Veterinary Parasitology 197 (2013) 665669

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Short communication

First evidence of autochthonous cases of Leishmania

(Leishmania) infantum in horse (Equus caballus) in the
Americas and mixed infection of Leishmania infantum and
Leishmania (Viannia) braziliensis
Isabel R. Soares a , Soraia O. Silva b , Filipe Moraghi Moreira a ,
Luan Gavio Prado a , Priscila Fantini a , Renata de Pino Albuquerque Maranho a ,
Jos Monteiro da Silva Filho a , Maria Norma Melo b , Maristela S. Palhares a,
a
Departamento de Clnica e Cirurgia Veterinrias, Escola de Veterinria, Universidade Federal de Minas Gerais, Av. Antnio Carlos 6627,
Pampulha, CEP: 30123-970, Belo Horizonte, MG, Brazil
b
Departamento de Parasitologia, Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais, Av. Antnio Carlos 6627,
Pampulha, CEP: 30123-970, Belo Horizonte, MG, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study reports the rst evidence of infection by Leishmania infantum in Equus caballus in
Received 14 December 2012 Americas and the rst mixed infection of L. infantum/Leishmania braziliensis on this mam-
Received in revised form 28 May 2013
malian species in the world. The diagnoses was based on presence of parasites in lesions
Accepted 12 June 2013
and bone marrow aspirates, their identication by using specic primers for L. infantum and
L. braziliensis complexes and also serological methods IFAT and ELISA. The analysis of the
Keywords:
PCR products suggested mixed infection in three animals. Further studies involving equine
Equine
Leishmania infantum leishmaniasis are carrying out in order to clarify the dynamic of Leishmania sp. in this mam-
Leishmania braziliensis malian specie and their role in the transmission of those parasites in urban endemic area
Mixed infection of Belo Horizonte, Minas Gerais State, Brazil.
Brazil 2013 Elsevier B.V. All rights reserved.

1. Introduction cutaneous and visceral leishmaniasis, this latest usually

Zoonotic leishmaniasis is present in the Old World and
in the Americas, infecting humans and other mammalian
species. It is a vector-borne disease, transmitted by the
bites of Phlebotomine sand ies and caused by the protozoa
of the genus Leishmania (Alvar et al., 2012).
Depending on the species, in humans, Leishmania can
cause both tegumentary (TL) and visceral leishmania-
sis (VL), producing a wide spectrum of diseases, from
the localized cutaneous form, mucocutaneous, diffuse
Corresponding author. Tel.: +55 31 3409 2284.
E-mail addresses: bebelsoares@yahoo.com.br (I.R. Soares),
rsoaresisabel@yahoo.com.br (M.S. Palhares).
fatal if untreated (Silveira et al., 2004).
Canids, rodents and humans are the major affecting
species, especially concerning about zoonotic and urban
cycle. However a large number of mammalian species
have been record as Leishmania hosts (Brando-Filho et al.,
2003). The records in domestic species, other than dogs,
such as felines (Schubach et al., 2004; Maia and Campino,
2011) and horses (Barbosa-Santos et al., 1994; Mller et al.,
2009) have been increasing wide World.
In Brazil, equine leishmaniasis have been reported at
States from Cear (Alencar, 1959), Bahia (Vexenat et al.,
1986), Rio de Janeiro (Aguilar et al., 1986), Esprito Santo
(Falqueto et al., 1987), So Paulo (Yoshida et al., 1988), Per-
nambuco (Brando-Filho et al., 2003), Paran (Vedovello
Filho et al., 2008) and Minas Gerais (Soares et al., 2012).

