Veterinary Parasitology 149 (2007) 280–284

www.elsevier.com/locate/vetpar

Short communication
The first records of Leishmania (Leishmania) amazonensis in dogs
(Canis familiaris) diagnosed clinically as having canine visceral
leishmaniasis from Araçatuba County, São Paulo State, Brazil
José E. Tolezano a,*, Sı́lvia R.B. Uliana b, Helena H. Taniguchi a,
Maria F.L. Araújo a, José A.R. Barbosa c, José E.R. Barbosa c,
Lucile Maria Floeter-Winter d, Jeffrey J. Shaw b,*
a
Seção de Parasitoses Sistêmicas, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 351 88 andar, 01246-902 São Paulo, SP, Brazil
b
Departamento de Parasitologia, Instituto de Ciências Biomédicas da Universidade de São Paulo, Av. Professor Lineu Prestes 1374,
05508-000 Cidade Universitária, São Paulo, SP, Brazil
c
Serviço de Biotério, Instituto Adolfo Lutz, São Paulo, SP, Brazil
d
Departamento de Fisiologia, Instituto Ciências Biomédicas da Universidade de São Paulo, São Paulo, Brazil
Received 23 March 2007; received in revised form 30 April 2007; accepted 5 July 2007

Abstract
Two cases of Leishmania (Leishmania) amazonensis are reported in the domestic dog (Canis familiaris). These are the first
records of this parasite in this species. The animals lived in the endemic visceral leishmaniasis area of Araçatuba, São Paulo State,
Brazil and were initially diagnosed, on clinical grounds, as having visceral leishmaniasis. Attempted parasite isolation from
inguinal lymph node aspirates was unsuccessful and the indirect immunofluorescent test for visceral leishmaniasis was negative in
both cases. Parasites were seen in cytological preparations of their lymph nodes and the DNA obtained from these same tissues
produced the expected fragment in a Leishmania specific rDNA based PCR assay. The products only hybridized with the L. (L.)
amazonensis specific probe S8. No human cases of L. (L.) amazonensis have been reported in this region. These results suggest that
L. (L.) amazonensis is being transmitted in the peridomestic habitat and that this parasite is responsible for a clinical condition that is
similar to visceral leishmaniasis caused by L. (L.) i. chagasi that is present in the same area.
# 2007 Published by Elsevier B.V.

Keywords: Visceral leishmaniasis; Serology; PCR; Natural infections; Transmission; Leishmania (Leishmania) amazonensis

1. Introduction Cutaneous leishmaniasis (CL) in dogs in the

In Latin America the domestic dog (Canis familiaris)
is intimately linked with leishmaniasis, but its role as a
source of infection for man depends on the species of
Leishmania.
* Corresponding authors. Tel.: +55 11 3068 2891;
fax: +55 11 62512249.
E-mail addresses: tolezano@hotmail.com (J.E. Tolezano),
jeffreyj@usp.br (J.J. Shaw).
Americas has been reported since 1913 (Pedrosa,
1913) but it is difficult to know what the parasites were
(Lainson and Shaw, 1979). Improvements in identifica-
tion methods now make it possible for us to say that the
following cutaneous Leishmania occur in dogs in the
Americas: L. (Viannia) braziliensis (Mayrink et al.,
1979), L. (V.) peruviana (Herrer, 1951), L. (V.)
panamensis (Herrer and Christensen, 1976) and L.
(V.) colombiensis (Delgado et al., 1993). However, it is
controversial as to whether the dog is a reservoir or

0304-4017/$ – see front matter # 2007 Published by Elsevier B.V.

