- Email: [email protected]
Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs
Accepted Manuscript Title: Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs Authors: Gustavo F. Paz, Jeronimo M.N. Rugani, Andreza P. Marcelino, C´elia M.F. Gontijo PII: DOI: Reference:
S0001-706X(18)30112-8 https://doi.org/10.1016/j.actatropica.2018.03.018 ACTROP 4619
To appear in:
Acta Tropica
Received date: Accepted date:
29-1-2018 10-3-2018
Please cite this article as: Paz, Gustavo F., Rugani, Jeronimo M.N., Marcelino, Andreza P., Gontijo, C´elia M.F., Implications of the use of serological and molecular methods to detect infection by Leishmania spp.in urban pet dogs.Acta Tropica https://doi.org/10.1016/j.actatropica.2018.03.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs
Gustavo F. Paz
a*,
Jeronimo M. N. Rugani a, Andreza P. Marcelino b, Célia M. F.
Grupo de Estudos em Leishmanioses, Instituto René Rachou, Fundação Oswaldo
SC R
a
IP T
Gontijo a
Cruz, 30190-002, Belo Horizonte, MG, Brazil b Serviço
de Doenças Parasitárias, Divisão de Epidemiologia e Controle de Doenças,
U
Fundação Ezequiel Dias,
A
N
30510-010, Belo Horizonte, MG, Brazil
M
* Corresponding author: Tel./fax: +55 31 3349 7721.
ED
E – mail address: [email protected] (G. F. Paz)
PT
Abstract
The aim of this study was to evaluate the relationship between naturally
CC E
occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum
A
samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that
amplify a 300-350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4
IP T
(4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania
SC R
amazonensis, and their serum samples tested positive by IFAT and DPP. These
findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the
U
first record of Leishmania (Leishmania) amazonensis confirmed by a specific
M
A
N
molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.
Keywords: Leishmania braziliensis, Leishmania amazonensis, Leishmania infantum,
PT
1. Introduction
ED
dogs, serological assays
CC E
Canine visceral leishmaniasis (CVL) is a vector-borne disease caused by Leishmania infantum (syn. Leishmania chagasi) (Kinetoplastida: Trypanosomatidae). In contrast to Europe, where Leishmania infantum is the only species found in dogs
A
with CVL, three Leishmania species have been found in dogs with CVL from several localities in Brazil: Leishmania infantum, Leishmania braziliensis and Leishmania amazonensis (Costa et al., 2015; Pires et al., 2014; Quaresma et al., 2009; Dias et al., 2009; Tolezano et al., 2007).
2
In Brazil, control of zoonotic visceral leishmaniasis focuses on the detection and elimination of infected dogs. Positivity to serum anti-Leishmania IgG antibodies is the principal criterion for culling of dogs (Ministério da Saúde, 2006). Immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunochromatographic devices are the most commonly used
IP T
techniques for detecting anti-leishmanial antibodies. IFAT is one of the most
frequently used tests (Morales-Yuste, 2012), and is considered to be the “gold
SC R
standard” of serological methods and has been the established WHO reference
technique since the 1980s (WHO, 1984) to the present day (WHO, 2010). For these reasons, IFAT was selected as the serological reference technique in this study.
U
False positive results due to serological cross-reactivity with other pathogens
N
have been described for all of the above-mentioned serological techniques,
A
particularly with Trypanosoma cruzi infection in North, Central and South America,
M
other species of Leishmania and tests using whole-parasite crude antigens (Ferreira
ED
et al., 2007; Solano-Gallego et al., 2009). Cross-reactions are less likely to occur when using recombinant peptides such as rA2, rK9, rK26 and rK39 (Solano-Gallego
PT
et al., 2009).
Until 2012, CVL was diagnosed using IFAT, which was the recommended
CC E
method for confirming positive cases detected by ELISA. Recently, in order to improve the accuracy of diagnosing CVL, the Brazilian Ministry of Health has
A
recommended the use of an immunochromatographic rapid test consisting of rK26, rK9 and rK39 recombinant antigens, the Dual-Path Platform (DPP; BioManguinhos/Fiocruz, Rio de Janeiro, Brazil) to screen infected dogs, and ELISA (BioManguinhos/Fiocruz) to confirm positive results (Peixoto et al., 2015).
3
The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs and the practical implications of the use of serological and molecular methods to confirm diagnoses.
2. Materials and methods
IP T
The study was approved by the Ethics Committee for Research with Animals
of the Oswaldo Cruz Foundation (CEUA/Fiocruz) under protocol number LW-76/12.
SC R
All procedures involving experimental animals were conducted according to the
guidelines of the Brazilian College for Experiments with Animals (Colégio Brasileiro de Experimentacão Animal/COBEA).
U
The study was conducted during the summer of 2012 in an urban area in
N
northeastern Belo Horizonte (latitude: 195501500 S; longitude: 43 5601600 W) that is
A
endemic for canine visceral leishmaniasis. This area was selected on the basis of
M
environmental and socio-economical characteristics (Buss and Pelegrini, 2007), and
ED
comprised a relatively flat, densely populated urban district containing predominantly one- or two-story buildings with woody vegetation between lots. The annual
department.
PT
prevalence of CVL in the local study area is about 10%, according to the local health
CC E
The canine population within the study area consisted of 120 domiciled
domestic dogs, corresponding to almost every resident dog in the area during the
A
study period. Only 96 (80%) of these dogs could be surveyed, the remaining dogs could not be evaluated because either the owners refused to participate in the study or residents were not present in their homes at the start of the study. Blood samples were collected by venipuncture and transferred to tubes without anticoagulant.
