fatal cytauxzoonosis in captive-reared lions in brazil
TRANSCRIPT
Veterinary Parasitology 145 (2007) 383–387
www.elsevier.com/locate/vetpar
Short communication
Fatal cytauxzoonosis in captive-reared lions in Brazil
P.V. Peixoto a,*, C.O. Soares b, A. Scofield c, C.D. Santiago d,T.N. Franca e, S.S. Barros f
a Departamento de Nutricao Animal e Pastagem, Instituto de Zootecnia, Universidade Federal Rural do Rio de Janeiro (UFRRJ),
Km 47, 23890-000 Seropedica, RJ, Brazilb Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA)—Centro Nacional de Pesquisa de Gado de Corte,
Mato Grosso do Sul, Brazilc Curso de Medicina Veterinaria, Universidade Federal do Para (UFPA), Castanhal, PA, Brazil
d Medico Veterinario, Volta Redonda, RJ, Brazile Curso de Medicina Veterinaria, Universidade Estacio de Sa (UNESA), Rio de Janeiro, Brazil
f Departamento de Patologia, Faculdade de Veterinaria, Universidade Federal de Pelotas (UFPEL), Rio Grande do Sul, Brazil
Received 11 September 2006; received in revised form 20 December 2006; accepted 27 December 2006
Abstract
Fatal cytauxzoonosis is described in a captive reared lioness (Panthera leo) and its 6-month-old cub. Clinical signs in the lioness
included loss of weight, depression, anaemia, loss of hair, dark discolored urine, tachypnoea, nystagmus, deaphness and staggering
gait. The cub died after a short period of depression. In the lioness, laboratory examination revealed normochromic normocytic
anaemia, neutrophilia, lymphopenia, monocytosis, eosinopenia, thrombocytopenia, proteinuria, pyuria, haematuria and increased.
At necropsy the lioness showed marked pulmonary edema and slight gelatinous translucent edema in the mediastinum, petechiae
and echymosis disseminated in the serosae, and the intestinal content was red and semiliquid. The cub presented hemothorax,
endocardial and pulmonary edema, petechiae in the cardiac serosae, hepatic and splenic congestion and segments of the small
intestine with blood stained fluid contents and reddish mesenteric lymph nodes. Histopathological examination of liver, spleen,
heart, lungs, intestines, pancreas, mesenteric lymph nodes, kidneys, skeletal muscle, brain and skin revealed large number of
intravascular macrophages with their cytoplasm filled with various schizogonic stages of a Theileriidae. Electron microscopy
confirmed the presence of schizonts in endothelial-associated macrophages. The diagnosis was established by the finding of the
pathognomonic schizonts in macrophages within blood vessels in several organs and tissues from both lions. This is the first report
of feline cytauxzoonosis in P. leo and of a confirmed infection by Cytauxzoon felis in felidae in South America.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Cytauxzoon felis; Cytauxzoonosis; Lions; Brazil
1. Introduction
The feline Cytauxzoonosis, disease of domestic and
wild felids infected by the protozoan Cytauxzoon felis,
* Corresponding author. Tel.: +55 21 26821081;
fax: +55 21 26821081.
E-mail addresses: [email protected], [email protected]
(P.V. Peixoto).
0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2006.12.023
was reported for the first time in domestic cats from rural
areas of Missouri’s southeast, USA (Wagner, 1976).
Since that time, fatal cases of the disease have been
registered in domestic cats (Meier and Moore, 2000),
Florida panther (Kier et al., 1982) and white tiger (Garner
et al., 1996). A non-symptomatic form of the cytaux-
zoonosis has been recognized in lynx (Glenn et al., 1982;
Blouin et al., 1984), Texas panthers (Rotstein et al.,
1999), Florida panthers (Butt et al., 1991) and cheetahs
P.V. Peixoto et al. / Veterinary Parasitology 145 (2007) 383–387384
(Zinkl et al., 1981). Outside North America, the feline
cytauxzoonosis has been registered only in a Bengal
Tiger in a zoo in Germany (Jakob and Wesemeier,
1996). The etiologic agent, from the Theileriidae family,
goes through a stage with binary division in erythrocytes
and a schizogonic stage in macrophages (Kocan et al.,
1992). Lynx (Glenn et al., 1982) and the Florida panther
(Butt et al., 1991) are considered as the definitive hosts
and natural reservatories of this parasite, whilst the
domestic cat is regarded as an accidental or dead-end host
(Wagner et al., 1976; Blouin et al., 1984). The ixodid
ticks Amblyomma americanum, Amblyomma macula-
tum, Ixodes scapularis, Ixodes affinis and Dermacentor
variabilis were observed parasitizing animals naturally
infected by C. felis (Wehinger et al., 1995), but only D.
