Veterinary Parasitology 197 (2013) 649652

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

Short communication

Longitudinal analysis of serological tests ofcially adopted by

the Brazilian Ministry of Health for the diagnosis of canine
visceral leishmaniasis in dogs vaccinated with Leishmune
Mary Marcondes a, , Valria Marcal Flix de Lima a ,
Maria de Ftima Lereno de Arajo b , Roberto Mitsuyoshi Hiramoto b ,
Jos Eduardo Tolezano b , Rafael F.C. Vieira c , Alexander W. Biondo d,e
a
Department of Clinics, Surgery and Animal Reproduction, School of Veterinary Medicine, So Paulo State University,
Aracatuba, So Paulo 16050-680, Brazil
b
Department of Parasitology and Mycology, Instituto Adolfo Lutz, So Paulo 01246-000, Brazil
c
Department of Veterinary Sciences, Universidade Federal da Paraba, Areia, Paraba 58397-000, Brazil
d
Department of Veterinary Medicine, Universidade Federal do Paran, Curitiba, Paran 80035-050, Brazil
e
Department of Veterinary Pathobiology, University of Illinois, IL 61802, USA

a r t i c l e

i n f o

Article history:
Received 19 February 2013
Received in revised form 3 July 2013
Accepted 9 July 2013
Keywords:
Leishmania infantum
ELISA
IFAT
DPP CVL rapid test

a b s t r a c t
Development of vaccines against canine visceral leishmaniasis (CVL) may provide a prophylactic barrier, but antibody response detected by standard diagnostic techniques may
not separate vaccinated from naturally infected dogs. Moreover, anti-Leishmania antibody
levels in vaccinated dogs may be detectable for months. Accordingly, the aim of the present
study was to comparatively evaluate an in-house ELISA with three serological tests ofcially adopted by the Brazilian Ministry of Health for the diagnosis of CVL in dogs vaccinated
with Leishmune . A total of 18 mongrel dogs were submitted to a complete protocol of the
vaccine, monitored and evaluated in 5 times (T0T4) up to 180 days after T0. Twenty-one
days after the rst dose (T1), 50% of the dogs were seropositive by the in-house ELISA and
5.5% by IFAT, while by the ofcial ELISA and DPP CVL rapid test all dogs tested negative.
At time T2, 42 days after of the rst dose, 100%, 83.3%, 11.1%, and 5.5% of the dogs were
seropositive by the in-house ELISA, IFAT, ofcial ELISA kit and the DPP CVL rapid test,
respectively. Ninety days after the rst dose (T3), 100%, 83.3%, 72.2% and 33.3% of the dogs
were seropositive by the in-house ELISA, ofcial ELISA kit, IFAT, and the DPP CVL rapid
test, respectively. Finally, at time T4, 88.8%, 33.3%, 11.1% and 5.5% of the dogs were seropositive by the in-house ELISA, ofcial ELISA kit, DPP CVL rapid test and IFAT, respectively.
In conclusion, dogs vaccinated with Leishmune cross-react by an in-house ELISA and by
the three ofcial Brazilian serological tests for the diagnosis of canine visceral leishmaniasis up to six months after the rst vaccine dose, and may be mistakenly diagnosed and
removed.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Corresponding author at: Departamento de Clnica, Cirurgia e
Reproduco Animal, Universidade Estadual Paulista Jlio de Mesquita
Filho, Faculdade de Medicina Veterinria de Aracatuba, Rua Clvis Pestana, 793, Jardim Dona Amlia, Aracatuba, So Paulo 16050-680, Brazil.
Tel.: +55 18 36361415; fax: +55 18 3636 1401.
E-mail addresses: marcondes@fmva.unesp.br,
marcondes.mary@gmail.com (M. Marcondes).
0304-4017/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2013.07.013

Visceral leishmaniasis (VL) is an important re-emergent

worldwide zoonosis caused by Leishmania infantum in
Latin America with domestic dogs playing an important
role as agent reservoirs (Dantas-Torres, 2009). The VL
Brazilian control program have associated vector control and human treatment to dog culling, following

