An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (São Paulo, Brazil)

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Veterinary Parasitology 150 (2007) 283–290 www.elsevier.com/locate/vetpar

An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (Sa˜o Paulo, Brazil) Maria Emı´lia Bodini Santiago a, Rosemeri O. Vasconcelos b, Karina Reinaldo Fattori a, Danı´sio Prado Munari c, Aparecida de Fa´tima Michelin a, Vale´ria Marc¸al Felix Lima a,* a

Departamento de Clı´nica, Cirurgia e Reproduc¸a˜o Animal, Faculdade de Odontologia, Curso de Medicina Veterina´ria, Universidade Estadual Paulista, Arac¸atub, SP, Brazil b Departamento de Patologia Veterina´ria, Faculdade de Cieˆncias Agra´rias e Veterina´rias, Universidade Estadual Paulista, Jaboticabal, SP, Brazil c Departamento de Cieˆncias Exatas, Faculdade de Cieˆncias Agra´rias e Veterina´rias, Universidade Estadual Paulista, Jaboticabal, SP, Brazil Received 21 June 2007; received in revised form 26 September 2007; accepted 26 September 2007

Abstract Blood and bone marrow samples were taken from 112 Didelphis spp., collected between March 2005 and February 2006, from urban and peri-urban areas of Bauru, Sa˜o Paulo State, Brazil, to evaluate the hypothesis that these animals might constitute a reservoir of Leishmania spp. Anti-Leishmania ssp. antibodies were screened in the serum samples using an enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR). PCR was performed on fragments of DNA samples from Leishmania spp. using primers 13A and 13B, and showed a positive outcome in 91.6% of the 112 samples tested. Of the 107 samples analyzed by ELISA, 71% were positive. Evidence of epidemiological risk factors such as a circulating parasite and freely moving vectors suggests that Didelphis spp. may participate in the transmission cycle of Leishmania spp. in Bauru. # 2007 Elsevier B.V. All rights reserved. Keywords: Leishmania spp.; Didelphis spp.; ELISA; PCR

1. Introduction Leishmanioses are diseases caused by a digenetic parasite, can appear in different clinical forms, depending on the Leishmania species involved (Gontijo and Carvalho, 2003). In Brazil, the dermotrophic form of the disease is caused by six different species of the

* Corresponding author. Present address: Faculdade de Odontologia de Arac¸atuba, Medicina Veterina´ria, Universidade Estadual Paulista, Rua Clo´vis Pestana 793, 16050-400 Arac¸atuba, SP, Brazil. Tel.: +55 18 36361422; fax: +55 18 36361403. E-mail address: [email protected] (V.M.F. Lima). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.09.026

Leishmania vianna complex (Basano and Camargo, 2004). The visceral form is caused by Leishmania chagasi of the Leishmania donovani complex (Gontijo and Melo, 2004). American cutaneous leishmaniosis was registered for the first time in the city of Bauru, Sa˜o Paulo State, Brazil, in 1908, during deforestation for the construction of the Brazilian Northwest Railway (Genaro, 2003). At the time, the disease was known as ‘‘Bauru ulcer’’. In 2002, the first cases of the visceral form of leishmaniosis were registered in dogs, and the first human cases were reported in 2003. Although the local government implemented control measures, the occurrence of human cases has increased: 65 cases and 4