4
Serum samples were obtained by centrifugation 2,500 rpm for 15 minutes and stored at -70 °C until use. To detect clinical signs of disease, each dog was subjected to one clinical examination conducted during the experimental period. All 96 sera were tested for the presence of anti-Leishmania immunoglobulin G
IP T
(IgG) antibodies using the following four commercial canine visceral leishmaniasis kits: immunofluorescence antibody test (IFAT) (Bio-manguinhos®/FIOCRUZ/MS),
SC R
enzyme-linked immunosorbent assay (ELISA) (Bio-Manguinhos®/FIOCRUZ/MS),
Dual Path Platform (DPP) (Bio-manguinhos®/FIOCRUZ/MS) and rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). Only dogs that tested positive by IFAT were
U
culled (according to the WHO).
N
An initial screening for the presence of anti-Leishmania immunoglobulin in
A
canine sera was performed using IFAT (Bio-manguinhos®/FIOCRUZ/MS) by the
M
local zoonoses control service. Sera were first diluted to 1:40 (the cut-off point) with
ED
1x phosphate buffered saline (PBS; pH 7.2) in order to establish which samples were seropositive for Leishmania spp. Positive samples were further diluted to 1:640 with
PT
1x PBS. Both procedures were performed and analyzed according to the criteria adopted by the Brazilian Ministry of Health (Ministério da Saúde, 2006). Confirmed
CC E
positive dogs were confiscated and euthanized by the local zoonoses control service following one of the VL control measures adopted by the Brazilian Ministry of Health
A
in 2002. Samples from mesenteric lymph nodes, spleen border, bone marrow, and ear skin were taken and submitted to DNA extraction. The DNA extraction was performed using Gentra Puregene QIAGEN (Germantown, MD, EUD) extraction kit, following the manufacturer’s specifications. PCR reactions were performed using primers that amplify a 300-350 bp fragment of
5
the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region; the primers used were LITSR 5’-CTG GAT CAT TTT CCG ATG-3’ and L5.8S 5’-TGA TAC CAC TTA TCG CAC TT-3’ (Schonian et al., 2003). Amplified products were visualized in a 2% agarose gel stained with ethidium bromide, with a 100 bp DNA Step Ladder provided as a molecular weight size standard, and analyzed using the L-PIX EX
used
as
a
positive
control,
which
included
Leishmania
IP T
Biotechnology Loccus photo-documentation system. A panel of reference strains was
amazonensis
SC R
(IFLA/BR/67/PH8), Leishmania braziliensis (MHOM/BR/75/M2903), Leishmania infantum (MHOM/BR/74/PP75) and Leishmania guyanensis (MHOM/BR/75/M4147). The ITS1 amplified products were analyzed by PCR-RFLP using HaeIII
U
(10U/µl) restriction endonuclease (New England Biolabs, Ipswich, MA, USA), and the
N
restriction profiles were visualized in 4% agarose gel stained with ethidium bromide
A
and compared with reference strains as previously described.
M
Positive samples were purified with QIAquick PCR Purification kit (QIAGEN,
ED
USA) according to manufacturer’s recommendations. To confirm the Leishmania species detected by PCR-RFLP, each purified sample was sequenced in duplicate
PT
using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with 20 ng of the purified PCR products and 3.3 pmol of the forward
CC E
and reverse primers to a final volume of 10 μL. Sequencing was performed using the Sanger Sequencing Platform at CPqRR/FIOCRUZ and a ABI 3730xl DNA Analyzer.
A
Sequences were analyzed in Genious 6.0 software (Biomatters Ltd.) and the BLAST algorithm
(www.ncbi.nlm.nih.gov/BLAST)
was
used
to
identify
homologous
sequences deposited in GenBank. 3. Results and discussion
6
Clinical examinations conducted at the time of blood sample collection revealed that none of the dogs exhibited any visible signs of disease. In endemic areas, serological tests, which are helpful tools in epidemiological studies, are useful for identifying infected dogs without clinical signs and for diagnosing chronic infections. The absence of clinical signs among the study population of dogs is of
IP T
limited value because it does not consider clinicopathological abnormalities and
disregards dogs that have widespread organ dysfunction without apparent visual
SC R
manifestations (Solano-Gallego et al., 2009).
Anti-Leishmania antibodies were detected by IFAT at a titration of 1:40 in 8.3% (8/96) of the animals comprising 5 males and 3 females. The seropositive animals
U
were euthanized and tissue samples were used for Leishmania DNA detection.
N
Analyses by ITS1-PCR revealed that all dogs were positive for the presence of
A
Leishmania DNA in at least one of the tissues analyzed. Table 1 summarizes the
M
results of the molecular tests used in this study. The infecting Leishmania species
ED
was identified in all samples by ITS1 PCR-RFLP and confirmed by sequencing. L. infantum DNA was identified in mesenteric lymph nodes, spleen border, bone
PT
marrow and, especially, ear skin. L. amazonensis and L. braziliensis DNA was identified in mesenteric lymph nodes and spleen. Surprisingly, the animals infected
CC E
with L. braziliensis and L. amazonensis (cutaneous strains) did not show skin parasitism. It is important to note that these two species have been previously
A
reported to infect visceral organs (Quaresma et al., 2009; Dias et al., 2010; Tolezano et al., 2007). The nucleotide sequences found in the samples from these dogs were deposited in GenBank with accession numbers KF985162, KF985164, KF985166, KF985169, KF985170 and KF985171, and were 96 to 100% similar to GenBank
7
sequences FJ753373, KC347301, KP274863, KC347300, KC347300, KC347300, as shown by MegaBlast analysis. Due to the low concentration of PCR products it was not possible to obtain accurate DNA sequences for species identification in 38.5% of the ITS1 PCR products of positive samples. Of the infected dogs, 25% produced positive reactions at a titration of 1:640.
