Piroplasma la Necunoscuta Brazilia
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Transcript of 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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213196
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 197
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213198
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,
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 199
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213200
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 201
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213202
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 203
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213204
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 205
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213206
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 207
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213208
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 209
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
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213210
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.
A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193–213 211
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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