variabilis has been experimentally demonstrated as a
vector capable of transmitting C. felis from lynx to
domestic cats in the USA (Blouin et al., 1984). Another
important fact was the observation of an agent morpho-
logically similar to C. felis parasitizing macrophages of a
non-human primate native to Panama (Gardiner, 1998,
personal communication).
The purpose of this paper was to describe a fatal
infection by C. felis in captive-reared lions in Brazil.
2. Material and methods
In 1998, a 6-month-old lion cub died inside its
exhibit in the Volta Redonda Municipal Zoo (VRMZ)
(altitude = 390 m, south latitude = 2283102300 and west
longitude = 4480601500), Rio de Janeiro State. The
necropsy was performed by professionals of the zoo.
Forty-five days later, its mother, an 8 years and 7 months
old lioness also died inside the same exhibit. Before its
death, the lioness was clinically examined by the zoo
veterinarian, who also collected material for seric
biochemistry, haematological and urinary analysis.
Fragments of liver, spleen, heart, lungs, intestines,
pancreas, mesenteric lymph nodes, kidneys, skeletal
muscle, brain and skin from both animals were fixed in
10–20% formaldehyde and processed using routine
methods, included in paraffin, sectioned in the thickness
of 5 mm and stained with hematoxylin–eosin (HE) for
examination in optic microscope. The descriptive
morphological characterization of the intrahistiocytic
parasites was carried out using an optic microscope
(Dialux 20 ES, Leitz) and the morphometric character-
ization with the aid of a micrometric ocular (pZo, Wild)
attached to the optic microscope for evaluation of the
parameters related to the minor diameter (md), greater
diameter (gd) and morphometric index (MI = md/gd) of
the parasitic forms. Fragments of brain, lungs and
kidneys, previously fixed in formalin and embedded in
paraffin blocks, after the paraffin melting, were again
processed for analysis through transmission electronic
microscopy (TEM). These samples were fixed in
sodium-caccodilate buffer solution 0.1 M, pH 7.3 with
the addition of 2.5% glutaraldehyde. The post-fixation
was performed with 2% osmium tetroxide (OsT4) and
the samples embedded in epoxy resin blocks. Ultrathin
sections of the material were contrasted with uranile
acetate and lead citrate and examined in transmission
electronic microscope (Zeiss EM-10) at 75 kV.
Ixodid ticks were collected in the lioness and
identified according to a key elaborated by Aragao and
Fonseca (1961). The adult ticks were dissected and
hemolymph smears were prepared. The smears were air
dried, fixed in methylic alcohol and stained with
Giemsa diluted in buffer Sorensen pH 6.8 (Lubinsky,
1960).
3. Results
In the VRMV the lion cub was born from the lioness
that died during this study and an approximately 8-year-
old lion. The cub’s father had died in the VRMZ, but the
definitive diagnosis of death was not conclusive. The
lioness, coming from the State of Minas Gerais had
been acquired along with two 45 days old cubs (one
male and one female), all from the same litter from a
private breeder.
The cub died after 24-h depression, and no additional
clinical observation was taken note of. The cub’s
necropsy, performed by the zoo veterinarian showed
hemothorax, endocardial and pulmonary edema, pete-
chiae in the cardiac serosae, congestion in the liver and
spleen, discrete splenomegaly, segments of the small
intestine with blood stained fluid contents and reddish
mesenteric lymph nodes. The lioness died 45 days later.