IP T
Titrations of 1:80 and 1:640 were found only in dogs infected with L. infantum. Titrations of 1:40 were found in dogs infected with L. braziliensis and L.
SC R
amazonensis. Table 2 summarizes the results of the different diagnostic tests used in
this study. Among the 96 serum samples submitted to serological assays, 4 (4.1%) were considered positive for Leishmania by ELISA, 2 (2.1%) by rK39 RDT and 7
U
(7.3%) by DPP. Only one dog was positive in all serological tests used. Our results
N
demonstrate that IFAT and DPP could detect antibodies in dogs infected with L.
A
braziliensis and L. amazonensis, unlike rK39 RDT and ELISA, which detected
M
antibodies only in dogs infected with L. infantum.
ED
Dogs are regarded as the principal reservoir of L. infantum (Deane 1956; Michalsky et al., 2007; Laurenti et al., 2013), and for this reason they are targets of
PT
control programs in some countries, such as Brazil. However, there is no scientificbased evidence supporting the participation of dogs in the zoonotic transmission
CC E
cycle of L. braziliensis and L. amazonensis, which is known to be maintained by small terrestrial mammals (Brandão Filho et al., 2003; Oliveira et al., 2005; Dantas-
A
Torres, 2007; Quaresma et al., 2011). In the present study, two L. braziliensis-infected dogs and two L.
amazonensis-infected dogs had positive serological results for IFAT and DPP. For this reason, the diagnosis of leishmaniasis in dogs should be confirmed using PCRbased methods or by the isolation and subsequent characterization of the parasite
8
using reference isoenzymatic methods. In this regard, the use of specific PCR-based methods should be used to distinguish dogs infected by L. braziliensis and L. amazonensis from those infected by L. infantum. To the best of our knowledge, there have been no previous studies comparing serological and molecular techniques in the diagnosis of VL in dogs infected with
IP T
different species of Leishmania. Even with advances in the understanding of the complex epidemiology of leishmaniasis and the use of these diagnostic methods, the
epidemiological differences are
important
SC R
interpretation of the results remains open for discussion. In Brazil, regional and challenges to
be considered
in
understanding this disease and for its control (Costa et al., 2015).
U
The accuracy of the serological techniques currently used by the program for
N
monitoring and control of CVL is questionable (Peixoto et al., 2015), especially when
A
dogs are infected by Leishmania species other than L. infantum.
M
The present investigation showed that 4 (50%) dogs were infected only by L.
ED
braziliensis or L. amazonensis and that when these serum samples were submitted to serological assays they were considered positive for CVL by IFAT and DPP.
PT
Infections by Leishmania infantum, Leishmania braziliensis and Leishmania amazonensis have been reported from several localities (Pires et al., 2014;
CC E
Quaresma et al., 2009; Dias et al., 2010; Tolezano et al., 2007), and infections by species from the Leishmania mexicana complex have been previously detected in
A
dogs from the municipality of Belo Horizonte, state of Minas Gerais, Brazil (Ferreira et al., 2015). 4 Conclusions These findings show for the first time that cross-reactivity between L. braziliensis and L. amazonensis infection in dogs occurs in the DPP serum test. This 9
study also reports the first confirmed record of Leishmania (Leishmania) amazonensis in the municipality of Belo Horizonte, state of Minas Gerais, Brazil.
Acknowledgments
IP T
We acknowledge Fundação de Amparo à Pesquisa do Estado de Minas
Gerais (FAPEMIG), Programa de Excelência em Pesquisa- PROEP, Centro de
SC R
Pesquisas René Rachou (CPqRR), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) grant number 401975/2012-6 and Programa de Apoio à Pesquisa (PAPES VI) da Fundação Oswaldo Cruz (FIOCRUZ) grant number
A
CC E
PT
ED
M
A
N
U
407529/2012-8. CMF Gontijo is research fellow of CNPq.
10
References Brandão-Filho, S.P., Brito, M.E., Carvalho, F.G., Ishikawa, E.A., Cupolillo, E., FloeterWinter, L., Shaw, J.J., 2003. Wild and synanthropic hosts of Leishmania (Viannia) braziliensis in the endemic cutaneous leishmaniasis locality of Amaraji,
IP T
Pernambuco State, Brazil. Trans. R. Soc. Trop. Med. Hyg. 97, 291-296. Buss, P. M. and F. Pelegrini. 2007. A saúde e seus determinantes sociais. Revista
SC R
de Saúde Coletiva 17: 77–93.
Costa, L.N., Borba, A.S., Castagna, C.L., Carvalho Filho, E.B., Marson, F.A., Sá Junior, F.F., Angerami, R.N., Levy, C.E., 2015. Evaluation of PCR in the diagnosis
U
of canine leishmaniasis in two different epidemiological regions: Campinas (SP)
N
and Teresina (PI), Brazil. Epidemiol. Infect. 143,1088-1095.
A
Dantas-Torres, F., 2007. The role of dogs as reservoirs of Leishmania parasites, with
M
emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia)
ED
braziliensis. Vet. Parasitol. 149,139-46.
Deane, L.M., 1956. Leishmaniose visceral no Brasil: Estudos sobre reservatórios e
PT
transmissores realizados no Estado do Ceará. Tese (Livre-Docência) – Universidade de São Paulo, São Paulo.
CC E
Dias, E.S., Regina-Silva, S., França-Silva, J.C., Paz, G.F., Michalsky, E.M., Araújo S.C., Valadão, J.L., de Oliveira Lara-Silva, F., de Oliveira, F.S., Pacheco, R.S.,
A
Fortes-Dias, C.L., 2010. Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil. Vet. Parasitol. 176,101-11.