0304-4017/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2013.06.014
666 I.R. Soares et al. / Veterinary Parasitology 197 (2013) 665669
Fig. 1. Clinical presentation of animal suspicious of leishmaniasis, presenting (A) low corporal condition and (B) ulcerative and circular with well-dened,
raised borders and a bed of granulation tissue.
When further investigations were possible on these Brazil-
ian cases, the parasite identied was always Leishmania
braziliensis.
Concerning the equine infection due Leishmania infan-
tum, it was detected exclusively in Europe, causing cuta-
neous problems. Cases were described as autochthonous
infections in Germany (Koehler et al., 2002), Spain (Solano-
Gallego et al., 2003) and Portugal (Rolo et al., 2005).
In this report, we described the rst autochthonous
cases of naturally-infected Equus caballus due to L. infan-
tum, in the Americas, and also the rst record, in the world,
of mixed infection with L. infantum and L. braziliensis in this
mammalian species.
2. Materials and methods
The animals were examined in the Equine Veterinary
Clinic at Veterinary School of the Federal University of
Minas Gerais, Belo Horizonte, Brazil.
The patient #1, 9-year-old male mixed breed, was pre-
sented at the veterinary clinic in March 2011. The owner
had noticed a large, ulcerated, granulomatous and exuda-
tive lesion, with a moderate degree of pruritus, measuring
6.5 cm 3.5 cm. A thorough physical examination revealed
no other clinical signs, a part the lesion and a low corpo-
ral condition (Fig. 1). The rst clinical suspicion was equine
pythiosis, which could not be conrmed due an inconclu-
sive histologic result. It was proceeded a new biopsy, a bone
marrow aspiration and a whole blood sample was extracted
from the jugular vein. The samples were destined to the
PCR test. In addition, serum was obtained by centrifugation
of whole blood sample and frozen until use to serological
tests.
The patient #2, 8-year-old female Mangalarga Mar-
chador breed, was presented with an extensive and
ulcerative lesion in the vulvar region as the major com-
plain. The complete physical examination did not present
any other disturbs. It was proceeded an incisional biopsy
and the analysis diagnosed a squamous cells carcinoma.
Due to this master research that was being developed at
the research group, focusing at the equine leishmaniasis,
a sample was examined to Leishmania PCR test and to an
imprint Giemsa-stained.
The animal #3, 7-year-old female mixed breed, had been
included in a prior master research, focusing at locomotors
problems in drafts horses. In her synovial liquid routine
exam was detected a structure that resembled an amastig-
ote. This suspicion could not be answered. One year later,
this mare was presented at veterinary clinic because of gas-
trointestinal problems in her foal. Because of the present
study, the owner authorized a bone marrow aspiration and
the sample was analyzed by direct examination and PCR
test. A whole blood sample was collected and serum pro-
cessed to serological analysis and PCR tests.
The direct examination was conducted in the imprint
of cutaneous lesions and also in the bone marrow
aspirates smears, Giemsa-stained. The presence of anti-
bodies IgG anti-Leishmania sp. was analyzed by the
serological reactions of the immunoenzymatic assay
(ELISA) and the indirect immunouorescent antibody test
(IFAT). Both tests were performed with a L. braziliensis
(MHOM/BR/75/M2904) antigen and needed standardiza-
tion to equine specie. Those were possible with a positive
control that presented active lesions on left pelvic limb,
which were biopsied to direct examination and PCR test,
showing positive results in both analyses (Soares et al.,
2012).
Briey, in ELISA test, polystyrene 96-well microtitre
plates were sensitized with 10 g/mL antigen. Optimal
concentrations of the sample sera and conjugate (Anti-
Horse IgG Peroxidase/Goat KPL) were established at
1:400 and 1:10,000, respectively. Cut-offs were deter-
mined separately for each plate, using sera (n = 6) of
2448 h old foals. The cut off was established at the mean
value plus three standard deviation of negatives sera. Reac-
tivity index (RI), the ratio between absorbance of the test
serum and the cut-off, was applied like criterion diagnosis.
The IRs higher than one, were considered positive.
The IFAT was carried out as described by Chiari et al.
(1973), with some modications. The promastigotes were
inactivated at 55 C per 8 min. The samples that showed
reaction in dilutions equal or higher than 1:40 were con-
sidered positive, according to the experiments in humans
(Pedras et al., 2003) and canines (Ferreira et al., 2007).
For molecular diagnosis, DNA was obtained using a
commercial kit (NucleoSpin Tissue Macherey-Nagel). The
 I.R. Soares et al. / Veterinary Parasitology 197 (2013) 665669 667

Fig. 2. Free amastigotes of Leishmania sp. showing a round nucleus and a kinetoplast surrounded by a clear halo (arrow). (A) Lesion imprint from patient
#2. (B) Smear of bone marrow aspirate from animal #1. Giemsa-stained (100).

