Download - Piroplasma la Necunoscuta Brazilia

Hemorrhagic disease in dogs infected with an unclassified

intraendothelial piroplasm in southern Brazil

Alexandre Paulino Loretti a,*, Severo Sales Barros b

a Section of Veterinary Pathology, Department of Veterinary Clinical Pathology, Faculty of Veterinary Medicine,

Federal University of Rio Grande do Sul (UFRGS), CEP 91540-000, Porto Alegre, RS, Brazilb Department of Animal Pathology, Faculty of Veterinary Medicine, Federal University of Pelotas (UFPel),

CEP 96010-900, Pelotas, RS, Brazil

Received 2 May 2005; received in revised form 4 July 2005; accepted 6 July 2005

www.elsevier.com/locate/vetpar

Veterinary Parasitology 134 (2005) 193–213

Abstract

A hemorrhagic disease affecting dogs in Brazil, referred to popularly as ‘‘nambiuvu’’ (bloody ears) and believed to be

transmitted by ticks, has been observed in animals infected with an organism described originally in 1910 as a piroplasm, and

known locally as Rangelia vitalli. In this series of 10 cases, the disease was characterized by anaemia, jaundice, fever, spleno-

and lymphadenomegaly, hemorrhage in the gastrointestinal tract, and persistent bleeding from the nose, oral cavity and tips,

margins and outer surface of the pinnae. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum infested

affected dogs from suburban and rural areas, respectively. Laboratory findings included regenerative anaemia, spherocytosis,

icteric plasma and bilirubinuria. Those intracellular organisms were found in bone marrow smears but not in blood smears.

Microscopically, zoites were seen within the cytoplasm of blood capillary endothelial cells. Parasitized and non-parasitized

endothelial cells were positive immunohistochemically for von Willebrand factor (vWF). Langhans-type multinucleate giant

cells were observed in the lymph nodes and choroid plexus. There was prominent erythrophagocytosis by macrophages in the

lymph node sinuses and infiltration of the medullary cords by numerous plasma cells. Ultrastructurally, this organism had an

apical complex that included a polar ring and rhoptries but no conoid. This parasite was contained within a parasitophorous

vacuole that had a trilaminar membrane with villar protrusions and was situated in the cytoplasm of capillary endothelial cells.

This organism tested positive by immunohistochemistry for Babesia microti. This pathogen was also positive by in situ

hybridization for B. microti. Tentative clinical diagnosis in these cases was based on the history, clinical picture, haemogram and

favorable response to therapy, and confirmed through microscopic examination of smears from the bone marrow or histological

sections of multiple tissues, especially lymph nodes where zoites were most frequently found. The disease was reproduced by

intravenous inoculation of blood from a naturally infected dog into an experimental dog. The authors demonstrate in this study

that this organism is a protozoa of the phylum Apicomplexa, order Piroplasmorida. This piroplasm seems to be different from

* Corresponding author. Present address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada

N1G 2W1. Tel.: +1 519 824 4120x54674; fax: +1 519 824 5930.

E-mail address: [email protected] (A.P. Loretti).

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2005.07.011

A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213194

Babesia since it has an intraendothelial stage. Molecular phylogenetic analysis is necessary to better characterize this parasite

and clarify its taxonomic status.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Rangelia vitalli; Protozoa; Apicomplexa; Piroplasmorida; Dog; Brazil

1. Introduction

‘‘Nambiuvu’’ (bloody ears), ‘‘peste de sangue’’

(bleeding plague) or ‘‘febre amarela dos caes’’ (yellow

fever of dogs) is a disease that commonly affects dogs

from rural and suburban areas in Brazil. Over the years,

this malady has been associated with an unclassified

organism that occurs within endothelial cells and

erythrocytes. The original reference to this disease

comes from 1908. In a short communication published

by Antonio Carini about the most common infectious

and parasitic diseases of domestic animals in Brazil at

that time, he mentioned observing a disease of dogs

called ‘‘nambiuvu’’. It was suspected to be caused by a

piroplasm since it was clinically similar to malignant

jaundice (canine babesiosis), a disease that hadn’t been

described yet in Brazil at that time (Carini, 1908). Two

years later, in 1910, Bruno Rangel Pestana published

two scientific papers characterizing the morphology of

this unusual protozoan parasite as seen under the light

microscope, and describing the epidemiological,

clinical and pathological aspects of the disease caused

by this atypical piroplasm. He proposed the new

taxonomic name Piroplasma vitalli since this was a

hitherto undescribed piroplasm. He also used this

parasite species name to pay a tribute of respect to his

mentor, the brazilian biomedical scientist Vital Brazil,

internationally renowned for the discovery of the

polyvalent anti-ophidic serum used to treat bites of

venomous snakes (Pestana, 1910a, 1910b). In 1914,

Antonio Carini and Jesuıno Maciel published a paper

together about this disease in which they proposed that

the name of this previously unidentified canine

piroplasm should be changed to Rangelia vitalli to

honor the investigator Bruno Rangel Pestana, who first

observed the presence of this organism within

endothelial cells and red blood cells of Brazilian dogs

affected by ‘‘nambiuvu’’. In this publication, they

reemphasized that this was a new species of canine

piroplasm, and that it should be included in a separate

genus (Carini and Maciel, 1914).

The popular name ‘‘nambiuvu’’ was coined in the

past by Brazilian aboriginal inhabitants in reference to

blood dripping continuously from the tips, margins

and outer surface of both pinnae, a clinical sign

usually observed in this illness. Categories of dogs

affected by this pathogen include hunting dogs,

herding dogs, search dogs, police dogs, guard dogs

and companion dogs. Ixodid ticks have been

implicated as the natural vectors of this organism

since cases of infection by this protozoa have been

consistently associated with the presence of these

ectoparasites on the host or in the environment (or

both). However, there are no published studies to date

showing that ticks can transmit this protozoal

pathogen to dogs. The disease may occur at any time

of the year, although peak occurrence is usually

observed during the summer and is associated with the

presence of large populations of ticks. Recovered

patients develop a strong immunity against this

protozoal parasite but still the organism can persist

for months in these clinically normal, cured animals

which can act as healthy carriers of the pathogen. The

host range of this protozoa seems to be restricted to

domestic dogs since other mammals, birds or

laboratory animals cannot be infected experimentally

by this parasite (Pestana, 1910a, 1910b; Carini and

Maciel, 1914).