Ferreira, E.C., de Lana, M., Carneiro, M., Reis, A.B., Paes, D.V., da Silva, E.S., Schallig, H., Gontijo, C.M., 2007. Comparison of serological assays for the
11
diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet. Parasitol. 146,235-241. Ferreira, E.C., Cruz, I., Cañavate, C., de Melo, L.A., Pereira, A.A., Madeira, F.A., Valério, S.A., Cunha, H.M., Paglia, A.P., Gontijo, C. M., 2015. Mixed infection of Leishmania infantum and Leishmania braziliensis in rodents from endemic urban
IP T
area of the New World. BMC Vet. Res .11,71.
Laurenti, M.D., Rossi, C.N., da Matta, V.L., Tomokane, T.Y., Corbett, C.E.,
SC R
Secundino, N.F., Pimenta, P.F., Marcondes, M., 2013. Asymptomatic dogs are
highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.Vet. Parasitol. 196, 296-300.
U
Morales-Yuste, M., Morillas-Márquez, F., Díaz-Sáez, V., Barón-López, S., Acedo-
N
Sánchez, C., Martín-Sánchez, J., 2012. Epidemiological implications of the use of
A
various methods for the diagnosis of canine leishmaniasis in dogs with different
M
characteristics and in differing prevalence scenarios. Parasitol. Res. 111,155-64.
ED
Michalsky, E.M., Rocha, M.F., Da Rocha Lima, A.C., França-Silva, J.C., Pires, M.Q., Oliveira, F.S., Pacheco, R.S, Dos Santos, S.L., Barata, R.A., Romanha, A.J.,
PT
Fortes-Dias, C.L., Dias, E.S., 2007. Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine
CC E
sand flies. Vet. Parasitol. 147, 67-76.
Ministerio da Saúde (2006). Manual de vigilância e controle da leishmaniose visceral.
A
Brasília: Editora do Ministério da Saúde. 120 p.
Oliveira, F.S., Pirmez, C., Pires, M.Q., Brazil, R.P., Pacheco, R.S., 2005. PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil. Vet. Parasitol. 129,219-227.
12
Peixoto, H.M., de Oliveira, M.R., Romero, G.A. 2014. Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis. Trop. Med. Int. Health. 20 ,334-352. Pires, M.Q., Madeira, M.F., Bittencourt, V.R., Pacheco, R.S., 2014. Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation
IP T
by specific PCR and RFLP-PCR assays. Rev. Soc. Bras. Med. 47,243-246.
Quaresma, P.F., Murta, S.M., Ferreira, E.C., Rocha-Lima, A.C., Xavier, A.A., Gontijo,
SC R
C.M., 2009. Molecular diagnosis of canine visceral leishmaniasis: identification of
Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta. Trop. 111, 289-294.
U
Quaresma, P.F., Rêgo, F.D., Botelho, H.A., da Silva, S.R., Moura, A.J., Neto, R.G.T.,
N
Madeira, F.M., Carvalho, M.B., Paglia, A. P., Melo, M.N., Gontijo, C. M.F., 2011.
A
Wild, synanthropic and domestic hosts of Leishmania in an endemic area of
M
cutaneous leishmaniasis in Minas Gerais State, Brazil. Trans. R. Soc. Trop. Med.
ED
Hyg. 105: 579-585.
Schonian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H.D.F.H.,
PT
Presber, W., Jaffe, C.L. 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn. Microbiol. Infect. Dis. 47, 349-358.
CC E
Solano-Gallego, L., Koutinas, A., Miró, G., Cardoso, L., Pennisi, M.G. , Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G. 2009. Directions for the diagnosis, clinical
A
staging, treatment and prevention of canine leishmaniosis. Vet. Parasitol. 165,118.
Tolezano, J.E., Uliana, S.R., Taniguchi, H.H., Araújo, M.F., Barbosa, J.A., Barbosa, J.E., Floeter-Winter, L.M., Shaw, J.J. 2007. The first records of Leishmania (Leishmania)amazonensis in dogs (Canis familiaris) diagnosed clinically as having
13
canine visceral leishmaniasis from Aracatuba County, São Paulo State Brazil. Vet. Parasitol. 149,280– 284. WHO. 1984. La leishmaniosis. Serie de Informes Técnicos no. 793. Geneva. WHO .2010. Control of the leishmaniases. Report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, 22–26 March 2010. WHO Technical
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Report Series no 949. Geneva. pp 1-186.
14
TABLE 1 - Positivity of molecular methods in clinical samples from seropositivy dogs
lymph nodes
spleen
bone marrow
skin
L. amazonensis/ 2
+
+
-
-
+
+
-
-
+
-
-
-
-
+
-
-
+
-
+
-
-
+
-
-
-
-
+
-
4 (50%)
4 (50%)
U
+ + -
2 (25%)
3 (37.5%)
A
CC E
PT
ED
M
Positive (n=8)
+
N
L. infantum/4
A
L. braziliensis/2
SC R
Leishmania spp/nºdogs
IP T
Samples
15
TABLE 2 - Results of the different serological methods tests used in this study
IFAT
ELISA
DPP
rK39 RDT
L. amazonensis/ 2
1:40
-
+
-
1:40
-
+
-
1:40
-
-
-
1:40
-
+
-
1:640
+
+
1:40
+
+
1:80
+
+
1:640
+
+
8 (100%)
4 (50%)
U
-
7 (87.5%)
-
+ 2 (25%)
A
CC E
PT
ED
M
Positive (n=8)
+
N
L. infantum/4
A
L. braziliensis/2
SC R
Leishmania spp/nºdogs
IP T
Serological methods
16
S0001-706X(18)30112-8 https://doi.org/10.1016/j.actatropica.2018.03.018 ACTROP 4619
To appear in:
Acta Tropica
Received date: Accepted date:
29-1-2018 10-3-2018
Please cite this article as: Paz, Gustavo F., Rugani, Jeronimo M.N., Marcelino, Andreza P., Gontijo, C´elia M.F., Implications of the use of serological and molecular methods to detect infection by Leishmania spp.in urban pet dogs.Acta Tropica https://doi.org/10.1016/j.actatropica.2018.03.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs
Gustavo F. Paz
a*,
Jeronimo M. N. Rugani a, Andreza P. Marcelino b, Célia M. F.
