and had partial shade on the top. The floor
was covered with gravel and there were ornamental plants in each aviary.
The feeders were made of concrete and were placed at a height of
approximately 1 m. Water was provided ad libitum in small shallow
depressions in the floor. All psittacines received a daily diet of a specific
ration (MegaZoo, Vale Verde, Minas Gerais, Brazil), fruits, and boiled
eggs 1 time/wk. The Luzon bleeding-heart pigeon was fed grains and
mash similar to a chicken diet. Biosecurity was considered good, except
for some loose wire mesh, an exposure to wind and rain, and the
presence of wild birds roosting on the roof.
Pathology. All birds were necropsied and gross lesions were recorded.
Portions of lungs, hearts, livers, spleens, kidneys, intestines, pancreas,
skeletal muscles, proventriculus, gizzards, and brains were collected,
fixed in formalin, embedded in paraffin, sectioned at 7 mm, stained with
hematoxylin and eosin (H&E), and examined by bright field
microscopy. Selected tissues were also stained by MacCallum–Good-
pasture gram stain, periodic acid-Schiff (PAS), and Warthin-Starry,
according to the methods described (17).
Immunohistochemistry (IHC). Analysis of all formalin-fixed tissues,
submitted for IHC for detection of S. falcatula, was performed in a
slightly modified method, as described (19). IHC was performed using
the Envision Plus–horseradish peroxidase labeled polymer (Dako Corp.,
Carpintaria, CA) and a rabbit polyclonal serum raised against S.
falcatula. Paraffin-embedded sections, cut at 3 mm thickness, were
deparaffinized, quenched with 3% hydrogen peroxide for 10 min,
antigen-retrieved with 0.4% pepsin in 1N Hydrochloric acid for 15 min
at 37 C, rinsed in 0.5% Tween-Tris buffered saline (TTBS), and
blocked with 0.5% casein in TTBS for 10 min. The primary antibody
was applied for 30 min, rinsed in TTBS, incubated with the polymer for
30 min, then rinsed in TTBS and deionized water. The substrate
chromogen 3-amino-9-ethyl carbazole was applied for 10 min and then
rinsed in deionized water. Mayer’s hematoxylin was used as a counter-
stain. Sections were covered with an aqueous mounting media and cover
glass. All incubations were at room temperature, except where noted.
The optimal dilution of the S. falcatula serum was 1:1600, in 1% casein
in TTBS. Positive- and negative-control tissues for S. falcatula were
used, along with duplicate test slides that received normal rabbit serum
as a substitute for the S. falcatula primary antibody.
RESULTS
Clinical signs. The first bird to die, the Luzon bleeding-heart
pigeon, did so without any clinical signs. Four months later, a young
female ring-necked parakeet was found dead in her enclosure, followed
one day later by another ring-necked parakeet from a different
enclosure. In the next 10 days, three more ring-necked parakeets from
the same enclosure as the first one died, all without any clinical signs.
Meanwhile, all the birds from the same enclosure were treated with
Enrofloxacin (Bayer, Sao Paulo, Brazil). A male cockatoo and the
African grey parrot died 20 and 30 days later, respectively. The African
grey parrot was recumbent and had polyuria for three days before
death. One more female cockatoo and the last ring-necked parakeet
died 3 mo after the first bird’s death. Except for the African grey
Parrot, none of the birds exhibited any clinical signs before they died.
Pathology. Necropsy revealed that all birds were in good body
condition and many had food in their crops. The most prominent
gross lesions exhibited as moderate- to severely-congested and
edematous lungs (Fig. 1). The liver was moderately enlarged and
congested in most birds. A few ring-necked parakeets had petechiae
and ecchymosis in several organs such as the lungs, air sacs, intestinal
mucosa, and dura mater. The pigeon had pale-yellow fibrin covering
the visceral pleura and dilation of the right ventricle; in addition, the
right ventricle was thinner than normal. The African grey parrot also
had opaque air sacs; the liver was congested and had multiple white
foci in the subcapsule that extended into the parenchyma.
Microscopically, most of the birds had moderate to severe lesions
in their lungs and livers. Lungs were moderately to severely
congested, and edematous, in most birds. There was mild to
moderate perivascular infiltration of lymphocytes, a few plasma cells,
and similar cells in the interstitium—most prominently in the
pigeon. There were many sinuous schizonts; some that contained
merozoites were present in the capillaries of the lungs (Figs. 2, 3).