There is no consensus about the life cycle and

taxonomic status of this organism at this time. It is

described that its life cycle consists of an intraery-

throcytic developmental phase (blood stage), and an

extraerythrocytic phase occurring in the cytoplasm of

endothelial cells (tissue stage). It is uncommon to find

this protozoa in blood smears in both natural and

experimental cases. According to the literature, the

intraerythrocytic form of this parasite is most often

seen – in very low numbers – in blood smears if blood

is drawn during an episode of high fever in the acute

stage of the disease (Pestana, 1910a, 1910b; Carini

and Maciel, 1914). Some researchers have found this

organism only within parasitophorous vacuoles in the

A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 195

cytoplasm of endothelial cells from blood capillaries

but not within red blood cells (Krauspenhar et al.,

2003). This unclassified organism has been misiden-

tified as Toxoplasma gondii (Wenyon, 1926; Moreira,

1938) and as Leishmania donovani (Pocai et al., 1998)

in histological sections, and misdiagnosed as Babesia

canis (Wenyon, 1926; Moreira, 1938; Levine, 1973) in

blood films. Some authors claim that cases of

‘‘nambiuvu’’ of dogs described by other investigators

as being caused by R. vitalli (Pestana, 1910a, 1910b;

Carini and Maciel, 1914) were, in fact, dual infections

by B. canis and T. gondii, but provide no substantial

data to support this statement (Wenyon, 1926;

Moreira, 1938; Paraense and Vianna, 1948; Levine,

1973). Recently, this issue was revisited by a group of

investigators (Krauspenhar et al., 2003) as a retro-

spective study of cases of infection with this

unclassified organism that, during the 1980s and

1990s, were mistaken for cases of canine visceral

leishmaniasis (Pocai et al., 1998).

In areas where this disease is common, such as in

the State of Rio Grande do Sul (RS), southern Brazil,

any dog with high fever, anaemia, jaundice and

hemorrhages and infested by ticks is suspected of

being infected by this unclassified pathogen. There

may be sufficient justification for treatment even

without blood examination being made or in the

absence of demonstrable organisms in the peripheral

blood since this organism is very difficult to recover in

blood smears, especially in the chronic form of the

disease. In many cases, the diagnosis may need to be

based on the animal’s positive response to antiproto-

zoal therapy. Supportive evidence for a diagnosis of

‘‘nambiuvu’’ in dogs include lymphadenopathy,

splenomegaly, bleeding tendencies (Pestana, 1910a,

1910b; Carini and Maciel, 1914; Braga, 1935; Carini,

1948), a haemogram consistent with immune-

mediated haemolytic anaemia (IMHA) (Krauspenhar

et al., 2003), increased amounts of bilirubin in the

serum, and the presence of R. sanguineus or A.

aureolatum on the coat of the affected dogs (Pestana,

1910a, 1910b; Carini and Maciel, 1914). In some

cases, tick infestation may be very light or the ticks

may have detached from the host by the time the

animal is examined. A definitive antemortem diag-

nosis of this protozoal infection is possible if

microscopic examination of blood films reveals this

organism in erythrocytes as well as extracellularly but

this is rarely achieved (Pestana, 1910a, 1910b; Carini

and Maciel, 1914; Rezende, 1976). There is no

published data about the use of fine needle aspiration

cytology of peripheral lymph nodes and bone marrow

in search for this parasite. However, in some cases of

‘‘nambiuvu’’ of dogs, this organism has been detected

in very low numbers in conventional cytological

preparations of kidney and lung aspirates (Carini and

Maciel, 1914).

In spite of the fact thatR. vitalliwas first described in

1910, this organism is yet poorly characterized. The

disease is not known by a wide range of readers since a

great deal of the information about this subject is

written in Portuguese and published in local, non-

indexed Brazilian scientific journals, especially during

the first half of the 20th century (1908–1948) (Carini,

1908, 1948; Pestana, 1910a, 1910b; Carini andMaciel,

1914; Braga, 1935; Rezende, 1976; Krauspenhar et al.,

2003).R. vitalli appears in the international literature as

a synonym for B. canis or B. vitalli (Wenyon, 1926;

Levine, 1973; Peirce, 2000). Considering the fact that

this organismwas characterized in 1910 based solely on

the morphology of the parasite as seen under the light

microscope in blood films, impression smears of tissues

and histological sections (Pestana, 1910a, 1910b), one

would question the validity of this unique species of

canine piroplasm that has been observed only in Brazil,

and argue that the namegiven to this parasite is a nomen

nudum. In fact, this proposed taxonomic name is not

considered valid because the group designated for this

parasite has been insufficientlydescribed and illustrated

in the literature to allow recognition, and has no

nomenclatural status. The taxonomy of this protozoan

parasite lacks credibility since there are no ultrastruc-

tural, immunohistochemical or molecular studies

published about this organism at this time. Without

these studies, it is not possible to assign a specific

identity to this pathogen.

The purposes of the present study are: (1) to

describe the epidemiology, clinical picture and

pathology of 10 cases of infection with this protozoan

parasite of dogs in the State of Rio Grande do Sul,

southern Brazil, diagnosed during 2000–2003; (2)

characterize the morphology of this organism under

the transmission electron microscope; (3) based on

ultrastructural, immunohistochemical and in situ

hybridization studies, compare this protozoa with

other known apicomplexan parasites.


2. Materials and methods

2.1. Signalment, history, clinical picture,

laboratory tests, therapy, follow-up and outcome

Ten affected dogs referred to a local university

veterinary hospital (HCV-UFRGS, Porto Alegre, RS,

Brazil) were included in this study. History, clinical

findings, management and outcome of the disease

were retrieved from veterinarians and owners.

Description of the clinical picture was also based

on the authors’ observations during regular visits to

the hospital. Tentative clinical diagnosis of ‘‘nam-

biuvu’’ was based on the history, clinical signs,

haemogram and favorable response to treatment,

which consisted of immunosuppressive therapy with

corticosteroids, administration of an antiprotozoal

drug (imidocarb dipropionate or doxycycline) and

blood transfusion. Diminazene aceturate was also used

to treat this atypical protozoal infection and was

administered in a single dose or in two injections given

on alternate days within a week. Giemsa-stained thin

blood smears were examined microscopically for

blood pathogens, i.e. the causal agent of ‘‘nambiuvu’’,

Ehrlichia canis and B. canis in those cases presented

for consultation at the hospital.

2.2. Experimental transmission

Artificial infection of a susceptible dog with

infective blood was performed by drawing 3 ml of

heparinized whole blood from the cephalic vein of one

naturally affected dog and inoculating this sample into

the cephalic vein of a non-castrated, non-splenecto-

mized experimental dog 4 h after collection. The

infected experimental dog was housed and cared for

according to conventional laboratory animal practices,

and the investigation was conducted in accordance

with the guidelines for experimental procedures as set

forth in the law 11.915 of May 21st, 2003 (Code for

Animal Well-Being) in the State of Rio Grande do Sul

(RS), southern Brazil. A thorough clinical examina-

tion was performed within 48 h of the arrival of this

dog to our unit. No ticks were detected on the coat of

this animal before the experimental procedure or

during the experiment or by the end of the clinical

trial. This animal had no history of exposure to ticks

and had no history of tick-borne diseases. This

incoming dog was monitored closely during the whole

experiment every 24 h. Blood smears for detection of

blood pathogens were made immediately before IV

inoculation in order to rule out the possibility that the

animal might be incubating an infectious or parasitic

blood-borne disease which might be manifested

clinically during the experiment, and then every

24 h during the whole trial period (19 days). On day 17

after inoculation, the popliteal lymph nodes and the

bone marrow of the experimental animal were

surgically removed and sampled for light and electron

microscopy.