Grupo de Estudos em Leishmanioses, Instituto René Rachou, Fundação Oswaldo
SC R
a
IP T
Gontijo a
Cruz, 30190-002, Belo Horizonte, MG, Brazil b Serviço
de Doenças Parasitárias, Divisão de Epidemiologia e Controle de Doenças,
U
Fundação Ezequiel Dias,
A
N
30510-010, Belo Horizonte, MG, Brazil
M
* Corresponding author: Tel./fax: +55 31 3349 7721.
ED
E – mail address: [email protected] (G. F. Paz)
PT
Abstract
The aim of this study was to evaluate the relationship between naturally
CC E
occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum
A
samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that
amplify a 300-350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4
IP T
(4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania
SC R
amazonensis, and their serum samples tested positive by IFAT and DPP. These
findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the
U
first record of Leishmania (Leishmania) amazonensis confirmed by a specific
M
A
N
molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.
Keywords: Leishmania braziliensis, Leishmania amazonensis, Leishmania infantum,
PT
1. Introduction
ED
dogs, serological assays
CC E
Canine visceral leishmaniasis (CVL) is a vector-borne disease caused by Leishmania infantum (syn. Leishmania chagasi) (Kinetoplastida: Trypanosomatidae). In contrast to Europe, where Leishmania infantum is the only species found in dogs
A
with CVL, three Leishmania species have been found in dogs with CVL from several localities in Brazil: Leishmania infantum, Leishmania braziliensis and Leishmania amazonensis (Costa et al., 2015; Pires et al., 2014; Quaresma et al., 2009; Dias et al., 2009; Tolezano et al., 2007).
2
In Brazil, control of zoonotic visceral leishmaniasis focuses on the detection and elimination of infected dogs. Positivity to serum anti-Leishmania IgG antibodies is the principal criterion for culling of dogs (Ministério da Saúde, 2006). Immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunochromatographic devices are the most commonly used
IP T
techniques for detecting anti-leishmanial antibodies. IFAT is one of the most
frequently used tests (Morales-Yuste, 2012), and is considered to be the “gold
SC R
standard” of serological methods and has been the established WHO reference
technique since the 1980s (WHO, 1984) to the present day (WHO, 2010). For these reasons, IFAT was selected as the serological reference technique in this study.
U
False positive results due to serological cross-reactivity with other pathogens
N
have been described for all of the above-mentioned serological techniques,
A
particularly with Trypanosoma cruzi infection in North, Central and South America,
M
other species of Leishmania and tests using whole-parasite crude antigens (Ferreira
ED
et al., 2007; Solano-Gallego et al., 2009). Cross-reactions are less likely to occur when using recombinant peptides such as rA2, rK9, rK26 and rK39 (Solano-Gallego
PT
et al., 2009).
Until 2012, CVL was diagnosed using IFAT, which was the recommended
CC E
method for confirming positive cases detected by ELISA. Recently, in order to improve the accuracy of diagnosing CVL, the Brazilian Ministry of Health has
A
recommended the use of an immunochromatographic rapid test consisting of rK26, rK9 and rK39 recombinant antigens, the Dual-Path Platform (DPP; BioManguinhos/Fiocruz, Rio de Janeiro, Brazil) to screen infected dogs, and ELISA (BioManguinhos/Fiocruz) to confirm positive results (Peixoto et al., 2015).
3
The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs and the practical implications of the use of serological and molecular methods to confirm diagnoses.
2. Materials and methods
IP T
The study was approved by the Ethics Committee for Research with Animals
of the Oswaldo Cruz Foundation (CEUA/Fiocruz) under protocol number LW-76/12.
SC R
All procedures involving experimental animals were conducted according to the
guidelines of the Brazilian College for Experiments with Animals (Colégio Brasileiro de Experimentacão Animal/COBEA).
U
The study was conducted during the summer of 2012 in an urban area in
N
northeastern Belo Horizonte (latitude: 195501500 S; longitude: 43 5601600 W) that is
A
endemic for canine visceral leishmaniasis. This area was selected on the basis of
M
environmental and socio-economical characteristics (Buss and Pelegrini, 2007), and
ED
comprised a relatively flat, densely populated urban district containing predominantly one- or two-story buildings with woody vegetation between lots. The annual
department.
PT
prevalence of CVL in the local study area is about 10%, according to the local health
CC E
The canine population within the study area consisted of 120 domiciled
domestic dogs, corresponding to almost every resident dog in the area during the
A
study period. Only 96 (80%) of these dogs could be surveyed, the remaining dogs could not be evaluated because either the owners refused to participate in the study or residents were not present in their homes at the start of the study. Blood samples were collected by venipuncture and transferred to tubes without anticoagulant.