Signs such as weight loss, depression, anaemia, pale
mucosae, emaciation, hair loss and brown colored urine
were observed 5 days before its death. This animal still
showed tachypnea, nystagmus, motor incoordination,
deafness, ocular and nasal discharge. The haematolo-
gical examination showed normocytic normochromic
anaemia, neutrophilia, monocytosis, lymphopenia,
eosinopenia and thrombocytopenia. The seric biochem-
istry revealed an elevated nitrogen concentration
coming from the urea in the blood (BUN); creatinin,
aspartate aminotransferase (AST), alanine aminotrans-
ferase (ALT) and creatinine kinase (CK) were normal.
The urinalysis showed pyuria, hematuria and signi-
ficant proteinuria. At necropsy, it was possible to
observe pronounced pulmonary edema and congestion,
P.V. Peixoto et al. / Veterinary Parasitology 145 (2007) 383–387 385
Fig. 1. Schizogonic forms of Cytauxzoon felis occluding the lumen of
a venule in the renal cortex of the lioness. HE, 200�.Fig. 3. Mature schizonts of Cytauxzoon felis free in the lumen of a
small blood vessel in the liver of the lioness. HE, 1000�.
bronchus and bronchiole with mucoid contents of
grayish coloration, slight gelatinous and translucent
edema in the mediastinum, petechiae in the epicardium
and echymosis in the endocardium, brown-reddish
stomach contents and intestinal fluids, petechiae and
echymosis disseminated through the intestinal serosae
and slight congestion of the kidneys. In both animals,
the histological examination, showed a great number of
macrophages containing, in its cytoplasm, schizogonic
forms in different development stages (Figs. 1–4). The
macrophages, fulfilled by the schizonts, were increased
in size, partially or totally occluding the lumina of many
capillaries, venules and arterioles of all the examined
tissues. The macrophage’s nucleus were commonly
eccentric, pycnotic or kariolytic. The basophilic,
pleomorphic, rounded and oval schizonts, visualized
in the macrophages cytoplasm, measured, in average,
17.493 � 6.561 mm � 13.100 � 5.216 mm, with aver-
age IM of 0.747 � 0.050. Each schizont contained, in
average, 63.444 � 64.873 basophilic, oval to elongated
Fig. 2. Mature schizonts of Cytauxzoon felis free and adhered to the
endothelium of the blood vessel in the central nervous system of the
lioness. HE, 400�.
merozoites (Fig. 2), measuring, in average, 1.260 �0.506 mm � 1.011 � 0.423 mm with average IM of
0.801 � 0.032. Cytomeres Fig. 3, and the formation of
a residual body were occasionally observed Fig. 1. No
regressive alterations were observed in the examined
organs.
By electron microscopy, it was verified that the
schizonts of C. felis, delimited by a thin double
cytoplasmatic membrane, were found inside the
cytoplasm of macrophages, sometimes kariopycnotic
or kariolytic, especially when the schizonts developed
until fulfilling all the cytoplasm. The cytomere,
delimited by a thick cytoplasmatic membrane, had
developing merozoites that were involved by a thin and
electrodense cytoplasmatic membrane and showed a
well-developed nucleus, surrounded by a similarly
double nuclear membrane. In the merozoites cytoplasm,
non-typical untucked mytocondria and peripherical
alimentary vacuole were observed. The ticks collected
in the lion were identified as Amblyomma cajennense.
Fig. 4. Schizonts of Cytauxzoon felis in different stages of develop
ment, free in the lumen of a pulmonary artery of the lioness. HE
1000�.
-
,
P.V. Peixoto et al. / Veterinary Parasitology 145 (2007) 383–387386
By microscopic evaluation of the hemolymph and of
different tissue macerated smears, as well by the
histological material prepared from ticks organs, no
hematozoan form was observed.
