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Experimental Parasitology 128 (2011) 347–352
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Experimental Parasitology
journal homepage: www.elsevier .com/locate /yexpr
Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia,
biological cycle, clinical-pathological aspects and treatment
Aleksandro S. Da Silva a,⇑, Raqueli T. França b, Marcio M. Costa b, Carlos B. Paim b, Francine C. Paim b,
Guilherme L. Dornelles b, João F. Soares c, Marcelo B. Labruna c, Cinthia M. Mazzanti b, Silvia G. Monteiro a,
Sonia T.A. Lopes b
a Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Brazil
b Department of Small Animals, Universidade Federal de Santa Maria, Brazil
c Department of Preventive Veterinary Medicine and Animal Health, Universidade de São Paulo, Brazil
a r t i c l e i n f o
Article history:
Received 23 March 2011
Received in revised form 16 April 2011
Accepted 28 April 2011
Available online 6 May 2011
Keywords:
Rangeliosis
Canine
Biological cycle
Treatment
0014-4894/$ - see front matter � 2011 Elsevier Inc. A
doi:10.1016/j.exppara.2011.04.010
⇑ Corresponding author. Address: Departamento de
da UFSM. Faixa de Camobi – Km 9, Campus Univer
97105-900 Santa Maria – RS, Brazil. Fax: +55 55 3220
E-mail address: aleksandro_ss@yahoo.com.br (A.S.
a b s t r a c t
Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathoge-
nicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to
natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clin-
ical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and
also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12
young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected
(n = 5), and Group B consisted of animals infected with R. vitalii (n = 7). After infection, the animals were
monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days
post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of cir-
culating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan
was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at
the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit
of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evalu-
ation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and
hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen
and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed
the treatment of dogs in Group B (n = 6) with a single dose of diminazene aceturate, which showed a
curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs.
� 2011 Elsevier Inc. All rights reserved.
1. Introduction
Rangelia vitalli is a member of the protozoan phylum Apicom-
plexa, order Piroplasmorida (Loretti et al., 2003; Spagnol et al.,
2003). It causes a tick-borne disease referred as ‘‘nambiuvú’’
(=blood dribbling down from the ear margins) or ‘‘peste de sangue’’
(=bleeding plague) (Krauspenhar et al., 2003; Loretti and Barros,
2005; França et al., 2010). It is a disease that commonly affects
dogs from rural and suburban areas in southern Brazil, possibly
being transmitted by the ticks Rhipicephalus sanguineus and
Amblyomma aureolatum (Loretti and Barros, 2005). Over the years,
this malady has been associated with an unclassified organism
which occurs within endothelial cells, leukocytes and erythrocytes.
ll rights reserved.
Microbiologia e Parasitologia
sitário, Prédio 20, Sala 4232,
8958.
Da Silva).
A recent genetic study showed that R. vitalii is phylogenetically
close-related to species of the Babesia spp. sensu stricto group of
piroplasmas (Soares et al., 2011).
R. vitalii primarily affects young dogs (Pestana, 1910a); how-
ever, adult dogs can also be affected less frequently (Carini,
1948). The disease is characterized by anemia, jaundice, fever,
splenomegaly, lymphadenopathy, hemorrhage along the gastroin-
testinal tract, and persistent bleeding through the tips of the pin-
nae, external surface of the ears, nose, and oral cavity (Pestana,
1910a,b; Loretti and Barros, 2005; França et al., 2010; Fighera
et al., 2010). One study on the pathogenesis, clinical aspects, hema-
tological and pathological findings of 35 dogs naturally infected
with R. vitalii showed that it is a hemolytic disease, exclusively ex-
tra-vascular with immune-mediated origin, and with typical clini-
cal signs that include regenerative anemia, jaundice, splenomegaly,
and lymphoid hyperplasia in multiple organs (Fighera et al., 2010).