4
Serum samples were obtained by centrifugation 2,500 rpm for 15 minutes and stored at -70 °C until use. To detect clinical signs of disease, each dog was subjected to one clinical examination conducted during the experimental period. All 96 sera were tested for the presence of anti-Leishmania immunoglobulin G
IP T
(IgG) antibodies using the following four commercial canine visceral leishmaniasis kits: immunofluorescence antibody test (IFAT) (Bio-manguinhos®/FIOCRUZ/MS),
SC R
enzyme-linked immunosorbent assay (ELISA) (Bio-Manguinhos®/FIOCRUZ/MS),
Dual Path Platform (DPP) (Bio-manguinhos®/FIOCRUZ/MS) and rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). Only dogs that tested positive by IFAT were
U
culled (according to the WHO).
N
An initial screening for the presence of anti-Leishmania immunoglobulin in
A
canine sera was performed using IFAT (Bio-manguinhos®/FIOCRUZ/MS) by the
M
local zoonoses control service. Sera were first diluted to 1:40 (the cut-off point) with
ED
1x phosphate buffered saline (PBS; pH 7.2) in order to establish which samples were seropositive for Leishmania spp. Positive samples were further diluted to 1:640 with
PT
1x PBS. Both procedures were performed and analyzed according to the criteria adopted by the Brazilian Ministry of Health (Ministério da Saúde, 2006). Confirmed
CC E
positive dogs were confiscated and euthanized by the local zoonoses control service following one of the VL control measures adopted by the Brazilian Ministry of Health
A
in 2002. Samples from mesenteric lymph nodes, spleen border, bone marrow, and ear skin were taken and submitted to DNA extraction. The DNA extraction was performed using Gentra Puregene QIAGEN (Germantown, MD, EUD) extraction kit, following the manufacturer’s specifications. PCR reactions were performed using primers that amplify a 300-350 bp fragment of
5
the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region; the primers used were LITSR 5’-CTG GAT CAT TTT CCG ATG-3’ and L5.8S 5’-TGA TAC CAC TTA TCG CAC TT-3’ (Schonian et al., 2003). Amplified products were visualized in a 2% agarose gel stained with ethidium bromide, with a 100 bp DNA Step Ladder provided as a molecular weight size standard, and analyzed using the L-PIX EX
used
as
a
positive
control,
which
included
Leishmania
IP T
Biotechnology Loccus photo-documentation system. A panel of reference strains was
amazonensis
SC R
(IFLA/BR/67/PH8), Leishmania braziliensis (MHOM/BR/75/M2903), Leishmania infantum (MHOM/BR/74/PP75) and Leishmania guyanensis (MHOM/BR/75/M4147). The ITS1 amplified products were analyzed by PCR-RFLP using HaeIII
U
(10U/µl) restriction endonuclease (New England Biolabs, Ipswich, MA, USA), and the
N
restriction profiles were visualized in 4% agarose gel stained with ethidium bromide
A
and compared with reference strains as previously described.
M
Positive samples were purified with QIAquick PCR Purification kit (QIAGEN,
ED
USA) according to manufacturer’s recommendations. To confirm the Leishmania species detected by PCR-RFLP, each purified sample was sequenced in duplicate
PT
using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with 20 ng of the purified PCR products and 3.3 pmol of the forward
CC E
and reverse primers to a final volume of 10 μL. Sequencing was performed using the Sanger Sequencing Platform at CPqRR/FIOCRUZ and a ABI 3730xl DNA Analyzer.
A
Sequences were analyzed in Genious 6.0 software (Biomatters Ltd.) and the BLAST algorithm
(www.ncbi.nlm.nih.gov/BLAST)
was
used
to
identify
homologous
sequences deposited in GenBank. 3. Results and discussion
6
Clinical examinations conducted at the time of blood sample collection revealed that none of the dogs exhibited any visible signs of disease. In endemic areas, serological tests, which are helpful tools in epidemiological studies, are useful for identifying infected dogs without clinical signs and for diagnosing chronic infections. The absence of clinical signs among the study population of dogs is of
IP T
limited value because it does not consider clinicopathological abnormalities and
disregards dogs that have widespread organ dysfunction without apparent visual
SC R
manifestations (Solano-Gallego et al., 2009).
Anti-Leishmania antibodies were detected by IFAT at a titration of 1:40 in 8.3% (8/96) of the animals comprising 5 males and 3 females. The seropositive animals
U
were euthanized and tissue samples were used for Leishmania DNA detection.
N
Analyses by ITS1-PCR revealed that all dogs were positive for the presence of
A
Leishmania DNA in at least one of the tissues analyzed. Table 1 summarizes the
M
results of the molecular tests used in this study. The infecting Leishmania species
ED
was identified in all samples by ITS1 PCR-RFLP and confirmed by sequencing. L. infantum DNA was identified in mesenteric lymph nodes, spleen border, bone
PT
marrow and, especially, ear skin. L. amazonensis and L. braziliensis DNA was identified in mesenteric lymph nodes and spleen. Surprisingly, the animals infected
CC E
with L. braziliensis and L. amazonensis (cutaneous strains) did not show skin parasitism. It is important to note that these two species have been previously
A
reported to infect visceral organs (Quaresma et al., 2009; Dias et al., 2010; Tolezano et al., 2007). The nucleotide sequences found in the samples from these dogs were deposited in GenBank with accession numbers KF985162, KF985164, KF985166, KF985169, KF985170 and KF985171, and were 96 to 100% similar to GenBank
7
sequences FJ753373, KC347301, KP274863, KC347300, KC347300, KC347300, as shown by MegaBlast analysis. Due to the low concentration of PCR products it was not possible to obtain accurate DNA sequences for species identification in 38.5% of the ITS1 PCR products of positive samples. Of the infected dogs, 25% produced positive reactions at a titration of 1:640.