protocol was followed as recommended by the manufac-
turer. A PCR test targeted to the parasite DNA polymerase
gene was conducted to assess the Leishmania sp. genus
infection using primers (forward, 5 -TGT CGC TTG CAG
ACC AGA TG-3 , reverse, 5 -GCA TCG CAG GTG TGA GCA
C-3 ) designed to amplify a 90 bp fragment (Bretagne et al.,
2001). PCR was performed using a mixture of 5 L of Mix
green (Green Master Mix-M7122, Promega), and 5 pmol of
the each primer and 2 L of DNA.
The same extracted DNA was analyzed for species
of parasites identication. Infection by L. infantum was
assessed by primers designed from kDNA minicircle
sequences LV1 (5 -ACG AGG TCA GCT CCA CTC C-3 ) and
LV2 (5 -CTG CAA CGC CTG TGT CTA CG-3 ) described for
Piarroux et al. (1993) which present an amplied prod-
uct about 100120 bp whenever positive. To identify the
L. braziliensis infection, primers B1 (5 -GGG GTT GGT GTA
ATA TAG TGG-3 ) and B2 (5 -CTA ATT GTG CAC GGG GAG
G-3 ) and the methodology described by Bruijn and Barker
(1992) were used, expecting a fragment about 750 bp on
positive samples.
DNA of L. infantum MHOM/BR/1975/PP75 and of L.
braziliensis MHOM/BR/1975/M2904 were used as positive
controls. Negative controls were included: PCR reagents
without DNA; DNA blood and skin of a healthy foal without
leishmaniasis from serological and PCR negative reac-
tion for L. infantum and L. braziliensis. PCR products were
analyzed by gel electrophoresis on a 5% non-denaturing
polyacrylamide gel in 89 mM Trisborate buffer (pH 8.0)
containing 2 mM EDTA. Fragments were visualized by sil-
ver nitrate staining.
3. Results
In direct examination, animal #2 presented amastig-
otes in lesion imprints (Fig. 2). This exam was negative in
imprints of the lesion, but positive in smears of the bone
marrow aspirate from animal #1, with scarce presence of
amastigotes (Fig. 2). In serological tests, both tested ani-
mals, #1 and #3, had anti-Leishmania antibodies. In IFAT,
the titres were 80 and 40, and in ELISA the IRs were 1148
and 1260, respectively.
PCR revealed L. braziliensis and L. infantum DNA in all
three animals. A PCR product of 90 bp was obtained with
DNA from the lesions biopsies material (patients #1 and
#2) and from bone marrow aspirates (animals #1 and #3)