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deaths were registered during 2006. One control measure, euthanasia, was performed in 4452 dogs of which 27% exhibited a positive diagnosis for leishmanioses, confirmed by laboratorial analysis. Fifty-five percent exhibited classical symptoms of visceral leishmaniosis while 18% suffered from other ailments (Gomieri, 2006 unpublished data). Bauru possesses areas of forest fragments that are undergoing severe anthropic pressures, which leads wild animals to occupy its urban areas. When these animals enter the urban environment and encounter abundant and easy food sources, they incorporate such areas into their habitat. Bauru also has many species of Lutzomyia which are principal vector of Leishmania spp. cycle. The marsupial opossums, Didelphis spp., known in Brazil as the ‘‘gamba´’’, are animals very well adapted to various environments, and are considered examples of evolutionary success (Legey et al., 1999). These opossums are found frequently in forested areas around Bauru and also within the urban environment, habiting the roofs of houses, hospitals, and industrial buildings. In Brazil, Didelphis spp. have been found naturally infected by Leishmania spp. in Manaus, Amazonas State (Arias and Naiff, 1981), in Barra de Guarituba, Rio de Janeiro State (Cabrera et al., 2003) and in Amaraji, Pernambuco State (Branda˜o-Filho et al., 2003). However, although human visceral leishmaniosis now occurs in Sa˜o Paulo State, and Leishmania spp. has been found among Didelphis marsupialis in this state (Yoshida et al., 1979, 1985) suggesting the role of opossums as reservoirs, the prevalence of the infection had not been investigated yet. Through the identification and knowledge of the biology of natural reservoirs, more efficient measures can be taken to control leishmanioses. The present study thus aims to evaluate the prevalence of the Leishmania spp. infection in species of the genus Didelphis spp. in urban and peri-urban areas of Bauru. 2. Material and methods 2.1. Study area Bauru city is located in the center-west region of Sa˜o Paulo State (228180 5300 S; 030 3800 W) and has a total area of 702 km2 with an urban perimeter of 120 km2 (17% of total area). The naturally preserved vegetation around Bauru consists of cerrado, cerrada˜o, and secondary forest, totaling 3771 hectares, which represents 14% of

the municipal area. Pluviometric indices vary from 33 to 286 mm per year, and the mean yearly temperature is 23.6 8C (Anon, 2006). 2.2. Opossum capture and sample collection Opossums were captured using a variety of different manners: with the help of specifically designed wooden traps (73 cm  28 cm  35 cm); by manual capture; by the voluntary delivery of animals by the Fire Brigade, and the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA). Between March 2005 and February 2006, traps baited with bananas, bread, and minced meat were set in the evening in forested areas, in streets, houses, and trees, and near public garbage cans, being checked before dawn. Prior to sample collection, captured animals were examined for the presence of wounds that might suggest leishmaniosis. These individuals then received a microchip implant to avoid duplicating sample collection. Samples were taken after anesthesia by intramuscular injection of ketamine (30 mg/kg) and xylazine (2 mg/kg); 1.0 ml blood was collected from the caudal vein, and 100 ml of bone marrow was collected from the femur under local anesthesia (lidocaine 2%), using a 40  12 needle and a 20 ml syringe. The material was transferred to sterile, 1.5 ml Eppendorf1 tubes containing EDTA and was held at 20 8C until processing. After recovery, each animal was returned to the place of capture or to a forest fragment. Capture was performed with permission from IBAMA (license #113/2005) and was approved by the Ethics Committee of the Universidade Estadual Paulista, Arac¸atuba campus. 2.3. Parasite isolation and detection of amastigotes Approximately 10 ml of bone marrow was transferred to tubes containing 3.0 ml of blood-based agar (BBA) culture medium, (Difco B45) and the same amount of RPMI-1640 (Sigma, USA) supplemented with 10% fetal bovine serum (v/v), antibiotics (100 IU/ ml penicillin and 100 mg/ml streptomycin), hemin (0.01 g/l), folic acid (0.02 g/l), and 2% human male urine, at 26 8C for 4 months and weekly checked under optical microscope to search for promastigote forms. In addition, the bone marrow samples were stained with rapid hematological staining (Laborclin, Brazil) and observed under optical microscope at a 100 magnification to search for amastigote forms of the parasite in the smear.

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2.4. Indirect enzyme-linked immunosorbent assay