IP T
Titrations of 1:80 and 1:640 were found only in dogs infected with L. infantum. Titrations of 1:40 were found in dogs infected with L. braziliensis and L.
SC R
amazonensis. Table 2 summarizes the results of the different diagnostic tests used in
this study. Among the 96 serum samples submitted to serological assays, 4 (4.1%) were considered positive for Leishmania by ELISA, 2 (2.1%) by rK39 RDT and 7
U
(7.3%) by DPP. Only one dog was positive in all serological tests used. Our results
N
demonstrate that IFAT and DPP could detect antibodies in dogs infected with L.
A
braziliensis and L. amazonensis, unlike rK39 RDT and ELISA, which detected
M
antibodies only in dogs infected with L. infantum.
ED
Dogs are regarded as the principal reservoir of L. infantum (Deane 1956; Michalsky et al., 2007; Laurenti et al., 2013), and for this reason they are targets of
PT
control programs in some countries, such as Brazil. However, there is no scientificbased evidence supporting the participation of dogs in the zoonotic transmission
CC E
cycle of L. braziliensis and L. amazonensis, which is known to be maintained by small terrestrial mammals (Brandão Filho et al., 2003; Oliveira et al., 2005; Dantas-
A
Torres, 2007; Quaresma et al., 2011). In the present study, two L. braziliensis-infected dogs and two L.
amazonensis-infected dogs had positive serological results for IFAT and DPP. For this reason, the diagnosis of leishmaniasis in dogs should be confirmed using PCRbased methods or by the isolation and subsequent characterization of the parasite
8
using reference isoenzymatic methods. In this regard, the use of specific PCR-based methods should be used to distinguish dogs infected by L. braziliensis and L. amazonensis from those infected by L. infantum. To the best of our knowledge, there have been no previous studies comparing serological and molecular techniques in the diagnosis of VL in dogs infected with
IP T
different species of Leishmania. Even with advances in the understanding of the complex epidemiology of leishmaniasis and the use of these diagnostic methods, the
epidemiological differences are
important
SC R
interpretation of the results remains open for discussion. In Brazil, regional and challenges to
be considered
in
understanding this disease and for its control (Costa et al., 2015).
U
The accuracy of the serological techniques currently used by the program for
N
monitoring and control of CVL is questionable (Peixoto et al., 2015), especially when
A
dogs are infected by Leishmania species other than L. infantum.
M
The present investigation showed that 4 (50%) dogs were infected only by L.
ED
braziliensis or L. amazonensis and that when these serum samples were submitted to serological assays they were considered positive for CVL by IFAT and DPP.
PT
Infections by Leishmania infantum, Leishmania braziliensis and Leishmania amazonensis have been reported from several localities (Pires et al., 2014;
CC E
Quaresma et al., 2009; Dias et al., 2010; Tolezano et al., 2007), and infections by species from the Leishmania mexicana complex have been previously detected in
A
dogs from the municipality of Belo Horizonte, state of Minas Gerais, Brazil (Ferreira et al., 2015). 4 Conclusions These findings show for the first time that cross-reactivity between L. braziliensis and L. amazonensis infection in dogs occurs in the DPP serum test. This 9
study also reports the first confirmed record of Leishmania (Leishmania) amazonensis in the municipality of Belo Horizonte, state of Minas Gerais, Brazil.
Acknowledgments
IP T
We acknowledge Fundação de Amparo à Pesquisa do Estado de Minas
Gerais (FAPEMIG), Programa de Excelência em Pesquisa- PROEP, Centro de
SC R
Pesquisas René Rachou (CPqRR), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) grant number 401975/2012-6 and Programa de Apoio à Pesquisa (PAPES VI) da Fundação Oswaldo Cruz (FIOCRUZ) grant number
A
CC E
PT
ED
M
A
N
U
407529/2012-8. CMF Gontijo is research fellow of CNPq.
10
References Brandão-Filho, S.P., Brito, M.E., Carvalho, F.G., Ishikawa, E.A., Cupolillo, E., FloeterWinter, L., Shaw, J.J., 2003. Wild and synanthropic hosts of Leishmania (Viannia) braziliensis in the endemic cutaneous leishmaniasis locality of Amaraji,
IP T
Pernambuco State, Brazil. Trans. R. Soc. Trop. Med. Hyg. 97, 291-296. Buss, P. M. and F. Pelegrini. 2007. A saúde e seus determinantes sociais. Revista
SC R
de Saúde Coletiva 17: 77–93.
Costa, L.N., Borba, A.S., Castagna, C.L., Carvalho Filho, E.B., Marson, F.A., Sá Junior, F.F., Angerami, R.N., Levy, C.E., 2015. Evaluation of PCR in the diagnosis
U
of canine leishmaniasis in two different epidemiological regions: Campinas (SP)
N
and Teresina (PI), Brazil. Epidemiol. Infect. 143,1088-1095.
A
Dantas-Torres, F., 2007. The role of dogs as reservoirs of Leishmania parasites, with
M
emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia)
ED
braziliensis. Vet. Parasitol. 149,139-46.
Deane, L.M., 1956. Leishmaniose visceral no Brasil: Estudos sobre reservatórios e
PT
transmissores realizados no Estado do Ceará. Tese (Livre-Docência) – Universidade de São Paulo, São Paulo.
CC E
Dias, E.S., Regina-Silva, S., França-Silva, J.C., Paz, G.F., Michalsky, E.M., Araújo S.C., Valadão, J.L., de Oliveira Lara-Silva, F., de Oliveira, F.S., Pacheco, R.S.,
A
Fortes-Dias, C.L., 2010. Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil. Vet. Parasitol. 176,101-11.