2.3. Pathology

At necropsy of the eight naturally affected dogs and

one experimental animal, samples of multiple tissues

including the liver, gallbladder, spleen, kidney, urinary

bladder, peripheral and visceral lymph nodes, bone

marrow, lungs, tonsils, nasal turbinates, adrenal

glands, thyroid glands, skin (from the neck, dorsum

and tip of the pinnae), skeletal muscle, tongue,

esophagus, trachea, jugular vein, carotid artery,

thoracic and abdominal aorta, heart, stomach, eyes,

small and large intestines, brain and spinal cord were

collected for histology, fixed in 10% neutral buffered

formalin for 24–48 h, routinely processed, embedded

in paraffin, sectioned at 5 mm and stained with

haematoxylin and eosin (HE) and Periodic Acid Schiff

(PAS). Smears from tissue samples made during the

necropsy (bone marrow, lymph node, spleen, liver,

kidney, choroid plexus of the fourth ventricle and

blood) were stained with Giemsa and Panoptic. In one

of the cases in which marked jaundice and widespread

hemorrhage were observed, a direct fluorescent

antibody test (FAT) using a multivalent Leptospira

fluorescent antibody conjugate [1098-LEP-FAC,

National Veterinary Services Laboratories (NVSL),

Ames, IA, USA] was done on impression smears of

kidney collected at necropsy as described elsewhere

(Pescador et al., 2004).

2.4. Immunohistochemistry

Immunohistochemical stainings for von Willebrand

factor (vWF) (anti-human factor VIII-related antigen

polyclonal antibody, rabbit origin, Dako Corp.,

Carpinteria, CA, USA), B. microti (anti-B. microti


polyclonal antibody, hamster and monkey origins), L.

chagasi [anti-L. chagasi polyclonal antibody, rabbit

origin, courtesy of Dr. Luciana R.Meireles, Laboratory

of Protozoology, Institute of Tropical Medicine of Sao

Paulo, University of Sao Paulo (USP), Sao Paulo, SP,

Brazil], Neospora caninum [anti-N. caninum poly-

clonal antibody, goat origin, Veterinary Medical

Research and Development (VMRD), Inc., Pullman,

WA, USA] and T. gondii (anti-T. gondii polyclonal

antibody, goat origin, VMRD, Inc., Pullman, WA,

USA) were done on a peripheral lymph node, kidney,

adrenal gland and thyroid gland of one natural case and

the experimental case as described elsewhere (Chehter

et al., 2001; Corbellini et al., 2002; Qin et al., 2003;

Torres-Velez et al., 2003). For vWF immunohisto-

chemistry, unstained slides (recuts) were submitted to

Dr. Josepha P. DeLay, Animal Health Laboratory

(AHL), Laboratory Services Division (LSD), Univer-

sity of Guelph, Guelph, ON, Canada, and for B. microti

immunohistochemistry recuts were sent to Dr. Jeann-

ette Guarner, Infectious Disease Pathology Activity,

National Center for Infectious Disease, Centers for

Disease Control and Prevention (CDC), Atlanta, GA,

USA.

2.5. In situ hybridization

The same set of tissues used in the immunohis-

tochemistry was also tested with an in situ hybridiza-

tion method for B. microti as described elsewhere

(Sledge et al., 2004). For B. microti in situ

hybridization, recuts were sent to Dr. Fernando J.

Torres-Velez, Department of Pathology, College of

Veterinary Medicine, University of Georgia, Athens,

GA, USA.

2.6. Transmission electron microscopy

Surgical and necropsy samples collected for

transmission electron microscopy from the experi-

mental animal included a peripheral lymph node, bone

marrow, choroid plexus of the fourth ventricle,

cerebral cortex above the thalamus, kidney, heart,

liver, spleen and tonsil. These tissues were fixed in 2%

glutaraldehyde in phosphate buffered saline (pH 7.4)

for 48 h, postfixed in 1% osmium tetroxide buffered in

0.4 M sodium cacodylate (pH 7.4) and embedded in

Epon 812. Semi-thin sections were stained with

methylene blue. Ultrathin sections were stained with

lead citrate and uranyl acetate and examined with an

EM 109 Zeiss transmission electron microscope at

80 kV. Formalin-fixed samples of a peripheral lymph

node from a natural case were similarly processed for

ultrastructural studies.

2.7. Taxonomy of ticks

Ticks were sampled from the coat of dogs affected

by ‘‘nambiuvu’’ (clinical cases from the university

hospital and necropsy cases), and, in those areas in the

State of Rio Grande do Sul (RS), southern Brazil,

where there were anecdotal reports, clinical cases or

necropsy cases of this protozoal infection, from dogs

(healthy and affected ones) and from the environment

where these animals used to wander or live. The study

area included the city of Porto Alegre (30820S,518130W), and the nearby counties of Caxias do

Sul, Gravataı, Nova Petropolis and Viamao. These

ticks were submitted to Dr. Joao Ricardo S. Martins,

Center of Veterinary Research Desiderio Finamor,

FEPAGRO, Eldorado do Sul, RS, Brazil, for

taxonomic classification.

It is beyond the scope of the current paper to do

experimental transmission studies in order to identify

vectors and establish the life cycle of this protozoan

parasite in the host, to search for the developmental

stages of this organism in the collected ticks, and to

develop phylogenetic studies based on other molecular

methods, i.e. characterization of the pathogen by PCR.

3. Results

3.1. Epidemiology, clinical signs and laboratory

findings

Infection with an atypical protozoan parasite, the

causal agent of ‘‘nambiuvu’’, was observed in 10 dogs

in the State of Rio Grande do Sul, southern Brazil,

during the year 2000 and fromMay 2002 to December

2003; 8 males and 2 females, of the breeds Boxer (1

case), Fila Brasileiro (Brazilian Fila) (2 cases) and

Weimaraner (1 case), and mixed-breed dogs (6 cases),

ranging in age from 1 to 2.5 years, were affected.