doi:10.1016/j.vetpar.2007.07.008
 J.E. Tolezano et al. / Veterinary Parasitology 149 (2007) 280–284
accidental host for these different parasites (Reithinger
and Davies, 1999). In some domestic situations, such as
in the highlands of Peru, there is statistical evidence
indicating that it is a source of infection for man
(Reithinger et al., 2003).
The dog is considered to be the major reservoir of
infection of visceral leishmaniasis (VL) for man
(Chagas et al., 1938; Deane, 1956) but in some areas
of Brazil there is evidence (Costa et al., 2000) that man
himself may serve as a source of infection. Visceral
leishmaniasis represents one of the major emergent
parasitological diseases in Brazil and in the late 1990’s
it became established in one of the country’s most
densely populated States, São Paulo. To date control
measures have not impeded the expansion of the
disease. Its presence in Araçatuba was signalled in 1997
(da Costa et al., 1997) by the finding of the vector,
Lutzomyia longipalpis, which was followed by the death
of a young child in 1999 (Neves, 2004). Subsequent
studies revealed the presence of infected dogs. Since
then the disease has spread south following the major
rail and road transportation routes to Bauru and Marı́lia
(Neves, 2004).
Animals infected with L. (L.) i. chagasi have also
been recorded in the municipality of Cotia (Super-
intendência de Controle de Endemias, 2005; Savani
et al., 2004), a locality that is 245 km away from Bauru
but Cotia is only 35 km from São Paulo city, which is
the state’s capital and the biggest metropolitan region in
Latin America. Autochthonous human VL is a rare
disease in Greater Sao Paulo. The first case was
recorded in 1979 and a second one in 1982 (Iversson
et al., 1982,1979). However, neither infected dogs nor
sand flies were found in the two areas where the cases
occurred. The diagnosis of canine VL in São Paulo has
been at the generic level even in PCR tests (Langoni
et al., 2005). In the present study we compared the
results of three leishmanial diagnostic techniques on 15
sick dogs from the Araçatuba municipality (218120 S/
508250 E) that were diagnosed clinically as visceral
leishmaniasis. An rDNA polymerase chain reaction
(PCR) test was used to determine the species of
Leishmania involved.
2. Materials and methods
As part of a survey of the canine population of the
Araçatuba region, the blood from 457 dogs and five
foxes was collected on filter paper (Whatman No.1).
The dogs were from 12 different areas of the Araçatuba
urban region. They were selected because of the
presence of Lu. longipalpis or suspected canine VL
281
cases. The five foxes had been confiscated by IBAMA
(Instituto Brasileiro do Meio Ambiente e dos Recursos
Naturais Renováveis) and kept in the local zoo. Both
dog and fox eluates were tested by an indirect
immunofluorescent antibody test (IFAT) using the
IFA kit supplied by Biomanguinhos, Oswaldo Cruz
Institute, Rio de Janeiro. This kit is presently used
throughout Brazil by the public health laboratories for
the diagnosis of visceral leishmaniasis. It consists of an
anti-dog IgG conjugate and an antigen prepared from
promastigotes of L. (L.) major.
Buffy coat, lymph node aspirates and biopsies of
skin, liver and spleen were collected from 15 dogs, with
clinical signs of VL (allopathy, weakness, cachexia,
long nails, skin lesions, splenomegaly, dermatitis,
conjunctivitis). Giemsa stained smears of these tissues
were examined for the presence of amastigotes. In vitro
culture of tissue samples was performed in biphasic
Blood Agar base medium (Walton et al., 1977) in which
the overlay was Brain Heart infusion. Cultures were
examined periodically over a period of 4 weeks. Lymph
node, spleen and liver samples were triturated in 0.85%
saline and 0.2 mL was inoculated intraperitoneally into
hamsters. These animals were sacrificed for parasito-
logical examination 4–6 months after being inoculated.
DNA was purified from the above mentioned canine
tissue samples and buffy coat by the method described
(Uliana et al., 1991). Small subunit ribosomal RNA
(SSU rRNA) sequence amplification was performed as
previously described, using primers S12/S4 (Uliana
et al., 1994). Positive and negative control reactions
were included in all assays. Positive control reactions
were performed with genomic DNA purified from
axenic cultures of L. (L.) amazonensis, L. (V.)
braziliensis or L. (L.) i. chagasi and negative controls
without added DNA. The amplified product, corre-
sponding to an approximately 500-bp fragment corre-
sponding to the 30 end of Leishmania SSU rDNA, was
slot-blotted and hybridized to oligonucleotide probes
specific for: L. (L.) amazonensis (S8 50 -TGCTATTC-
TATGGGCAATTC-30 ); the subgenus L. (Viannia) (S10
50 -TGCTATCCTATGGACAATTC-30 ); and L. (L.)
infantum chagasi (S16 50 -TCCCAAGTGAGTCTAT-
GTG-30 ) (Uliana et al., 1994).
3. Results and discussion
Seventy-one (15.54%) of 457 sera tested by the IFAT
were positive at dilutions equal or greater than 1:40.
Only one fox serum was reactive at 1:40 using the anti-
dog IFA conjugate. Table 1 summarizes the results of
the parasitological and IFAT tests performed on 15 dogs
282 J.E. Tolezano et al. / Veterinary Parasitology 149 (2007) 280–284