Sarcocystis infection in birds 707

These schizonts were long, elongated, and ranged in size from 15 to
20 mm in length and from about 7 to 10 mm in diameter. Some of
the schizonts were immature (Fig. 2), but most had mature
merozoites (Fig. 3). Some of the merozoites were arranged
concentrically at the periphery, and were most evident on the
cross-section (Fig. 2). In addition, there was a moderate amount of
fibrin in the lumen of air spaces and parabronchioles. IHC
demonstrated large numbers of schizonts of S. falcatula in the lungs
(Fig. 4) of most birds.
The liver had moderately to severely increased cellularity of
mononuclear inflammatory cells in the sinusoids, as well as mild
lymphoplasmacytic periportal infiltration (Fig. 5). In the heart, there
was mild to moderate, perivascular and random, infiltration of
lymphocytes and plasma cells. Occasionally, there were a few long,
elongated schizonts, without any evidence of inflammation, scattered
here and there within the heart; these were most evident by IHC
(Fig. 6). There were increased numbers of macrophages and fibrin
exudation in the vascular sinuses of the spleen. Kidneys had
multifocal mild infiltration of lymphocytes and a few plasma cells in
the interstitium. In random birds, the brain exhibited mild
perivascular cuffing and gliosis. Schizonts were sparse and difficult
to identify on H&E in most organs, except in the lungs. Skeletal
muscles in ring-necked parakeets had mild, multifocal infiltration of
macrophages and lymphocytes, but no sarcocysts were detected.
By IHC, other organs such as the liver, spleen, kidney, intestine,
pancreas, and brain were positive for a few schizonts of Sarcocystis in
the endothelial cells. In a few birds, even though there were some
schizonts in the above organs, they stained only faintly with IHC.
Fig. 1. Lung from a ring-necked parakeet infected with Sarcocystis
sp., showing diffuse congestion and edema.
Fig. 2. Photomicrograph of lung from an infected ring-necked
parakeet with congestion and edema, immature schizonts (arrows), and a
mature schizont with merozoites of Sarcocystis sp. lining the periphery
(arrow head). H&E.
Fig. 3. Photomicrograph of lung from the pigeon infected with
Sarcocystis sp., with congestion, edema, and mature schizonts (arrows) of
Sarcocystis sp. H&E.
Fig. 4. Photomicrograph of lung from a ring-necked parakeet,
positive for S. falcatula by IHC, showing a few schizonts (arrows) and a
schizont with merozoites lining the periphery (arrow head). Envision
Plus–horseradish peroxidase.
708 R. Ecco et al.

Lungs from ring-necked parakeets, stained with PAS, Gram’s, and
Warthin-Starry silver stains, were negative for fungi and bacteria.
DISCUSSION
Clinical history, gross and microscopic lesions, and demonstration
of schizonts (meronts) of Sarcocystis in various organs by
histopathology and confirmation by IHC, suggests that these birds
had sarcocystosis. Most of the birds did not have clinical signs prior
to death, which is not unusual, as some birds can die without any
signs of sarcocystosis (2,26,30). The primary reason for the lack of
clinical signs is the severe acute pulmonary congestion and edema
that resulted in death (2,30). It is well known that the clinical
diagnosis of the acute pulmonary form of the disease can be difficult
(2). Clinical signs of sarcocystosis in birds found ill prior to death
included severe dyspnea, polyuria, anorexia, lethargy, loss of weight,
and neurologic signs (2,16,24).
Pulmonary edema and congestion were the most prominent gross
and microscopic findings in all birds examined in this study. These
lesions are directly related to the pathogenesis of Sarcocystis infection,
because endothelial cell invasion by schizonts occurs during asexual
multiplication of the protozoan parasite (7,25,26). However, the
number of cysts that developed in the endothelium, and the organs
involved, differed among bird species (14). Schizogony in all bird
species usually begins in the endothelium of capillaries and venules
in the lamina propria of the small intestine. In parrots and parakeets,
pulmonary schizogony is more intense than in other organs. In
contrast, schizogony in pigeons is more intense in the liver (27,30);
the lesions in the liver of the pigeon were more intense compared to
other birds in the present study. There are various reasons for these
differences. It is known that host cellular immune response for
controlling Sarcocystis infection differs among bird species (28). In
vitro studies have shown that the type of host cell infected also
influences the growth and persistence of S. falcatula merozoites (12).
Severity of lesions is well correlated with the amount of sporocysts
ingested by a bird (14).