Those animals came from rural areas or suburban

areas, and all had a history of tick exposure. Seven


Fig. 1. Ixodid ticks found in dogs naturally infected with an unclassified intraendothelial piroplasm: (a) Rhipicephalus sanguineus (‘‘the brown

dog tick’’) and (b) Amblyomma aureolatum (‘‘the yellow dog tick’’). The ruler is in millimeters.

cases were observed from November to March during

the hot season when ticks were abundant in the

environment, one in April, one in May and one in July.

For those cases occurring in suburban areas, the

animals had access to areas heavily contaminated by

ticks. On one occasion, numerous ticks were found

walking on the outside walls of the owner’s home. In

one hunting dog, the first clinical signs of the disease

were observed a few weeks after a hunt on a field

infested with ticks. A few to numerous ticks were

observed on the coat of the affected dogs. The ixodid

ticks Rhipicephalus sanguineus (the ‘‘brown dog

tick’’) and Amblyomma aureolatum (the ‘‘yellow dog

tick’’) were consistently found infesting dogs from

suburban areas and rural areas, respectively (Fig. 1).

The same species of ticks were also recovered from

the environment where these dogs used to roam.

The clinical picture in the naturally infected dogs

was characterized by marked pallor of oral and

conjunctival mucous membranes (anaemia), or yellow

discoloration of mucosae, abdominal skin and inner

surface of the pinnae (jaundice), dehydration, depres-

sion, undulating fever, chronic weight loss, weakness,

lymphadenomegaly and splenomegaly. There was

widespread petechiation of the oral and vaginal

mucosae, bleeding from the nose (epistaxis) and oral

cavity, haematemesis, and pasty, dark, blood-stained

feces or bloody, watery diarrhea with matted hairs on

the perineum. Persistent or intermittent bleeding from

the skin of the tips, margins and outer aspect of both

pinnae was also observed. Multiple areas of coagu-

lated blood formed on the outer surface and margins of

the pinnae (Fig. 2). Blood oozed profusely from

venipuncture sites.

Laboratory findings included severe regenerative

anaemia, spherocytosis, icteric plasma and bilirubi-

nuria. Peripheral blood smears examined for the

unclassified protozoan parasite being characterized in

this study were consistently negative, and no other

blood parasites or rickettsial agents were observed

within erythrocytes or leukocytes.

Eight animals died spontaneously. Of those, three

were jaundiced and died acutely approximately 1

week after the first clinical signs were observed.

Therapy with doxycycline was started at a late stage of

the disease in one of the icteric dogs and was

unsuccessful. One animal that died after a protracted

clinical course of 2–3 months had anaemia. One

animal recovered approximately 48 h after therapy

with imidocarb dipropionate and blood transfusion,


Fig. 2. Dog, natural case. Massive bleeding from the skin covering the outer surface of the pinnae.

and another one was cured after treatment with

doxycycline and glucocorticoid therapy. Three ani-

mals died 24–48 h after therapy with diminazene

aceturate.

3.2. Pathology

3.2.1. Gross lesions, cytology and histopathology

Necropsy findings in eight natural cases included

diffuse pallor or yellowish discoloration of the carcass

and internal organs, two to threefold increase in the

size of the spleen (splenomegaly), generalized

enlargement of the peripheral and visceral lymph

nodes, large amounts of coagulated blood in the lumen

of the gastrointestinal tract, especially in the intestines

(enterorrhagia), enlarged and reddened tonsils, and

vivid red, pasty bone marrow. On cut surface, the

lymph nodes were wet (edematous), discolored red or

dark brown, and had multifocal to coalescing white

foci (Fig. 3). The cut surface of the spleen had

prominent lymphoid follicles embedded in a bulging

and dark red pulp. The liver was moderately enlarged,

diffusely yellow or orange, had rounded edges and an

accentuated lobular pattern. The gallbladder was

markedly distended with thick, inspissated, dark green

bile. The lungs were diffusely red, wet, heavy, and

failed to collapse. There was a moderate amount of

white foam in the lumen of the trachea and bronchi.

The kidneys were diffusely pale. Mild to moderate

hydrothorax, hydropericardium and ascites were

observed. There was also yellow, diffuse subcutaneous

edema of both hindlimbs, and interlobular pancreatic

edema. Multiple pinpoint hemorrhages were observed

on the serosa of many organs and tissues.

Histologically, zoites were found in the endothelial

cells of blood capillaries from many organs (Figs. 4

and 5). The intracytoplasmic organisms were round,

homogeneous and basophilic when stained with HE.

The cytoplasm of these protozoan parasites was pale

and inconspicuous and the nucleus was prominent,

basophilic and eccentrically located (Fig. 4, inset).

Individual organisms measured 2.5 mm. These para-

sites were PAS-negative and were most numerous in

sections of peripheral lymph nodes, bone marrow,

kidneys, and choroid plexus; 20–30 organisms were

found within the cytoplasm of each capillary


Fig. 3. Lymph nodes, dog, natural case. Enlarged and swollen (edematous) lymph nodewith multifocal to coalescent white foci (arrowheads) on

cut surface that correspond microscopically to hyperplastic (reactive) lymphoid nodules. Bar = 0.6 cm.

Fig. 4. Lymph node, dog, natural case. Numerous organisms are seen in the cytoplasm of endothelial cells of blood capillaries. HE stain.

Bar = 74 mm. The inset shows a higher magnification of the zoites within an endothelial cell. HE stain. Bar = 17 mm.


Fig. 5. Lymph node, dog, natural case. Zoites (arrows) are seen within the cytoplasm of endothelial cells of a blood capillary. The medullary

cords are infiltrated by numerous plasma cells. Resin. Methylene blue. Bar = 16 mm.

endothelial cell. The parasitized endothelial cells were

markedly enlarged (up to 30 mm) due to the presence

of large numbers of intracytoplasmic organisms.

These swollen endothelial cells protruded into the

lumena of the capillaries. Few parasites were found in

the tissues of the dog that died spontaneously despite

treatment with doxycycline. These organisms were not

found in the endothelium of arteries, arterioles,

venules or veins. The follicles of the lymph nodes

and spleen were hyperplastic with prominent germinal

centers (follicular reactive hyperplasia). Marked

erythrophagocytosis was observed in the medullary

sinuses of the lymph nodes (Fig. 6), and the medullary

cords of the lymph nodes were populated by numerous

plasma cells (Fig. 5). Langhans-type multinucleated

giant cells were seen in the lymph nodes and choroid

plexus, especially around the capillaries (Fig. 7).