Severe anemia and thrombocytopenia were also reported in seven
cases of natural infection (França et al., 2010).
http://dx.doi.org/10.1016/j.exppara.2011.04.010
mailto:aleksandro_ss@yahoo.com.br
http://dx.doi.org/10.1016/j.exppara.2011.04.010
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348 A.S. Da Silva et al. / Experimental Parasitology 128 (2011) 347–352
Currently, the diagnosis of R. vitalii relies on typical clinical
signs (Loretti and Barros, 2005; França et al., 2010), observation
of parasites in peripheral blood smear (França et al., 2010), histopa-
thology (Fighera et al., 2010), and molecular identification of the
parasite (Soares et al., 2011). The favorable response to treatment
is also considered by veterinarians a form of diagnosis (Loretti
and Barros, 2004). Therapy with corticosteroids, doxycycline, and
dipropionate imidocarb in dogs naturally infected with R. vitalii
had a curative efficacy of 85.7% (6/7 dogs – França et al., 2010).
As mentioned by other researchers (Loretti and Barros, 2005;
Fighera et al., 2010; França et al., 2010), an experimental study
on the pathogenesis of R. vitalii infection in dogs is urgently
needed. In this regard, the present study aimed to evaluate the par-
asitemia, biological cycle, and clinical-pathological findings in dogs
experimentally infected with R. vitalii in the acute phase of disease,
and also aimed to test a therapeutic protocol based on the dimina-
zene aceturate.
2. Material and methods
2.1. Animals
Twelve female mongrel dogs between 6 and 12 months old,
weighing between 4 and 7 kg, were used. The animals were kept
in cages and placed in an experimental room with controlled tem-
perature and humidity (25 �C and 80% RH), and had free access to
water and commercial ration chow throughout the experiment. All
animals were treated with anti-helminthic, vaccinated, and were
submitted to a period of 30 days for adaptation. Complete hemo-
gram, hepatic and renal biochemical panel were performed three
times at 15 day intervals. After 30 days (day 0 of the experiment),
the evaluated patterns showed normal values according to canine
reference values (Feldman et al., 2000). Before starting the experi-
ment, all animals were shown to be free of Babesia spp., R. vitalii,
Hepatozoon spp., and Ehrlichia spp. infection by testing their blood
by a battery of PCR protocols as described elsewhere
(Criado-Fornelio et al. 2006; Labruna et al. 2007; Spolidorio et al.
2009; Soares et al., 2011).
The present study has been approved by the Animal Welfare
Committee of the Universidade Federal de Santa Maria (UFSM),
protocol number 15/2010, in accordance to the Brazilian laws
and ethical principles published by the Colégio Brasileiro de Exper-
imentação Animal (COBEA).
Fig. 1. Kinetics of parasitemia in seven dogs infected with R. vitalii. We quantified forms o
the animals infected with R. vitalii were treated with diminazene aceturate.
2.2. Rangeliavitalii isolate
The R. vitalii strain used in this study was obtained from a nat-
urally infected dog in Santa Maria, state of Rio Grande do Sul
(Soares et al., 2011). A fresh blood sample of this animal was inoc-
ulated (2 mL trough the jugular vein) in another dog (Dog 13: male,
five months old) for maintenance of the isolate in the laboratory.
This procedure was used because information about the biological
cycle of this parasite are lacking, and successful use of other meth-
ods of maintaining this parasite in the laboratory remains
unknown.
2.3. Experimental design
The animals were divided into two groups: Group A consisted of
five healthy animals (not infected), used as a control group; Group
B consisted of seven dogs experimentally infected with R. vitalii.
Each of the seven Group B animals was infected through intrave-
nous inoculation of 2 mL of fresh blood collected from Dog 13,
which had been kept infected in the laboratory. At the moment
of inoculation, Dog 13 was shown to be infected by visualization
of the parasite within erythrocytes and leukocytes of blood smears
(procedure described below). It was not possible to quantify the
number of parasites inoculated, but Dog 13 showed to have an
average of six parasites per slide examined.
2.4. Estimation of parasitemia
After inoculation, blood smears were performed at two day-
intervals, starting at day 1 post-infection (PI). Peripheral blood
smears were performed with blood obtained at the tip of the ear
of dogs, with the help of a hypodermic needle. The smears were
Romanovsky stained, and examined under 1000� magnification
by optical microscopy. Quantification of parasites in the slides
was made by counting the parasites in 10 random fields, and divid-
ing the number of parasites quantified by 10. Therefore, the results
of parasitemia were presented in number of parasites/field. In the
blood smears, it was measured the extracellular and intracellular
parasites (erythrocytes and leukocytes). Length and width of the
organisms, and their nucleous, were determined using the Motic
Images Plus 2.0 program for analysis of images and morphometry,
fitted to an optical microscope (Olympus). The analysis of the
slides was performed in duplicate, by the same observer.
f R. vitalii in the cytoplasm and interior of erythrocytes and leukocytes. On day 21 PI,
Fig. 2. Forms of R. vitalii found in blood smears. The parasite was identified among the cells, extracellular milieu (A) and within erythrocytes (B–E) and leukocytes (F).