Pulmonary congestion and edema result from stenosis of the
capillaries and venules caused by the protozoan parasite, in addition to
micro-thrombi formation induced by endothelial lesions during
schizogony (25). Experimental oral infection with S. falcatula in the
budgerigar demonstrated clinical signs of infection 4 days after
inoculation, at which time parasites were present in the endothelial
cells of the capillaries, veins, and venules of the lungs. Protozoan
burden was intense during days 7–10, resulting in acute pulmonary
distress and death. Similar clinical signs and lesions were observed in
other psittaciformes and columbiformes (25). In contrast, galliformes,
passeriformes, and New World psittacines survived the acute
pulmonary phase and developed cysts in the skeletal muscles (2).
Several others psittacines, including native Brazilian species (New
World psittacines), and passeriforme and galliforme species that were
present in the zoo, were not affected by this disease in the present
outbreak. It is well known that Old World psittacines are more
sensitive to Sarcocystis infection than are New World psittacines (2,3).
Microscopic lesions in the pigeon were subacute, with involve-
ment of cardiac muscle, and were protozoan structures easily
identified by routine stains (H&E). On the other hand, due to the
acute nature of the infection, the diagnosis was challenging in some
of the ring-necked parakeets because it was difficult to identify the
schizonts in the H&E sections. In this case, IHC was required for a
definitive diagnosis. In some birds, even though there were many
schizonts revealed in the lungs by H&E, they stained only faintly by
IHC. One possible explanation for this is that the tissues were fixed
in formalin for more than 30 days; this might have destroyed most
of the antigen. It is known that sarcocysts can also be identified on
Giemsa-stained impression smears of lungs (10), but we did not
attempt this. Typical tissue cysts of Sarcocystis in the skeletal muscle
were not identified in any of the birds in our study because most of
the birds died acutely.
The most likely source of Sarcocystis spp. infection in this outbreak
was the opossum. The wild opossum (D. albiventris) inhabits the forest
surrounding the zoo, and they are frequently seen at night looking for
food near the enclosures. Unfortunately, the wire mesh covering a
portion of the enclosure facilitated the entry of opossum, resulting in
the contamination of the floor, food, and water with sarcocysts.
Sarcocystis falcatula sporocysts are shed in the feces over a long period of
time. As a result, a single infected opossum, regularly visiting the
enclosures, could have spread the infection (2). Cockroaches could
have also the carried the protozoa into the enclosures, as it is known
that cockroaches can act as mechanical transmitters of Sarcocystis spp.
(2). In one outbreak, the cause of acute pulmonary sarcocystosis was
the introduction of soil into the enclosure of captive pigeons, soil
Fig. 5. Photomicrograph of liver from the pigeon infected with
Sarcocystis sp., showing large numbers of lymphocytes in the
sinusoids. H&E.
Fig. 6. Photomicrograph of heart from a ring-necked parakeet
positive for S. falcatula by IHC. Envision Plus–horseradish peroxidase.
Sarcocystis infection in birds 709

contaminated with sporocysts of Sarcocystis (30). However, in the
present outbreak, there had been no introduction of soil, animal
fertilizer, or plants into the enclosures of the affected birds; therefore,
neither the soil nor the aviary floor were sampled for Sarcocystis.
It is most likely that the birds in the study were infected with S.
falcatula, based on the lack of clinical signs, the pathology, and the
IHC results. However, the South American opossums (D.
marsupialis and D. albiventris) can be the intermediate hosts for S.
falcatula,S. speeri, and S. falcatula-like protozoans (4,6,10). The S.
falcatula-like protozoan from one of the opossums (D. albiventris)
was later identified as a separate species, based on its structure and
molecular characterization; it was named S. lindsayi (4,11). In that
study, the schizonts of S. lindsayi reacted positively by IHC to the
polyclonal sera prepared against both S. lindsayi and S. falcatula (11).
In the present study, using IHC, the polyclonal antisera prepared
against S. falcatula that can also crossreact with S. lindsayi was used
to identify the sarcocysts in various tissues. Therefore, the species of
sarcocysts that infected the birds in the study could not be
determined and awaits further study.
This report demonstrates how easily Sarcocystis infection can
spread to birds from opossums and is an alert for veterinarians and
zoo workers toward preventing sources of infection. In addition,
sarcocystosis should be considered in the differential diagnosis of
sudden death and pneumonia in captive birds. More studies should
be carried out in order to better understand the epidemiology of the
disease and the possible wild reservoirs in Brazil. Although Sarcocystis
sp. has been demonstrated in the feces of opossums from Brazil, this
is the first documentation of disease associated with a Sarcocystis sp.
in psittacines and a pigeon.
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ACKNOWLEDGMENTS
We would like to thank Dr. Crespo, R. Gonzales, M. Manzer, K.
Swerlow, L. Alsing, and D. Ramirez for their technical assistance.
710 R. Ecco et al.