4. Discussion
The diagnosis was based on the clinical signs,
necropsy findings, and specially on the typical histo-
pathological picture confirmed by the ultrastructural
analysis compatible to those described for fatal sponta-
neous cytauxzoonosis observed in domestic cats
(Wagner, 1976; Meier and Moore, 2000) and in a captive
tiger in the USA (Garner et al., 1996) and another one in
Germany (Jakob and Wesemeier, 1996). The histological
and macroscopic findings, on both lions, were consistent
with those reported in the cases of fatal feline
cytauxzoonosis and considered, by itself, sufficient to
establish of the diagnosis of cytauxzoonosis, because the
histological observation of intrahistiocytic schizonts
partially or totally obstructing the blood vessels of many
organs is considered a pathognomonic lesion of
cytauxzoonosis (Wagner et al., 1976; Hauck et al.,
1982). Ultrastructurally, the intracytoplasmatic schizonts
inmacrophages are identical to those reported indomestic
cats (Kier et al., 1987) and wild felidae (Garner et al.,
1996; Jakob and Wesemeier, 1996). Although Cytaux-
zoon silvicaprae, Cytauxzoon strepsicerosi and Cytaux-
zoon taurotragi are also morphologically similar to C.
felis (Kier et al., 1987), there is no report of felids infected
by these three species. Additionally, 15 days after the
lioness death, intraerythrocytic forms of a Theileridae
were observed in blood smears of two additional felidae
(Panthera onca and Leopardus tigrinus), in the same zoo.
The blood of both felidae was collected and storaged in
cryoprotective fluid at—196 8C in liquid nitrogen.
Recently, Scofield (2006) made the molecular character-
ization of the gene 18SrRNA from both isolates and
demonstrated 99% similarity between this gene (DQ
382276 and DQ 382277, respectively) and that from C.
felis deposited with the Genbank in USA (AF 399930—
http://www.ncbi.nlm.nih.gov/Blast.cgi). In our opinion
this results confirm C. felis as the etiologic agent in
both cases (captive-reared lions and Panthera onca/
Leopardus tigrinus).
The clinical evidence of lethargy, weight loss,
anaemia and pale mucosae found in the lioness is
similar to the symptoms observed in both domestic cats
(Wagner et al., 1976; Kier et al., 1987), and wild felidae
that died due to the infection caused by this protozoan
(Garner et al., 1996) in the EUA. It is possible that
the motor incoordination, nystagmus and deafness,
evidenced in the lioness, may also be a consequence of
the brain vessels occlusion with reduction of the blood
supply and low oxygenation of this tissue. Normo-
chromic normocytic anaemia, lymphopenia, eosinope-
nia and thrombocytopenia have also been frequently
observed in the feline cytauxzoonosis in domestic cats
(Garner et al., 1996; Rotstein et al., 1999). The anaemia
seems to be based on an immunomediated erytropha-
gocytosis, whilst the thrombocytopenia may be related
to the platelet aggregation formerly registered in felidae
with cytauxzoonosis in the USA (Kier et al., 1987;
Meier and Moore, 2000).
The lack of demonstration of intraerythrocytic forms
in the blood smears and in the electronic microscopy is
justifiable, since in 50% of the fatal feline cytauxzoo-
nosis cases, either from natural or experimental origin,
there is no occurrence of parasites in erythrocytes (Kier
et al., 1987). Blood smears and imprints of organs were
not prepared during the necropsy procedure, since at
that occasion we had no suspicion of cytauxzoonosis.
It was not possible to determine how the lions
became infected. Although Amblyomma cajennense
specimens were found in these animals exhibit, there is
no proof that these ixodids are the ones who transmitted
C. felis to the lions, because the parasite was not found
neither in the hemolymph nor in the histological
sections of the ticks collected in the lions cage. Anyway,
it cannot be disregarded that A. cajennense, as well as
other ixodid species that parasite felidae in Brazil, may
be eventual vectors of C. felis in our environment. It is
not possible to precisely determine the transmission
route of C. felis in these cases in Brazil, since lions are
transported and dislocated from one continent to
another through different ways and motives, such as
for zoological collection, artistic attraction or scientific
research. In the same manner, other animals parasitized
by ticks or possibly by exotic pathogens move or are
transported among distinct physiographycal regions. In
this paper, the first occurrence of cytauxzoonosis in
Panthera leo and the first confirmed record of C. felis in
South America are reported.
Acknowledgments
To Conselho Nacional de Desenvolvimento Cientı-
fico e Tecnologico—CNPq for financial support.
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