Table 1
The results of three diagnostic methods for leishmaniasis performed on tissues of 15 dogs from an endemic area of visceral leishmaniasis situated in
the Araçatuba municipality, São Paulo State, Brazil that were diagnosed on clinical grounds as having visceral leishmaniasis
Dog’s number Culture IFAT5 PCR S.8* S.10* S.16* Material examined
11 + + +   + Lymph node aspirate
22   +   + Lymph node aspirate
33 Nt + +   + Skin lesion biopsy
4 + + +   + Lymph node aspirate
5 + + +   + Lymph node aspirate
6   + +   Lymph node aspirate6
7 Nt      Lesion aspirate
8      - Lesion aspirate
9 + + +   + Lymph node aspirate
10  + +   + Lymph node aspirate
11  + +   + Lymph node aspirate
12   + +   Lymph node aspirate6
13  + +   + Lymph node aspirate
14 + + +   + Lesion aspirate
154 + + +   + Lymph node aspirate
Total 6 10 13 2 – 11
Nt: not tested; *S.8: rDNA probe for Leishmania (Leishmania) amazonensis; *S.10: rDNA probe for subgenus Leishmania (Viannia); *S.16: rDNA
probe for Leishmania (Leishmania) infantum chagasi; 1–3heart, liver, spleen, cutaneous lesion and normal skin all positive with probe S.16; 4heart,
liver, spleen, bladder, cutaneous lesion and normal skin all positive with probe S16; 5the material examined was the eluate of blood collected on
Whatman no. 1 filter paper; 6Amastigotes detected in smears.