Scattered mononuclear infiltrates were seen in the

kidneys, especially around the glomeruli and asso-

ciated with the presence of R. vitalli in capillary

endothelial cells of the interstitium. Besides the

occurrence of multinucleate giant cells and the

presence of the intraendothelial protozoa, mild to

moderate lymphoplasmacytic inflammation was

observed in the stroma of connective tissue of the

choroid plexus. The bone marrow was hypercellular

and had many hemosiderin-laden macrophages. There

was extramedullary hematopoiesis in the liver and

spleen. The hepatic sinusoids were filled with

numerous nucleated round cells resembling metaru-

bricytes. Microthrombi were observed inside the

lumena of arterioles, capillaries and venules. There

was ischemic centrilobular hepatocellular coagulative

necrosis, diffuse fatty change of the hepatocytes, and

canalicular cholestasis. In one case, there was fibrinoid

necrosis of the lymphoid follicles of the splenic white

pulp.

No zoites were found in smears of bone marrow,

lymph node, spleen, liver, kidney, choroid plexus and

blood. None of these organisms were found histolo-

gically in the nasal cavity and skin of the tips of the

pinnae. Immunofluorescence (FAT) for Leptospira

spp. in kidney samples collected at necropsy from one

jaundiced dog was negative. Gross and histological

findings typical of diamidine poisoning, i.e. sym-

metric bilateral hemorrhagic encephalomalacia affect-

ing the brainstem were observed in the three animals

treated with diminazene aceturate. In these cases, the

history, clinical picture, gross findings and histological

lesions in multiple organs and tissues were consistent


Fig. 6. Lymph node, dog, natural case. Erythrophagocytosis by macrophages is observed in the sinuses of the lymph nodes. HE stain.

Bar = 90 mm. Inset: Higher magnification of a macrophage that has phagocytized several red blood cells. HE stain. Bar = 17 mm.

Fig. 7. Choroid plexus, lateral ventricle, dog, natural case. Intraendothelial zoites (arrowheads), moderate lymphoplasmacytic inflammation

and a Langhans-type multinucleated giant cell (arrow) are found in the stroma of connective tissue of the choroid plexus. HE stain.

Bar = 120 mm. Inset: Lymph node, dog, natural case. A multinucleated giant cell is seen around a blood capillary. Resin. Methylene blue.

Bar = 30 mm.


with the protozoal infection reported in the present

study, but no zoites were observed.

3.2.2. Immunohistochemistry and in situ

hybridization

The protozoan parasite described in this study

reacted positively with an anti-Babesia microti anti-

body in an immunohistochemical assay. Immunos-

taining for Leishmania chagasi, Neospora caninum

and Toxoplasma gondii was consistently negative.

Immunohistochemically, the endothelial cells of the

blood capillaries parasitized by R. vitalli and also

those from the other blood vessels not parasitized

stained positive for vWF. R. vitalli was positive by in

situ hybridization for B. microti (Fig. 8).

3.2.3. Experimental disease

A male, mixed-breed, 1-year-old dog was inocu-

lated intravenously with blood from a parasitized dog

referred to the veterinary teaching hospital for

consultation. The first clinical signs in the experi-

mental dog were observed 5 days after the inoculation,

and included fever, mucosal pallor and blood oozing

from the nose. Examination of bone marrow smears

donewith samples surgically removed on day 17 of the

Fig. 8. Adrenal medulla, dog, natural case. This unclassified piroplasm of

Bar = 60 mm.

experiment and during necropsy revealed numerous

zoites in the cytoplasm of endothelial cells (Fig. 9).

These intracellular organisms were round or pear-

shaped and had a weakly stained blue cytoplasm and a

pinpoint, eccentric basophilic nucleus. Both unin-

ucleate and binucleate zoites were present. Binucleate

organisms were interpreted as being the result of

schizogony. These parasites were also found in

histological sections of peripheral lymph nodes and

bone marrow in samples collected antemortem and

postmortem as well. No gross changes were observed

in these two biopsied tissues. The experimental animal

died unexpectedly 19 days after the onset of the

clinical signs despite emergency treatment for shock.

The clinical and pathological findings of the experi-

mental case were similar to those observed in the

natural cases of ‘‘nambiuvu’’ reported here including

mucous membrane pallor, fever, epistaxis, splenome-

galy, pale kidneys and the presence of the organisms in

the endothelial cells of blood capillaries from multiple

organs and tissues. The causal agent of this atypical

protozoal disease of brazilian dogs was positive in the

immunohistochemistry (Fig. 10) and in the in situ

hybridization for B. microti, and parasitized and non-

parasitized endothelial cells were positive immuno-

brazilian dogs stains positive by in situ hybridization for B. microti.


Fig. 9. Bone marrow, dog, experimental case. Smear. Many zoites are found in the cytoplasm of an endothelial cell. Panoptic.

Bar = 4.5 mm.

Fig. 10. Lymph node, dog, experimental case. This unclassified piroplasm is positive on immunohistochemistry for Babesia microti.

Bar = 120 mm.


Fig. 11. Lymph node, dog, experimental case. By immunohistochemistry, endothelial cells of blood capillaries parasitized by this unnamed

piroplasm, and those from other blood vessels not parasitized, stain positive for von Willebrand factor (vWF). Bar = 90 mm.

histochemically for vWF (Fig. 11), as observed in the

natural cases of the disease. No pathogens were found

in antemortem blood films and smears of lymph node,

and in postmortem blood films and smears of lymph

node, spleen, liver, kidney and choroid plexus.

Bleeding from the ears, mouth or gastrointestinal

tract was not observed, and this protozoan parasite was

not seen histologically in the nasal cavity.

3.2.4. Ultrastructural findings

Transmission electron microscopy studies showed

that zoites were situated within a single parasitophor-

ous vacuole (PV) in the cytoplasm of the endothelial

cells, i.e. tissue stage or intraendothelial form (Figs. 12

and 13). These organisms were round or oval, had a

polarized electron-dense nucleus, and were sur-

rounded by a trilaminar boundary pellicle which

was composed of two electron-dense laminae sepa-

rated by an electron-lucent one (Fig. 13). This

protozoan parasite had an apical complex consisting

of a polar ring, electron-dense rhoptries, ductules of

rhoptries and dense granules. No conoid was

observed. The nucleus consisted of a single, eccentric,

electron-dense, double-layered, round to oval struc-

ture situated near the posterior end of the parasite. The

ground substance of the electron-lucent cytoplasm

was filled with electron-dense granules and profiles of

mitochondria-like structures (Fig. 13). Large, elec-

tron-dense, crystalline inclusions were observed in the

cytoplasm of those organisms situated in the

endothelium of interstitial capillaries of the kidney

(Fig. 12). Some of these inclusions were pointed at one

end and rounded at the opposite end while others were

polygonal. A single, large, round, membrane-bound

bud protruded from the edge of one of those organisms

seen in the kidney. The PV membrane consisted of a

trilaminar structure, which had one central electron-

lucent layer and two peripheral, distinct electron-

dense layers. The PV membrane measured 30 nm.

Intravacuolar tubules and microvillus-like structures,

i.e. villar protrusions extended from the inner layer of

the PV and were parallel to each other. Cross sections

of these microvillus-like projections had electron-

lucent cores. These finger-like projections measured

40–50 nm in diameter and 0.5 mm in length (Fig. 14).