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M. Marcondes et al. / Veterinary Parasitology 197 (2013) 649652

seropositivity to enzyme-linked immunosorbent assay

(ELISA) and/or immunouorescence antibody test (IFAT)
(Brasil, 2006).
Development of vaccines against canine visceral leishmaniasis (CVL) may provide a prophylactic barrier,
nevertheless antibody response detected by standard
diagnostic techniques may not separate vaccinated from
naturally infected dogs, as previously shown (Marcondes
et al., 2011). Thus, the development of more sensitive
tests capable of differentiate infected from vaccinated animals is needed. In this scenario, a colloidal gold-based
immunochromatography assay (Dual Path Platform (DPP )
CVL rapid test, Bio-Manguinhos/Fiocruz, Rio de Janeiro,
Brazil), which uses a recombinant (r) rK26/rK39 fusion
protein of L. infantum as antigen, was recently developed
(Grimaldi et al., 2012) and adopted by the Brazilian Ministry of Health as the screening test for the diagnosis of
CVL. Since previous studies were developed using indirect in-house ELISA, the aim of the present study was to
comparatively evaluate the immunochromatographic test
with an in-house ELISA routinely used in an endemic area
and two standard serological tests ofcially adopted by the
Brazilian Ministry of Health for the diagnosis of CVL in dogs
vaccinated with Leishmune .
2. Materials and methods
The study was approved by the Ethics Committee in
Animal Experimentation and Animal Welfare at So Paulo
State University, Aracatuba, So Paulo State, Brazil and was
conducted according to the ethical principles of animal
experimentation, adopted by the Brazilian College of Animal Experimentation.
2.1. Animals
A total of 18 healthy mongrel dogs, negative for L.
infantum by ELISA, direct examination of stained smears
of bone marrow and lymph nodes aspirates, and PCR of
peripheral blood, lymph nodes and bone marrow, as previous described (Nunes et al., 2007), with ages ranging
from 2 to 6 years, were submitted to a complete 3-dose
protocol of a commercially available vaccine against CVL
(Leishmune , Fort Dodge, USA) as previously described
(Marcondes et al., 2011). Each Leishmune vaccine dose
was composed of lyophilized FML antigen adjuvanted with
saponin, reconstituted in 1 mL NaCl 0.9% sterile saline
solution at the moment of vaccination and administered
subcutaneously.
In order to avoid post-vaccination infection by
infected sandies, vaccinated dogs received individual
deltamethrin-impregnated collars (Scalibor Protector
Band, Intervet International, Summit, NJ, USA), which were
replaced every four months following the manufacturers
recommendations. Dogs were also evaluated at the time
of each blood sampling by bone marrow and lymph nodes
smears obtained by needle aspiration biopsy and by a PCR
protocol for the detection of Leishmania species in peripheral blood, lymph nodes and bone marrow samples, as
previous described (Nunes et al., 2007).