Fig. 3. (A) Amplication products of 90 bp are obtained when Leishmania DNA is amplied by PCR using primers described for Bretagne et al. (2001). (B)
Amplication products of 100 bp are obtained when L. infantum DNA is amplied by PCR using primers LV1 and LV2. M: DNA size marker; Lane 1: DNA
extracted from the bone marrow aspirate, patient #3; Lane 2: DNA extracted from the lesion biopsy material, patient #2; Lane 3: DNA extracted from
the lesion biopsy material, patient #1; Lane 4: DNA extracted from the bone marrow aspirate, patient #1; Lane 5: Positive control (2 ng of L. infantum
MHOM/BR/1975/PP75); Lane 6: Negative control (no DNA).
668 I.R. Soares et al. / Veterinary Parasitology 197 (2013) 665669
(Fig. 3). A PCR in whole blood from patient #1 was also
conducted, resulting positive. After the new reactions to
identication of species of parasites, the patient #1 showed
mixed infection at the lesion and bone marrow samples,
being positive only to L. infantum at the whole blood.
Patients #2 and #3 presented mixed infection at the lesions
samples and bone marrow aspirate, respectively.
4. Discussion
So far, L. infantum, the aetiological agent of visceral
leishmaniasis has been reported in equines exclusively in
the Old World. The cases described here, therefore, repre-
sent the rst record of E. caballus naturally-infected with L.
infantum in the Americas and also, the rst mixed infection
with L. infantum/L. braziliensis in the world.
In the Americas, equine leishmaniasis is quite common
and cutaneous leishmaniasis in horses, mules and don-
keys has been diagnosed in South (Aguilar et al., 1987),
Central (Ramos-vara et al., 1996) and even North Amer-
ican (Reuss et al., 2012) countries. In Venezuela, equids
acts as reservoirs of L. braziliensis in rural and suburban
areas (Aguilar et al., 1984) being described in E. caballus
(Bonfante-Garrido et al., 1981) and E. asinus (Morales et al.,
2010).
In Brazil the equine leishmaniasis with cutaneous
lesions has been described since the late fties and more
frequently after the eighties with the urbanization of cuta-
neous leishmaniasis caused by L. braziliensis. More than ten
reports in Brazil had been cited and in the studies that fur-
ther characterization could be done, the aetiological agent
identied was L. braziliensis.
In our study, it was described by specics primers that
one of the species involved at the lesions was L. brazilien-
sis. It is uncertain if this was the only cause of the lesion,
because of the proved mixed infection and, mainly in
animal #2 who was diagnosed with squamous cells car-
cinoma. Even though, the TL is an endemic infection for
humans at the study area and the veterinarians should be
aware that leishmaniasis can and does occur in horses and
should be considered in the differential diagnosis of any
papulonodular and/or ulcerated lesion in the horse.
The disease in horses probably remained undiagnosed,
mainly in areas where leishmaniasis in man and dogs are
endemic. Besides, associated with the other descriptions
of equine leishmaniasis, the results of the present study
suggest that Leishmania infection in horse is capable of pro-
ducing a variety of cutaneous lesions in horse, as well being
asymptomatic.
In Europe, equine leishmaniasis caused by L. infantum
has been described in Germany (Koehler et al., 2002), in
an endemic area in Spain (Solano-Gallego et al., 2003),
and in Portugal (Rolo et al., 2005). All these cases were
a skin-dwelling disease and the infrequency of visceral
leishmaniasis in horses in endemic areas and the low level
(Fernndez-Bellon et al., 2006) or lack (Koehler et al., 2002)
of specic Leishmania antibodies shown by infected ani-
mals, suggested that cutaneous leishmaniasis is the only
clinical form in horses and argued against a visceral infes-
tation (Koehler et al., 2002).
However, Rolo et al. (2005) detected, by the CIE
technique, anti-Leishmania antibodies and suggested a con-
comitant visceral involvement. The authors support this
hypothesis by the comparison with the characteristics of
the disease at the canine hosts, whom present the sys-
temic form accompanied by cutaneous lesions, which are
frequently the rst clinical sign and the most common
manifestation.
Fernndez-Bellon et al. (2006) found positive serologi-
cal results in healthy horses from endemic areas in Spain,
and this immune prole are the same as the found in the
animal #3, who presented no clinical signs. These resem-
ble what is observed in many healthy dogs and humans
infected by L. infantum, which, in many times, mount
a specic immune response without developing clinical
symptoms (Alvar et al., 2004).
The animals present in our study had positive serology
(IFAT and ELISA), with moderate to high concentration level
of immunoglobulin. These characteristics could be explains
by the previous contact with the parasite, since they are
from endemic area. Even though, the positive result of the
PCR from material biopsied from skin lesions, bone marrow
aspirate and whole blood sample conrm the presence of
both species L. infantum and L. braziliensis.
The results achieved in these cases, conrmed the circu-
lating of both Leishmania species among the equines living
in this area as well increase the hypothesis of a concomitant
systemic involvement and the risk of horses are participat-
ing at the epidemiological chain of this parasite.
Another interesting point is the visceralization of
the L. braziliensis. Although Reithinger et al. (2003),
studying dogs infections, suggested that the hematoge-
nous metastasis in the parasitism by this species is a
common phenomenon, this is one of the many charac-
teristics unknown about the Leishmania sp. infection in
equids.
Vedovello Filho et al. (2008) detected DNA from the sub-
genus L. (Viannia) in blood samples of 7.1% (3/42) horses
in Parana State. These ndings corroborate our results and
increase the possible involvement of horses in the trans-
mission of TL, becoming more infective to the vectors.
However, the importance of this specie on the maintenance
of the parasite cycle and as a reservoir need to be accessed
by xenodiagnoses and environmental studies.
The mixed infection with two different Leishmania
species was already described in humans (Martinez et al.,
2002) and dogs (Silva et al., 2011), include in Minas
Gerais States (Quaresma et al., 2011). In Brazil, pre-
vention measures and surveillance are totally distinct
between tegumentary and visceral leishmaniasis and the
understanding of these co-infections scenarios have such
importance, mainly in endemic areas with overlapping of
species (Silva et al., 2011).
As the tree horses described here were born at in
the Metropolitan Region of Belo Horizonte, and never
left this area, these represent autochthonous cases and
corroborated the underdiagnosed idea and suggested the
adaptation of L. infantum at this new mammalian host.
Epidemiological studies are in progress to determine the
role of horses in both visceral and cutaneous leishmania-
sis, in order to clarify the clinical forms and the importance
 I.R. Soares et al. / Veterinary Parasitology 197 (2013) 665669
of the horse as reservoir of different parasite species in
endemic urban areas.
Likewise, the isolation is mandatory for a more rened
genotyping, biological and biochemical characterization of
the strains infecting horses in the urban area. However
these results could not be achieved for while and further
researches are in process for this goal.
Acknowledgements
This work has been nancially supported by Fundaco
de Amparo a Pesquisa de Minas Gerais (FAPEMIG),
Conselho Nacional de Desenvolvimento Cientco e Tec-
nolgico (CNPq) and Coordenaco de Aperfeicoamento
de Pessoal de Nvel Superior (CAPES), Brazil.
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