A.S. Da Silva et al. / Experimental Parasitology 128 (2011) 347–352 349
2.5. Clinical examination and hematocrit
Signs of the disease were monitored for 30 days PI through eval-
uations of cardiorespiratory frequency, temperature, weight,
mucosal, and capillary perfusion at three day intervals. Hematocrit
values were determined through the jugular vein at the days 0, 10,
20, and 30 PI.
2.6. Pathological findings
After 21 days of infection, one of the dogs from Group B (dog 7)
was anesthetized (ketamine at 0.08 mL kg�1, and xylazine at
0.05 ml kg�1), and then euthanized using tetracainchloride
(T-61). At the same day it was performed the necropsy of the ani-
mal, when fragments of internal organs, such as spleen, liver, kid-
ney, lymph nodes, brain, intestines, heart, lungs, veins, and arteries
were collected. The material was stored in 10% formalin. For
histopathology, sagittal sections were done with an interval of
3 mm between regions to mount the slides, which were stained
with hematoxylin and eosin.
Pathological analysis of this animal in the acute phase of the
disease, when clinical signs were not severe (different from natu-
rally infected dogs), had the purpose to investigate the disease
pathogenesis, as well as the macro and microscopically histological
injuries produced by the parasite.
2.7. Treatment
At day 21 PI, the remaining six Group B dogs received a single
intramuscular dose of diminazene aceturate (3.5 mg kg�1), in order
to evaluate the drug efficacy against infection by R. vitalii in dogs.
The experiment ended at 30 days PI, because this was the experi-
mental period requested in the research project that has been ap-
proved by the committee of ethics and animal welfare.
Fig. 3. Hematocrit of dogs experimentally infected with R. vitalii. Asterisk shows
statistically significant difference between groups not-infected and infected
(⁄⁄Pin all dogs from group B. In these infected ani-
mals, there was a significant (P2010). Treatment with diminazene aceturate in cases of
rangeliosis has been questioned, due to the toxicity of this drug.
For this fact, researchers have not recommended this drug for
the treatment of R. vitalii (Loretti and Barros, 2004). In our study,
we observed that the employed diminazene aceturate dose was
effective in the treatment of R. vitalii during the early phase of dis-
ease. The animals showed no adverse clinical effects due to drug
intoxication. Therefore, we believe that diminazene aceturate
may be a drug of choice in the treatment of R. vitalii, but veterinar-
ian must evaluate the patient’s health before applying the drug, to
prevent worsening of disease and death of the animal by
intoxication.
This study allowed us to make the following conclusions about
the infection by R. vitalii in dogs: (1) the parasite erythrocyte cycle
occurs during the acute phase of the disease; (2) after the peak of
parasitemia in erythrocytes, the protozoan invades vascular endo-
thelial cells and leukocytes; (3) clinical signs and pathological find-
ings observed in experimental infection are similar to those
described in natural infections by R. vitalii; (4) the therapeutic pro-
tocol based on diminazene aceturate was 100% effective in clean-
ing R. vitalii from blood of infected dogs.
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http://dx.doi.org/10.1016/j.vetpar.2011.03.024
	Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment
	1 Introduction
	2 Material and methods
	2.1 Animals
	2.2 Rangelia vitalii isolate
	2.3 Experimental design
	2.4 Estimation of parasitemia
	2.5 Clinical examination and hematocrit
	2.6 Pathological findings
	2.7 Treatment
	2.8 Molecular diagnosis
	2.9 Statistical analysis
	3 Results
	3.1 Parasitemia
	3.1.1 Measurement of extracellular parasites
	3.1.2 Measurement of parasites within erythrocytes
	3.1.3 Measurement of parasites within leukocytes
	3.2 Clinical signs and hematocrit
	3.3 Pathological findings
	3.4 Treatment
	4 Discussion
	References