2.5. Fucose–mannose ligand–ELISA

The samples were analyzed by ELISA assay using total antigen from lysed promastigotes. The antigen was prepared from Leishmania spp. promastigotes. The parasites were grown at 28 8C in RPMI 1640 (Gibco, Pisley, UK), supplemented with 100 IU/ml of penicillin, 100 mg/ml of streptomycin, 2 mM L-glutamine and 10% heat-inactivated fetal calf serum (Gibco, Pisley, UK). After reaching the stationary phase, the parasites were harvested, washed in phosphate buffered saline (PBS), and lysed by repetitive freeze–thaw cycles until completely disintegrated as determined by microscopic inspection. The protein concentration in the lysed parasites was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL). The ELISA assay was based on the protocol provided by Lima et al. (2003). Micro-plates (Greiner-bio One Microloan 600, Germany) were coated with 20 mg/ml total antigen from L. chagasi (MHOM/ BR00/MERO2) in 100 ml buffer (0.05 M carbonate buffer and pH 9.6) and incubated overnight at 4 8C. The plates were then washed in washing buffer (PBS + 0.05% Tween 201) and blocked for 1 h at room temperature with 150 ml PBS containing 10% fetal bovine serum. After a further wash, 100 ml of opossum serum sample diluted in PBS containing 10% fetal bovine serum and 0.05% Tween 201 (concentration 1:200) were added and the plates were incubated for 1.5 h at room temperature. After incubation, the plates were washed again, and 100 ml of anti-IgG (produced in sheep and diluted 1:100 in PBS + 0.05% Tween 201) were added and incubated for 30 min. The washing process was repeated and 100 ml peroxidaseconjugated protein A (Sigma St. Louis, MO), diluted 1:1000 in the same solution were added. After 30 min at 37 8C, the plates were washed again and the substrate added (o-phenylenediamine and H2O2 in phosphate– citrate buffer). The reaction was stopped by the addition of 50 ml 1N HCl. Absorbance was measured at 490 nm using an automatic reader (Spectra Count, Packard1). The samples were analyzed in duplicate and a blank well (PBS + 0.05% Tween 201 solution) was included in all plates. Owing to the lack of reactivity of the opossum serum to proteins A and G, an anti-IgG opossum antibody was produced. This antibody was isolated from the pooled sera of previously immunized sheep after precipitation with sodium sulfate, purification by ion exchange DEAE chromatography and dialysis (Laborato´rio de Zoonoses e Doenc¸as Transmitidas por Vetores, PMSP).

Isolation and chemical characterization of FML obtained from stationary growth phase promastigotes of L. donovani Sudan (LD IS/MHOM/SD/00-strain IS) was performed as previously described (Palatnik de Sousa et al., 1989). The FML (80 mg/ml) was solubilized in carbonate buffer (pH 9.6) and used to coat flat-bottom 96-well plates (Cornig Incorporated, USA). The plate was incubated for 1 h at room temperature and then overnight at 4 8C. The plates were then washed in washing buffer (PBS + 0.05% Tween 201) and blocked for 1 h at room temperature, with 100 ml PBS containing 5% skimmed milk. After a further wash, 50 ml of opossum’s serum samples diluted in PBS containing 5% skimmed milk and 0.05% Tween 201 (1:50) were added and the plates were incubated for 1 h at 37 8C. After the incubation, the plates were washed again and incubated another hour at 37 8C with 50 ml of anti-IgG, produced in sheep and diluted 1:100 in PBS containing 5% skim milk and 0.05% Tween 201. The washing process was repeated and 50 ml peroxidase-conjugated protein A (Sigma St. Louis, MO) diluted 1:1000 in the same solution was added. After 1 h at 37 8C, the plates were washed again and the substrate added (o-phenykenediamine and H2O2 in phosphate–citrate buffer). The reaction was stopped by the addition of 50 ml 1N HCl. Absorbance was measured at 490 nm using an automatic reader (Spectra Count, Packard1). The samples were analyzed in duplicate and in all plates were included a blank well (PBS + 5% skimmed milk + 0.05% Tween 201 PBS 201 solution). The cut-off was established using sera from negative opossum (PCR and ELISA against total antigen from Leishmania spp. negative), employing the mean value plus 2sd obtained for the group as a reference. 2.6. PCR protocols DNA obtained from the bone marrow aspirate was extracted after lysing by freezing and thawing three times, and washing in 1 SSC buffer solution (NaCl 3 M, sodium citrate 0.3 M, and pH 7.0). For digestion, 300 ml of lysing solution were added (10% SDS in 0.2 M sodium acetate) together with 20 mg/ml proteinase K. Samples were incubated at 56 8C for 2 h and the DNA was extracted using the phenol/chloroform/isoamyl alcohol method (25:24:1) according to Sambrook et al. (1989). After extraction, DNA was re-suspended in 50 ml TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, and pH 8.0) and incubated for 3 min at 60 8C. The material was held at