Ferreira, E.C., de Lana, M., Carneiro, M., Reis, A.B., Paes, D.V., da Silva, E.S., Schallig, H., Gontijo, C.M., 2007. Comparison of serological assays for the
11
diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet. Parasitol. 146,235-241. Ferreira, E.C., Cruz, I., Cañavate, C., de Melo, L.A., Pereira, A.A., Madeira, F.A., Valério, S.A., Cunha, H.M., Paglia, A.P., Gontijo, C. M., 2015. Mixed infection of Leishmania infantum and Leishmania braziliensis in rodents from endemic urban
IP T
area of the New World. BMC Vet. Res .11,71.
Laurenti, M.D., Rossi, C.N., da Matta, V.L., Tomokane, T.Y., Corbett, C.E.,
SC R
Secundino, N.F., Pimenta, P.F., Marcondes, M., 2013. Asymptomatic dogs are
highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.Vet. Parasitol. 196, 296-300.
U
Morales-Yuste, M., Morillas-Márquez, F., Díaz-Sáez, V., Barón-López, S., Acedo-
N
Sánchez, C., Martín-Sánchez, J., 2012. Epidemiological implications of the use of
A
various methods for the diagnosis of canine leishmaniasis in dogs with different
M
characteristics and in differing prevalence scenarios. Parasitol. Res. 111,155-64.
ED
Michalsky, E.M., Rocha, M.F., Da Rocha Lima, A.C., França-Silva, J.C., Pires, M.Q., Oliveira, F.S., Pacheco, R.S, Dos Santos, S.L., Barata, R.A., Romanha, A.J.,
PT
Fortes-Dias, C.L., Dias, E.S., 2007. Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine
CC E
sand flies. Vet. Parasitol. 147, 67-76.
Ministerio da Saúde (2006). Manual de vigilância e controle da leishmaniose visceral.
A
Brasília: Editora do Ministério da Saúde. 120 p.
Oliveira, F.S., Pirmez, C., Pires, M.Q., Brazil, R.P., Pacheco, R.S., 2005. PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil. Vet. Parasitol. 129,219-227.
12
Peixoto, H.M., de Oliveira, M.R., Romero, G.A. 2014. Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis. Trop. Med. Int. Health. 20 ,334-352. Pires, M.Q., Madeira, M.F., Bittencourt, V.R., Pacheco, R.S., 2014. Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation
IP T
by specific PCR and RFLP-PCR assays. Rev. Soc. Bras. Med. 47,243-246.
Quaresma, P.F., Murta, S.M., Ferreira, E.C., Rocha-Lima, A.C., Xavier, A.A., Gontijo,
SC R
C.M., 2009. Molecular diagnosis of canine visceral leishmaniasis: identification of
Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta. Trop. 111, 289-294.
U
Quaresma, P.F., Rêgo, F.D., Botelho, H.A., da Silva, S.R., Moura, A.J., Neto, R.G.T.,
N
Madeira, F.M., Carvalho, M.B., Paglia, A. P., Melo, M.N., Gontijo, C. M.F., 2011.
A
Wild, synanthropic and domestic hosts of Leishmania in an endemic area of
M
cutaneous leishmaniasis in Minas Gerais State, Brazil. Trans. R. Soc. Trop. Med.
ED
Hyg. 105: 579-585.
Schonian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H.D.F.H.,
PT
Presber, W., Jaffe, C.L. 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn. Microbiol. Infect. Dis. 47, 349-358.
CC E
Solano-Gallego, L., Koutinas, A., Miró, G., Cardoso, L., Pennisi, M.G. , Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G. 2009. Directions for the diagnosis, clinical
A
staging, treatment and prevention of canine leishmaniosis. Vet. Parasitol. 165,118.
Tolezano, J.E., Uliana, S.R., Taniguchi, H.H., Araújo, M.F., Barbosa, J.A., Barbosa, J.E., Floeter-Winter, L.M., Shaw, J.J. 2007. The first records of Leishmania (Leishmania)amazonensis in dogs (Canis familiaris) diagnosed clinically as having
13
canine visceral leishmaniasis from Aracatuba County, São Paulo State Brazil. Vet. Parasitol. 149,280– 284. WHO. 1984. La leishmaniosis. Serie de Informes Técnicos no. 793. Geneva. WHO .2010. Control of the leishmaniases. Report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, 22–26 March 2010. WHO Technical
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Report Series no 949. Geneva. pp 1-186.
14
TABLE 1 - Positivity of molecular methods in clinical samples from seropositivy dogs
lymph nodes
spleen
bone marrow
skin
L. amazonensis/ 2
+
+
-
-
+
+
-
-
+
-
-
-
-
+
-
-
+
-
+
-
-
+
-
-
-
-
+
-
4 (50%)
4 (50%)
U
+ + -
2 (25%)
3 (37.5%)
A
CC E
PT
ED
M
Positive (n=8)
+
N
L. infantum/4
A
L. braziliensis/2
SC R
Leishmania spp/nºdogs
IP T
Samples
15
TABLE 2 - Results of the different serological methods tests used in this study
IFAT
ELISA
DPP
rK39 RDT
L. amazonensis/ 2
1:40
-
+
-
1:40
-
+
-
1:40
-
-
-
1:40
-
+
-
1:640
+
+
1:40
+
+
1:80
+
+
1:640
+
+
8 (100%)
4 (50%)
U
-
7 (87.5%)
-
+ 2 (25%)
A
CC E
PT
ED
M
Positive (n=8)
+
N
L. infantum/4
A
L. braziliensis/2
SC R
Leishmania spp/nºdogs
IP T
Serological methods
16