A thin layer of cytoplasm from the host cell bordered

the external layer of the PV (Fig. 12). In some tissues,

two adjoining endothelial cells shared the same PV.


Fig. 12. Electron micrograph. Kidney, dog. Experimental case. Zoites are situated within a parasitophorous vacuole in the cytoplasm of an

endothelial cell of a renal interstitial blood capillary. A thin layer of cytoplasm from the host cell surrounds the external layer of the

parasitophorous vacuole. Large, electron-dense, crystalline inclusions are observed in the cytoplasm of these protozoan parasites. Some of these

intracytoplasmic inclusions are pointed at one end and rounded at the opposite end while others are polygonal. Bar = 3.7 mm. The inset depicts

organisms within a parasitophorous vacuole in the cytoplasm of an endothelial cell of a glomerular capillary. Bar = 2.6 mm.

Thick fibrin strands and clumpedmasses of fibrin were

trapped in the lumena of some blood vessels. The

presence of free zoites in the lumena of the blood

capillaries was attributed to sampling artifact. These

organisms were not found inside erythrocytes.

4. Discussion

Based on our ultrastructural, immunohistochemical

and in situ hybridization findings, we suggest that the

organism partly characterized in this study is a

protozoan parasite of the phylum Apicomplexa, order

Piroplasmorida. We will have to call it an unclassified

piroplasm until a proper name can be proposed for this

parasite based on additional molecular studies.

Interestingly enough, the idea that the organism

studied here was a new, yet to be classified piroplasm,

was originally considered approximately 1 century

ago, as early as 1910, when the wide array of

techniques currently used for the classification of

parasites were not available (Pestana, 1910a, 1910b).

This unnamed piroplasm seems to be different from

Babesia since it has an intraendothelial stage. This is

important from a practical perspective since protozoal

and infectious diseases are diagnosed routinely by

veterinary pathologists and parasitologists based on

the morphology of the organism under the light

microscope, and location of the pathogen in the

tissues, i.e. cell type affected by the organism.

R. vitalli has been referred to in the international

literature as Babesia vitalli of the brazilian dog

(Wenyon, 1926), and considered as a synonym for B.

canis (Levine, 1973; Peirce, 2000). It is postulated by

some investigators that the intracellular form of

Rangelia found inside the endothelial cells is possibly

Toxoplasma, and that the tissue stage of Rangelia has

no connection to the blood stage which, according to

them, is in reality B. canis (Wenyon, 1926; Moreira,

1938; Paraense and Vianna, 1948). It is stated in one


Fig. 13. Electron micrograph. Lymph node, dog. Experimental case. A single zoite is shown. Rhoptries (arrows), apical polar ring (arrowhead),

nucleus (N), outer trilaminar plasma membrane (pm), and lumen (pv) and wall (w) of the parasitophorous vacuole. Bar = 0.5 mm.

publication that the masses of R. vitalli merozoites

described originally as being within the cytoplasm of

the endothelial cells were, in fact, agglomerates of

these organisms accumulated inside the lumena of the

capillary blood vessels (Levine, 1973). The author of

another publication argues that probably the masses of

intracytoplasmic protozoan parasites described as

being within the endothelial cells merely represent

phagocytized organisms circulating in the blood

(Wenyon, 1926). Although this organism was not

completely characterized in this study, we demon-

strated that the fine structure of this unnamed

protozoan parasite and parasitophorous vacuole are

similar to those of other apicomplexan protozoa

including the piroplasms (Buttner, 1968; Aikawa and

Sterling, 1974; Scholtyseck, 1979; Mehlhorn and

Schein, 1984; Cheville, 1994). This unclassified

parasite shares the following morphological and

biological features with other apicomplexan protozoa

including the piroplasms: (1) an apical complex

reduced to a polar ring and rhoptries, and that does not

have a conoid, (2) a trilaminar PV membrane with

villar protrusions and (3) asexual reproduction by

schizogony (Levine, 1973; Mehlhorn and Schein,

1984; Mehlhorn, 2001). B. canis (Buttner, 1968;

Mehlhorn and Schein, 1984), Theileria parva (Mehl-

horn and Schein, 1984) and C. felis (Kier et al., 1987;

Simpson et al., 1985) are examples of piroplasms that

can be compared to this unnamed protozoan parasite

in terms of structure and biology. Our transmission

electron microscopy and immunohistochemistry also

showed that this unclassified piroplasm is situated

within the cytoplasm of the endothelial cells and not in

macrophages as previously stated by other authors

(Wenyon, 1926; Moreira, 1938; Paraense and Vianna,

1948; Levine, 1973; Pocai et al., 1998), and that this

organism is not Toxoplasma gondii or Leishmania spp.

as mentioned in previous publications (Wenyon, 1926;

Moreira, 1938; Pocai et al., 1998). Other apicomplex-

ans that, as the unnamed protozoan parasite studied

here, have been observed in the cytoplasm of

endothelial cells include Sarcocystis spp. (Corner

et al., 1963; Lane et al., 1998), Haemoproteus

columbae (Levine, 1973), Plasmodium gallinaceum

(Levine, 1973), Karyolysus sp. (Barta, 2000), Hemo-

livia stellata (Barta, 2000) and Hepatozoon boigae


Fig. 14. Electron micrograph. Lymph node, dog. Natural case. The parasitophorous vacuole situated in the cytoplasm of an endothelial cell is

limited by a trilaminar structure (arrowheads), which has one central electron-lucent layer and two peripheral, distinct electron-dense layers.

Intravacuolar microvillus-like structures (villar protusions) with electron-lucent cores (arrows) arise from the internal vacuolar membrane.

Formalin-fixed tissue. Bar = 1 mm.

(Jakes et al., 2003). This unclassified piroplasm was

positive in the immunohistochemistry for B. microti

and also positive in the in situ hybridization for B.

microti. The positive results in these two tests provide

additional evidence that this organism belongs to the

same group of Babesia, Theileria and Cytauxzoon.

Cross-reactivity of the polyclonal anti-B. microti

antibody with other Babesia species, e.g. Babesia

WA-1 strain has been documented. However, this

antibody does not cross react with B. bigemina of

cattle and also does not cross react with Plasmodium

spp. (P. vivax, P. falciparum, P. knowlesi) of non-

human primates. Reactivity of this antibody against

other Apicomplexa that infect cells of different organs

and tissues has not been tested (Torres-Velez et al.,

2003). The riboprobe developed for the in situ

hybridization method for B. microti (Sledge et al.,

2004) that was used in this study has a high degree of

homology with other piroplasms, i.e. C. felis (96% of

homology), B. rodhaini, T. equi (B. equi), B. felis, and

B. leo (above 95% of homology each) (F.J. Torres-

Velez, 2004, unpublished data).