with clinical conditions compatible with visceral
leishmaniasis. The in vitro culture was positive for 6
of the 15 dogs (40%). 86.66% of the samples inoculated
into hamsters produced clinical VL and amastigotes
were seen in cytological preparations. Amastigotes
were seen in the smears of the lymph nodes of the L. (L.)
amazonensis PCR positive dogs but hamsters inoculated
with the same tissue were negative. This is not
surprising since this Leishmania does not produce
visceral infections in hamsters and when inoculated
intraperitoneally cutaneous lesions only appear after
12–18 months.
The PCR was positive for 13/15 dogs indicating the
presence of Leishmania. Eleven of the amplified
fragments hybridized with the L. (L.) i. chagasi probe
(S 16) and two with the S8 oligonucleotide that is
specific for L. (L.) amazonensis. The latter result
confirms that amastigotes seen in the lymph node
cytological preparations were not L. (L.) i. chagasi.
Positive and negative controls were respectively
positive and negative. This test is very specific and
besides being negative with tissues from uninfected
individuals it is negative with other closely related
pathogenic trypanosomatids, such as Trypanosoma
(Schizotrypanum) cruzi (Uliana et al., 1991).
Apart from being the first records of L. (L.)
amazonensis in the domestic dog the present results
raise a number of important questions in relation to the
diagnosis of canine visceral leishmaniasis. The first is
that clinical symptoms that are normally contributed to
L. (L.) i. chagasi could be caused by other Leishmania.
It might be argued that these two animals were also
infected with L. (L.) i. chagasi but there is no diagnostic
evidence to support this.
The serology of the two L. (L.) amazonensis infected
dogs was negative but it was also negative for a dog that
had a L. (L.) i. chagasi positive PCR as well as two other
dogs whose Leishmania tests were all negative. The
present results show that leishmaniasis cannot be
eliminated as a possible diagnosis because of a negative
serology. Similar results have been found in other
studies that compared serological and PCR methods
(Ashford et al., 1995). A study in Spain (Solano-
Gallego et al., 2001) found that a symptomatic dogs
may have negative PCRs and positive ELISAs and vice
versa. This indicates that one test is insufficient for the
diagnosis of canine VL.
In a longitudinal study of a cohort of 126 dogs
exposed to natural infection in Marajó Island, Brazil
(Quinnell et al., 2001) the sensitivity of three diagnostic
methods (ELISA serology, kDNA and rDNA PCRs and
lymphocyte proliferation) were compared and asso-
ciated with different stages of the infection. The
conclusion of this study was that all infected dogs
apparently seroconverted indicating that serology was
more sensitive than the PCR tests, although in early
infections the latter was more sensitive. However, in a
study (Solano-Gallego et al., 2001) of L. (L.) i. infantum
 J.E. Tolezano et al. / Veterinary Parasitology 149 (2007) 280–284
in Spain the serology of 39/54 asymptomatic dogs was
negative but their PCRs were positive. Lack of
serological conversion in European dogs had been
noted previously (Dye et al., 1993). These two
contrasting results could be due to epidemiological
differences such as different transmission rates related
to vector density. In experimentally infected beagles it
has been shown that the positivity of different
diagnostic tests, such as the PCR, IFAT and the rK39
immunoassay, varies according to number of parasites
that are inoculated (Rosypal et al., 2005).
In the Marajó study 13 of the dogs that seroconverted
had no other positive test. The authors (Quinnell et al.,
2001) suggested that this could have been due to
serological cross-reactions or very low parasitaemias
associated with natural immunity. But what could have
caused these cross-reactions? Although L. (L.) amazo-
nensis has never been recorded in that study area it
seems quite likely that it occurs there. So could they
have been due to L. (L.) amazonensis? The primers used
in that study were specific for parasites of the infantum
complex so other Leishmania would have been missed.
In the present study the serology of both L. (L.)
amazonensis infected dogs was negative which does not
support the idea that the seroconversion of the Marajó
dogs was caused by L. (L.) amazonensis. However, in
that study the false-positive serology rate was 2.3%. In a
comparison of serological methods for the diagnosis of
canine VL we have found (Tolezano et al., unpublished
observations) that the false-positive rate is greater with
the ELISA test than the IFAT. There is thus a greater
possibility that cross-reactions could be occurring in the
ELISA with infections caused by other Leishmania. It
must be remembered, however, that the specificity of
the ELISA and IFAT tests depends on the composition
of the antigen as well as the species of Leishmania used.
The first record of L. (L.) amazonensis in a canid was
in 1979 in the crab eating fox, Dusicyon thous (Lainson
and Shaw, 1979). This infection was detected by the
intraperitoneal inoculation of spleen into hamsters and
manifested itself as cutaneous lesions on the paws that
appeared 12 months after inoculation. L. (L.) i. chagasi
was also isolated from foxes captured in the same
silvatic habitat (Lainson et al., 1969). L. (L.) amazo-
nensis was recently recorded (de Souza et al., 2005) in a
domestic cat (Catus felis) from the city of Campo
Grande, Mato Grosso do Sul, where visceral leishma-
niasis is endemic. These are two examples of the co-
transmission of L. (L.) amazonensis and L. (L.) i.
chagasi in the same ecological niche.
We conclude that clinical symptoms, similar to those
caused by L. (L.) i. chagasi in dogs, may be associated
283
with other Leishmania, such as L. (L.) amazonensis, and
that the diagnosis of canine visceral leishmaniasis in
Latin America should be performed with PCR tests that
are both generic and species specific. Present and past
results also suggest that the zoonotic L. (L.) amazo-
nensis occurs in peridomestic habitats. It remains to be
seen what its vector is.
Acknowledgements
We are grateful to Rui Larosa and Carlos Roberto
Elias for their technical help during the field work and to
the Health Department of Araçatuba municipality for
logistical support in the field. This work received
financial support from Conselho Nacional do Desen-
volvimento Cientı́fico e Tecnológico (CNPq) and from
the Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP).
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