2.2. Study design

Blood samples were obtained as previously described
(Marcondes et al., 2011) at ve time points (T) based on the
3-dose vaccination protocol of 21 days apart: T0 immediately before the rst dose; T1 immediately before the
second dose (21 days); T2 immediately before the third
dose (42 days); T3 90 days after the rst dose; and T4
180 days after the rst vaccine dose.
Detection of L. infantum antibodies was determined in
all 5 time points by an indirect in-house ELISA routinely
used at the Veterinary Teaching Hospital of So Paulo State
University, and by the ofcial serological methods adopted
by the Brazilian Ministry of Health for the diagnosis of
canine visceral leishmaniasis: indirect ELISA (ofcial ELISA
kit), IFAT and Dual Path Platform (DPP ) CVL rapid test.
2.3. Sampling
Dog blood samples (10 mL) were collected by venipuncture of jugular vein in tubes without anti-coagulant and
kept at room temperature (25 C) until visible clot retraction, centrifuged at 1500 g for 5 min and serum separated
and kept at 20 C until testing.
2.4. Detection of L. infantum antibodies by in house
indirect ELISA
Detection of anti-L. infantum IgG antibodies was performed by ELISA as previously described (Lima et al., 2003).
Each sample was tested in triplicate, the mean was determined, and two negative and three positive controls were
included in each plate. Values were expressed by serum
optical density (OD) mean. The cut-off value was dened
as being three standard deviations from the mean of 30
healthy dogs from a non endemic area. Accordingly, cut-off
value used was 0.218.
2.5. Detection of L. infantum antibodies by the ofcial
indirect ELISA kit
Detection of anti-Leishmania IgG antibodies was performed by an indirect ELISA kit (Bio-Manguinhos/
FIOCRUZ/MS, Rio de Janeiro, Brazil), which is based on
microtiter plates previously sensitized with soluble antigens of Leishmania major-like (MHOM/BR/76/JOF). Samples
showing OD values above the cut-off were considered to
be positive according to the manufacturers recommendations. Procedures were performed and analyzed according
to the criteria adopted by the Brazilian Ministry of Health
(Brasil, 2006).
2.6. Detection of L. infantum antibodies by IFAT
Detection of anti-Leishmania IgG antibodies was performed by IFAT kits (Bio-Manguinhos/FIOCRUZ/MS), which
is based on antigens of Leishmania major-like promastigote forms (MHOM/BR/76/JOF). Reaction was performed
according to the manufacturers protocol, with samples
presenting uorescence at dilutions of 1:40, 1:80 or higher

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651

Table 1
Comparative serological results longitudinally obtained by different methods in 18 healthy dogs submitted to a full vaccination protocol against visceral
leishmaniasis with Leishmune . Ofcial ELISA kit (Bio-Manguinhos/FIOCRUZ/MS), in-house ELISA (UNESP), immunouorescent antibody test (IFAT,
Bio-Manguinhos/FIOCRUZ/MS) and Dual Path Platform (DPP ) CVL rapid test (Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil).
Test

T0
N (%)

T1
N (%)

T2
N (%)

T3
N (%)

T4
N (%)

Ofcial ELISA
In-house ELISA
IFAT
DPP CVL rapid test

0
0
0
0

0
9 (50)
1 (5.5)
0

2 (11.1)
18 (100)
15 (83.3)
1 (5.5)

15 (83.3)
18 (100)
13 (72.2)
6 (33.3)

6 (33.3)
16 (88.8)
1 (5.5)
2 (11.1)

T0: prior to 1st dose of vaccine; T1: prior to 2nd dose of vaccine; T2: prior to 3rd dose of vaccine; T3: 90 days after the rst dose of vaccine; T4: 180 days
after the rst dose of vaccine.

considered positives. Procedures were performed and analyzed according to the criteria adopted by the Brazilian
Ministry of Health (Brasil, 2006).
2.7. Detection of L. infantum antibodies by Dual Path
Platform (DPP ) CVL rapid test
Detection of anti-Leishmania IgG antibodies was performed by Dual Path Platform (DPP ) CVL rapid test
(Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil), according
to the manufacturers instructions.
3. Results
When evaluating tests per time points, all dogs were
negative for Leishmania spp. at time T0 by the in-house
ELISA, and by the three ofcial serological methods: ELISA
kit, IFAT, and DPP CVL rapid test. At time T1, 9/18 (50%)
dogs were seropositive by the in-house ELISA, and 1/18
(5.5%) by IFAT. At time T2, 18/18 (100%), 15/18 (83.3%), 2/18
(11.1%), and 1/18 (5.5%) dogs were seropositive for Leishmania spp. by the in-house ELISA, IFAT, ofcial ELISA kit
and the DPP CVL rapid test, respectively. At time T3, 18/18
(100%), 15/18 (83.3%), 13/18 (72.2%), and 6/18 (33.3%) dogs
were seropositive for Leishmania spp. by the in-house
ELISA, ofcial ELISA kit, IFAT, and the DPP CVL rapid test,
respectively. Finally, at time T4, 16/18 (88.8%), 6/18 (33.3%),
2/18 (11.1%) and 1/18 (5.5%) dogs were seropositive for

Leishmania spp. by the in-house ELISA, ofcial ELISA kit,

DPP CVL rapid test and IFAT, respectively (Table 1). The
OD values for the in-house ELISA and ofcial ELISA kit
are shown in Fig. 1.