In the present study, intraerythrocytic forms of R.

vitalli were not found in blood smears, impression

smears of organs, histological sections, or under the

transmission electron microscope. Similarly, a study

based on light microscopy describes the presence of

this piroplasm within endothelial cells but not inside

red blood cells (Krauspenhar et al., 2003). These

results do not match with most of the earlier research

on this protozoan parasite in which the organism was

found within erythrocytes and also within endothelial

cells (Pestana, 1910a, 1910b; Carini andMaciel, 1914;

Braga, 1935; Carini, 1948; Rezende, 1976). Red blood

cells parasitized by this unclassified piroplasm are

considered as uncommon to rare findings in blood

smears (Pestana, 1910a, 1910b; Carini and Maciel,

1914) and this would explain our negative findings in

the blood. Similarly, some cases of B. gibsoni are also

difficult to diagnose since only small numbers of


parasites are present in peripheral blood smears (low-

level parasitemia) (Inokuma et al., 2005), and a single

negative set of blood smears does not rule out a

Babesia infection (Setty et al., 2003).

Babesia spp. are intraerythrocytic protozoan para-

sites of domestic animals and humans (Mehlhorn,

2001). In human beings, extraerythrocytic forms of

Babesia, described as large extracellular aggregates of

trophozoites, have been found occasionally in the

circulating blood (Setty et al., 2003). According to the

literature, extracellular forms of the unclassified

canine piroplasm described here can sometimes be

seen on blood smears as individual organisms or as

aggregates of parasites (Pestana, 1910a, 1910b; Carini

and Maciel, 1914; Rezende, 1976). It could hypothe-

sized that these organisms would be released into the

bloodstream after the rupture of a parasitized

endothelial host cell. It has been speculated that some

species of Babesia, e.g. B. microti might have an

exoerythrocytic stage similar to those seen in Theileria

that replicates inside red blood cells and lymphocytes

as well (Setty et al., 2003) but this has never been

rigorously proved. Babesia has not been identified as

occurring elsewhere in the body or invading cell types

other than erythrocytes (Mehlhorn, 2001) as described

in cases of ‘‘nambiuvu’’ of brazilian dogs in which this

unclassified piroplasm can infect endothelial cells and

also erythrocytes (Pestana, 1910a, 1910b; Carini and

Maciel, 1914; Braga, 1935; Carini, 1948; Rezende,

1976). The results of our study suggest that this

unnamed protozoan parasite has not only a tissue stage

but also a blood stage as described by other

investigators, since the disease was successfully

reproduced through intravenous inoculation of blood

from a naturally infected dog into an experimental

dog. In our cases, the finding of these organisms free

within the lumena of capillaries under the electron

microscope only was considered artifactual. Piro-

plasms are typically intracellular parasites, so

probably this finding is due to cellular damage

during tissue handling. Similar events have been

observed in severe cases of babesiosis and malaria, i.e.

red cells infected with mature forms of these

hemoprotozoan parasites can be very fragile and

rupture during preparation of blood films, giving the

wrong impression that mature organisms are extra-

cellular (http://www.path.cam.ac.uk/�schisto/Parasi-

tology_Practicals/Protozoology__Pract.html).

The tentative clinical diagnosis of the disease in

dogs caused by this unclassified piroplasm was based

on the history, clinical picture, haemogram and

favorable response to therapy. The diagnosis was

confirmed through necropsy, cytology and histo-

pathology. Infection with this unnamed protozoa

should be differentiated from other diseases com-

monly observed in dogs that cause fever, anaemia,

jaundice and bleeding tendencies. In Brazil, these

include (1) the acute form of leptospirosis caused by L.

interrogans serovar icterohaemorrhagiae, (2) babe-

siosis (B. canis), and (3) ehrlichiosis (E. canis). In our

cases, these three diseases were ruled out through

examination of blood smears, immunofluorescence

and histopathology.

This unclassified piroplasm of dogs causes a unique

disease that, to the best of our knowledge, has been

described only in Brazil (Carini, 1908, 1948; Pestana,

1910a, 1910b; Carini and Maciel, 1914; Braga, 1935;

Rezende, 1976; Krauspenhar et al., 2003). Some

analogies can be drawn between the infection with this

organism that affects dogs in Brazil and the infection

with a virulent strain of the flagellate protozoan

Trypanosoma vivax that affects cattle in Kenya and

causes widespread, severe hemorrhage (Gardiner

et al., 1989). The acute hemorrhagic disease in cattle

caused by this particular strain of T. vivax is

characterized by bleeding from external orifices,

including the nose and ears, pallor and petechiation

of the mucous membranes, high fever, autoimmune

anaemia, generalized hemorrhages, which are more

severe in the gastrointestinal tract, enlarged and

reactive lymph nodes, marked splenomegaly, and

interstitial mononuclear inflammatory infiltrates, most

frequently seen in the heart, kidneys and choroid

plexus (Gardiner et al., 1989). Erythrophagocytosis is

found in bonemarrow impressions (Connor, 1994) and

histological sections of the lymph nodes and spleen of

cattle infected by this strain of T. vivax (Gardiner et al.,

1989). Similar findings have been observed in cases of

this atypical protozoal infection of dogs (Pestana,

1910a, 1910b; Carini and Maciel, 1914; Braga, 1935;

Carini, 1948; Rezende, 1976; Krauspenhar et al.,

2003).

In this study, evidence for protozoan-induced

immune-mediated haemolytic anaemia included

severe regenerative anaemia, spherocytosis and

erythrophagocytosis which are the hallmarks of

http://en.wikipedia.org/wiki/Antivenin




immune-mediated (autoimmune) haemolytic anaemia

(Feldman et al., 2000; McCullough, 2003). Similar

findings and pathogenesis have been reported by other

authors in cases of infection with this unusual

protozoal parasite (Krauspenhar et al., 2003). Favor-

able response to corticosteroids in one of the cases

described here strengthens the hypothesis of IMHA.

The clinical picture observed in the infection with this

unnamed canine piroplasm reflects the autoimmune

hemolytic anaemia, i.e. pale or yellow mucous

membranes, weakness, lethargy and anorexia, and

the underlying immune-mediated destruction of red

blood cells, i.e. lymphadenopathy, splenomegaly and

pyrexia. Marked erythrophagocytosis in the lymph

nodes and prominent stores of hemosiderin within

phagocytic cells (hemosiderophages) in the bone

marrow are morphological findings consistent with

excessive erythrocyte breakdown in cases of IMHA

(Wellman and Radin, 1999; Feldman et al., 2000) as

observed here. Extramedullary hemopoiesis in the

liver and spleen and the presence of nucleated

erythrocytes (metarubricytes) in the circulation are

part of a regenerative response to chronic anaemia

(Valli contrib. Parry, 1993) as seen in our cases. In

these cases, other possible causes of IMHAwere ruled

out through history analysis, clinical examination,

hematology, necropsy and histopathology. IMHA,

although uncommon, has been described in canine

babesiosis (Wozniak et al., 1997; Inokuma et al.,

2005).