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20 8C until use. The 13A (30 -GTG GGG GAG GGG CGT TCT-50 ) and 13B (30 -ATT TTA CAC CAA CCC CCA GTT-50 ) primers were used (Rodgers et al., 1990) to amplify a 120 bp fragment in the constant region of the kinetoplast minicircles in all Leishmania species. The primers 13A and 13B were previously shown to be specific in Leishmania species (Reale et al., 1999). The polymerase chain reaction was undertaken in a 60 ml volume containing 30 pmol of each indicator (Invitrogen1), 0.2 mM DNTPs (Invitrogen1), 1.5 mM MgCl2 (Invitrogen1), 5 U Taq DNA polymerase (Invitrogen1), 50 nM buffer solution, milliQ water and 100–700 ng DNA. Amplification was performed in an Eppendorf1 Mastercycler Thermocycler gradient with initial heating to 95 8C for 5 min, followed by 33 cycles at 95–57–72 8C for 1.5, 1.5, and 2 min, respectively. Extension was carried out at 72 8C for 10 min and the final product was kept at 20 8C until analysis. For each reaction, a negative control (no DNA), a positive control with DNA extracted from a L. chagasi promastigote culture (MHOM/BR00/MER02) and two molecular weights were added. The amplified product was analyzed by electrophoresis in 2% agarose gel followed by ethidium bromide staining and visualization under UV light. 2.7. Statistical analysis McNemar’s non-parametric test and the Kappa coefficient were used to compare the positive indices furnished by the two diagnostic methods used, ELISA and PCR.

Fig. 1. Serum reactivity against total antigen from Leishmania (L.) chagasi of opossum from an endemic area for leishmaniosis. The horizontal line represents the cut-off value (0.349).

In indirect ELISA 107 samples were assay (5 samples collected were lost) the cut-off value of 0.349 was established using an ROC curve (MedCalc, Version 9.0.1.1). The ELISA assays demonstrated that 29% (31/107) of the sera were negative, and 71% (76/107) were positive for Leishmania antibodies. A comparative analysis of the 107 paired serum samples tested by ELISA and PCR showed that 67.3% were positive and 4.7% were negative by both tests; 24.3% were positive by PCR alone, and 3.7% by ELISA alone (Fig. 1). Analysis of the ELISA findings, considering the PCR finding as a gold test, gave 73.5% sensibility and 66.7% specificity (Table 1). 3.4. Fucose–mannose ligand–ELISA

3. Results 3.1. Captured animals Of the 112 opossums captured, 102 were Didelphis albiventris and 10 were Didelphis aurita. On clinical examination, no animal showed lesions that might suggest leishmaniosis. 3.2. Parasite isolation and detection of amastigotes

The positive samples in both PCR and indirect ELISA crude antigen were submitted to fucose– mannose ligand–ELISA and 25% were seropositive (Fig. 2).

Table 1 Performance of ELISA considering PCR as gold standard to diagnose Leishmania spp. in Didelphis spp. PCR

Flagellated parasite forms were found in 18.8% (21/112) of the cultures while 16.1% (18/112) of the smears contained amastigote forms. 3.3. PCR and indirect ELISA PCR of the bone marrow 112 samples tested, 91.6% (103/112) were positive and 8% (9/112) were negative.

Total

Positive

Negative

ELISA Positive Negative

59 21

12 15

71 36

Total

80

27

107

73.5% –

– 66.7%

Sensitivity Specificity

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Fig. 2. Fucose–mannose ligand (FML)–ELISA reactivity with sera of opossum from an endemic area for leishmanioses. The horizontal line represents the cut-off value (0.083).

