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: pfpeixoto@terra.com.br, peixotop@ufrrj.br

(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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