The pathogenesis of the characteristic clinical sign

of ear bleeding (‘‘nambiuvu’’) seen in cases of dogs

infected with this unclassified piroplasm is unclear. To

the authors’ knowledge, this intriguing clinical sign

has not been documented in other diseases of dogs.

Aural hematoma has been reported by some authors as

a clinical sign typically seen in dogs affected by E.

canis in endemic areas (Beugnet et al., 2002), but

hematoma of the ear is distinct from the ear bleeding

observed in cases of infection by this protozoan

parasite. Disseminated intravascular coagulation

(DIC) secondary to endothelial damage caused by

the replication of this organism inside host cells

should be considered here as one of the putative

pathogenetic mechanisms involved in the develop-

ment of hemorrhages in this protozoal disease of

Brazilian dogs. Dogs with IMHA can also develop

DIC (McCullough, 2003). Activation of the coagula-

tion cascade leading to the accumulation of fibrin

degradation products suggests that DIC may be an

important component of the terminal stages of the

disease. The presence of microthrombi in the lumena

of small blood vessels observed under the light

microscope that corresponds in the transmission

electron microscope to strands and clumps of fibrin

is consistent with this theory. DIC has also been

documented in feline cytauxzoonosis (Garner et al.,

1996). Thrombocytopenia, although rarely observed

in cases of infection with this unnamed organism

(Krauspenhar et al., 2003), and a protozoan-associated

blood clotting defect not yet characterized might also

be involved in the pathogenesis of these hemorrhages.

Trauma inflicted by blood-feeding flies, e.g. stable

flies (Stomoxys calcitrans) feeding on the tips of the

ears, and the vigorous scratching and compulsive head

shaking to alleviate the pain and irritation on these

areas may also contribute to the occurrence of frank

hemorrhage from the pinnae of dogs affected by this

protozoan-induced coagulopathy.

Infection with this unclassified piroplasm is

suspected to be transmitted by ticks that commonly

affect dogs in Brazil (Pestana, 1910a, 1910b; Carini

and Maciel, 1914; Braga, 1935; Carini, 1948). In the

present study, the ixodid ticks R. sanguineus and A.

aureolatum were consistently found infesting affected

animals, especially during the hot season. Interest-

ingly enough, in southern Brazil, in rural areas, adult

specimens of A. aureolatum have been found infesting

domestic dogs, while immature stages of this tick have

been recovered from wild carnivores including crab-

eating foxes (Cerdocyon thous – popular name:

‘‘graxaim-do-mato’’ – and Pseudalopex gymnocercus

– ‘‘graxaim-do-campo’’), the crab-eating raccoon

(Procyon cancrivorus – ‘‘guaxinim’’) and the opos-

sum (Didelphis albiventris), and also several families

of Passeriformes birds (Evans et al., 2000; Muller

et al., 2005). We speculate that a wild animal or a bird

could be a reservoir of this pathogen in rural areas,

while in suburban areas, R. sanguineus would be the

vector and also the reservoir of this apicomplexan

protozoa. Our hypotheses are based only on field

observations. Data to support the assumptions that this

organism is transmitted by ticks, and that this protozoal

infection is maintained in suburban areas by ticks, and

in rural areas in a cycle between ticks and native wild

vertebrates have not been published to date.


Currently, there is not an efficient laboratory test to

confirm suspected cases of infection with this

unclassified piroplasm in live, sick dogs. In general,

clinically affected animals do not have organisms

visible in blood smears since levels of parasitemia are

usually low. Therefore, these protozoa are difficult or

impossible to find, especially in chronic infections

(Pestana, 1910a, 1910b; Carini andMaciel, 1914). In a

case report of B. gibsoni infection in a dog, for

example, no parasites were found in blood smears of

the symptomatic animal, and the diagnosis of canine

babesiosis was based on molecular techniques only,

i.e. seminested PCR (Criado-Fornelio et al., 2003).

The same molecular approach can be pursued in the

future for the diagnosis of infection by this piroplasm

which identity remains to be determined. Currently, a

definitive diagnosis of infection with this unnamed

protozoa can be achieved only through microscopic

examination of bone marrow smears done at necropsy

or histological sections of multiple organs and tissues,

especially the lymph nodes where this organism is

most frequently found. Transfusion-associated infec-

tion by this unclassified piroplasm has also to be

considered here as a potential risk since screening of

potential blood donors is highly problematic because

this organism is usually not detected in blood smears.

This diagnostic problem may also impact dog

husbandry and intercountry transfer and importation

of dogs, if infected but clinically normal animals are

transported to regions with a suitable environment and

adequate vectors and reservoirs for the disease to

occur. Strict quarantine control measures and thor-

ough health examination are suggested here in order to

prevent the entrance of this protozoal parasite in other

countries apparently free of this pathogen.

Molecular methods are invaluable tools in the

classification and phylogenetic analysis of parasites,

i.e. molecular phylogeny. With the advent of PCR as

one of the most important innovations in molecular

biology over the last 20 years, now morphological

features only are not sufficient to fully characterize an

unidentified organism as in this case. Phylogenetic

studies based on other molecular methods such as PCR

amplification and DNA sequencing are necessary to

better characterize the unnamed apicomplexan pro-

tozoa studied here, clarify its taxonomic status and

assign a specific identity to it. The assignment of a

unique taxon for this parasite as a new piroplasm is

clearly tentative at this time in the absence of

molecular characterization. It is worthwhile mention-

ing that serious conflicts between molecular

approaches and more classical approaches, such as

those based on morphology or life cycle, have been

reported by other investigators when classifying

unknown species of protistan parasites. Combination

of the molecular characters to existing morphological

and biological characters should be attempted where

possible. Agreement of both morphological and

molecular features would provide increased confi-

dence and reliability to these studies (Barta, 2001).

Acknowledgements

We thank Irene Breitsameter, Andre Correa, David

Driemeier, Luciana de Oliveira, Caroline Pescador,

Marcia Regina Ilha, Murray Hazlett, Susan Lapos and

Sofie Tatarski for their technical help, Jeff Caswell and

Tony van Dreumel for their suggestions and correction

of the manuscript, and John Barta and M. Agnes

Fernando for reviewing the electron micrographies.

Alexandre Paulino Loretti is sponsored by Coorde-

nacao de Aperfeicoamento de Pessoal de Nıvel

Superior (CAPES), Brazil.

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