3.5. Statistics The McNemar test indicated a significant difference between the positivity values obtained by the ELISA and PCR methods. The k value (0.195) was considered poor. 4. Discussion Wild animals constitute important reservoirs of Leishmania spp. (Jacobson et al., 2003), and many mammalian species, especially rodents, edentates, carnivores, primates and marsupials carry Leishmania spp. (Shaw and Lainson, 1987; Grimaldi and Tesh, 1993). The opossum Didelphis spp. can harbor both the viscerotrophic and the dermotrophic strains of Leishmania (Franco, 1990). Experimental infection of D. marsupialis with L. chagasi reveals that this species can present the disease at a sub-clinical level (Travi et al., 1998). In this study, no animal showed lesion that might suggest leishmaniosis, but 71% of them were Leishmania seropositive by ELISA, and 91.6% by PCR. The ELISA test is usually employed in epidemiological studies and is also used to complement the diagnosis of various diseases, visceral leishmaniosis among them (Camargo-Neves et al., 2006). The use of ELISA to detect anti-Leishmania spp. antibodies in the opossum has not been performed before. Previous investigations employing different sorological tests in opossum have found 29% positivity in Barra de Guaratiba, Rio de Janeiro State, using an indirect immunofluorescent assay (Cabrera et al., 2003), and a 61.9% infection rate in animals from Manaus, Amazonas State, using the culture method of Arias and Naiff. The percentage positivities for the Bauru

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samples analyzed here are similar to those reported for Manaus. Although the ELISA method can be useful to identify animals with a positive profile, there are certain limitations to the technique such as cross-reactivity between Leishmania spp. and Trypanosoma (Rosa´rio et al., 2005; Rami et al., 2005; Nunes et al., 2006) and between Leishmania spp. and Babesia (Rosa´rio et al., 2005), creates false positives. Opossums of the genus Didelphis can serve as reservoirs for Trypanosoma (Deane et al., 1984; Legey et al., 1999; Bar et al., 1999; Grisard et al., 2000; Ruiz-Pin˜a and Cruz-Reyes, 2002; Gurgel-Gonc¸alves et al., 2004) and Babesia (Herrera and Urdaneta-Morales, 1991). Simultaneous infection of D. marsupialis by Trypanosoma cruzi and L. chagasi has been demonstrated (Travi et al., 1994). A further limitation to the ELISA technique in Didelphis seen here was the lack of reactivity by IgG to peroxidaseconjugated proteins A and G, an inconvenience that required the production of an opossum anti-serum in sheep. The fact that conventional serological techniques use crude antigens, considerably limits their specificity. To overcome this problem the antibody response in leishmaniosis has been studied using other antigens. The fucose–mannose ligand (FML) is a surface glycoprotein complex isolated from L. donovani promastigotes that has a sensibility of 100% and a specificity of 96% when used to diagnose kala-azar (infection by L. chagasi) in humans from Brazil. The specificity of FML is even higher (100%), still with same sensibility, when it was used to diagnose canine visceral leishmaniosis (Cabrera et al., 1999). In addition, no cross-reaction was observed in patients infected with other Leishmania spp. species, nor in those with Chagas disease (Palatnik de Sousa et al., 1995). The FML–ELISA showed 25% of seropositivity suggesting that other than viscerotrophic Leishmania species could be shelter by opossum. The percentage of animals from Bauru showing a positive profile (91.6%) is much greater than that reported by other investigators like Zulueta et al. (1999), who found 7% positivity by PCR testing in blood, liver, and spleen samples from opossums in Venezuela. Telleria et al. (1999) found no opossums with a positive profile in Cajuata (Bolivia) while Branda˜o-Filho et al. (2003), reported 13.3% positivity using spleen samples in Amaraji, Pernambuco State. The high positivity found in the present study may derive from the analysis of bone marrow rather than blood. Mello and Teixeira (1984) noticed that greater quantities of amastigotes could be found in bone