4. Discussion
The commercial vaccine used in the present study
(Leishmune , Fort Dodge, USA) is currently licensed
in Brazil and employs a strong glycoproteic complex (FML or fucosemannose ligand) for its protective
anti-immunogenic response (Borja-Cabrera et al., 2008).
Unfortunately, potential interference of the vaccine antibodies has been a growing concern (Palatnik-de-Sousa
et al., 2009), since vaccinated dogs may become indistinct
from naturally infected dogs (Da Silva et al., 2000; BorjaCabrera et al., 2002; Mendes et al., 2003).
The in-house ELISA overtime was the serological test
identifying the highest number of seropositive dogs, reaching 100% of dogs at T2 (42 days) and T3 (90 days), and
88.8% of the dogs at T4 (six months after the rst vaccine
dose). On the other hand, a decline in seropositive dogs was
observed six months after the rst dose of vaccine, nevertheless 88.8%, 33.3%, 11.1%, and 5.5% of dogs still remained
seropositive for Leishmania spp. by the in-house ELISA,
ofcial ELISA kit, DPP CVL rapid test and IFAT, respectively.
Cross-reactivity of Leishmune with immunological diagnostic tests has been controversial, since it was observed in

Fig. 1. Box plot of optical density (OD) values longitudinally obtained by an (A) in house ELISA and (B) ofcial ELISA kit, in 18 healthy dogs submitted to
a full vaccination protocol against visceral leishmaniasis with Leishmune , T0 prior to 1st dose of vaccine; T1: prior to 2nd dose of vaccine; T2: prior to
3rd dose of vaccine; T3: 90 days after the rst dose of vaccine; T4: 180 days after the rst dose of vaccine.

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M. Marcondes et al. / Veterinary Parasitology 197 (2013) 649652

48/68 (70.0%) (Mendes et al., 2003) and 39/39 (100%) dogs

(Grecco et al., 2009) with FML-ELISA, in 12/62 (19.3%) with
commercial IFAT (Arajo et al., 2009); 4/39 (10.3%) dogs
reacted with S7-recombinant ELISA and only 1/39 (2.6%)
with IFAT (Grecco et al., 2009). In addition, only 4/141
(2.8%) dogs reacted with S7-recombinant ELISA and IFAT
(Ristow and Perez Junior, 2009) and 76/5680 (1.3%) dogs
cross-reacted with ELISA and IFAT (Palatnik-de-Sousa et al.,
2009). More recently, a study based on in-house ELISA
has shown that antibody peak may delay to be observed
but persist up to 180 days and therefore the lack of follow
up monitoring after vaccination may have limited previous
studies (Marcondes et al., 2011). Thus, differences between
results in the present study and those from others (Mendes
et al., 2003; Grecco et al., 2009; Palatnik-de-Sousa et al.,
2009; Ristow and Perez Junior, 2009) may be accounted by
the overtime vaccinated dogs monitoring instead a single
pinpoint sampling following vaccination protocol.
Despite increased anti-Leishmania-IgG production due
to infection may allow the use of a wide range of serological diagnostic techniques with relatively high sensitivity
(Dantas-Torres et al., 2006), our present results have consistently shown that, as previously described only for
indirect ELISA (Marcondes et al., 2011), vaccinated animals
may not be separate overtime from naturally infected animals using different serological tests. As dog culling based
on serological results alone may lead vaccinated dogs to
be mistakenly diagnosed and systematically removed as
naturally infected by Leishmania spp., other testing such
as molecular approaches should be included to provide
a more reliable diagnosis and to avoid the elimination of
non-infected dogs.
5. Conclusion
Dogs vaccinated with Leishmune cross-react by an inhouse ELISA and three standard serological tests ofcially
adopted by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis up to six months after
the rst vaccine dose.
Acknowledgment
Dr. Vieira was a PhD student sponsored by a researcher
fellowship from the Brazilian National Council of Scientic and Technological Development (CNPq) at the time of
research.

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