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marrow and spleen tissues when compared with blood and other tissues. Our comparison of the two analytical methods revealed samples positive by PCR but negative by ELISA. Such findings may be a consequence of the small number of parasites present in the tissues analyzed, since a positive correlation between antibody titers in the serum and number of parasites has been reported in dogs (Reis et al., 2006). The PCR reaction can be positive when the parasite load is low. However, antibody production is stimulated only by a higher antigenic load. The positivity seen in samples by ELISA with corresponding negative results by PCR may be explained by the cross-reactivity of the antibodies to other antigens, not evaluated in the samples used. The difference observed in results obtained in PCR and ELISA assay might be associated with the technique. In PCR, primer that detected genera was used, the positivity observed includes different species, however in ELISA assay the crude antigen from Leishmania (L.) chagasi should be more sensitive in order to detect infection of this species, our results pointed that opossums are infected with other Leishmania spp. species, because FML–ELISA showed only 25% of seropositivity, suggesting that other than viscerotrophic Leishmania species could be sheltered by opossum. In fact, previous paper had been shown higher sensitivity for the detection of IgG in sera from dogs with visceral leishmaniosis when homologous antigen was used (L. (L.) chagasi) compared to heterologous antigens (L. (V.) braziliensis), and L. (L.) amazonensis) (Baleeiro et al., 2006), in American tegumentary leishmaniosis in dogs the autologous antigen also increase the sensibility (Ribeiro et al., 2007). In addition, several studies in human and dogs had been showed that the PCR detected higher positivity rates when compared to other diagnostic methods like anti-Leishmania serological testing, culture, and microscopic examination of lesion biopsy specimens (Riera et al., 2005; Rodrigues et al., 2002; Oliveira et al., 2005) similar to our finding. The high incidence of infection seen in opossums suggests that these species participate in the transmission cycle of Leishmania spp. This hypothesis is corroborated by a study demonstrating the feeding preference of the vector Lutzomyia evansi for D. marsupialis when compared with the squirrel Sciurus granatensis, and the rodents Heteromys anomalus and Zygodontomys brevicaudata (Adler et al., 2003). The high incidence of infection in opossums from an urban area suggests that these species may provide a natural

bridge between the wild and domestic cycles of the parasite owing to their co-existence with dogs. Considering the various relevant factors such as the presence of elevated numbers of Lutzomyia spp. in the city, the synatropic nature of the opossum, the presence of these animals in the urban environment in Bauru since 1999 (Santiago, 2006 unpublished data), the sharing of food by dogs and opossums, and the fact that the appearance of the disease in the dog precedes that in humans (Neves et al., 2001; Zapata, 2002), the hypothesis that the migration of opossums into the urban environment of Bauru has influenced the occurrence of visceral leishmaniosis in this city seems likely. In conclusion, this study reveals high levels of infection by Leishmania spp. in Didelphis spp., suggesting that opossums may participate in the epidemiological cycle of Leishmania spp. in Bauru, Sa˜o Paulo, Brazil. Acknowledgments This work was financed by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). We thank IBAMA and the 128 Grupamento de Bombeiros de Bauru. References Adler, G.H., Becerra, M.T., Travi, B.L., 2003. Feeding success of Lutzomyia evansi (Dı´ptera: Psychodidae) experimentally exposed to small mammal host in an endemic focus of Leishmania chagasi in northern Colombia. Biome´dica 23, 396–400. www.bauru.sp.gov.br/dadosgeograficos (Acesso 30 de outubro de 2006). Arias, J.R., Naiff, R.D., 1981. The principal reservoir host of cutaneous leishmaniasis in the urban areas of Manaus, central Amazon of Brazil. Mem. Inst. Oswaldo Cruz 76, 279–286. Baleeiro, C.O., Paranhos-Silva, M., dos Santos, J.C., Oliveira, G.G., Nascimento, E.G., de Carvalho, L.P., dos-Santos, W.L., 2006. Montenegro’s skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area. Vet. Parasitol. 139, 21–28. Bar, M.E., Alvarez, B.M., Oscherov, E.B., Damborsky, J.M.E., 1999. Contribucion al conocimiento de los reservorios del Trypanosoma cruzi (Chagas, 1909) em la Provı´ncia de Corrientes. Argent. Rev. Soc. Braz. Med. Trop. 32, 271–276. Basano, S.A., Camargo, L.M.A., 2004. Leishmaniose tegumentar americana: histo´rico, epidemiologia e perspectivas de controle. Rev. Braz. Epidemiol. 7, 328–337. Branda˜o-Filho, S.P., Brito, M.E., Carvalho, F.G., Ishikawa, E.A., Cupolillo, E., Floeter-Winter, L., Shaw, J.J., 2003. Wild and synanthropic host of Leishmania (Viannia) braziliensis in the endemic cutaneous leismaniasis locality of Amaraji, Pernambuco State, Brazil. Trans. Roy. Soc. Trop. Med. Hyg. 97, 291–296.

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