PCR Associated with Molecular Hybridization Detects Leishmania (Viannia) braziliensis in Healthy Skin in Canine Tegumentary Leishmaniasis Author(s): Guilherme Marx de Oliveira, Maria de Fatima Madeira, Fernanda S. Oliveira, and Raquel S. Pacheco Source: Journal of Parasitology, 101(1):91-93. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/14-567.1 URL: http://www.bioone.org/doi/full/10.1645/14-567.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
J. Parasitol., 101(1), 2015, pp. 91–93 Ó American Society of Parasitologists 2015
PCR ASSOCIATED WITH MOLECULAR HYBRIDIZATION DETECTS LEISHMANIA (VIANNIA) BRAZILIENSIS IN HEALTHY SKIN IN CANINE TEGUMENTARY LEISHMANIASIS Guilherme Marx de Oliveira, Maria de Fatima Madeira, Fernanda S. Oliveira, and Raquel S. Pacheco Laborat´ orio de Epidemiologia e Sistema´tica Molecular, Instituto Oswaldo Cruz, Funda¸c˜ao Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, 21040-360. Correspondence should be sent to: [email protected]
ABSTRACT:
Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis` a-vis the importance of the domestic dogs in endemic areas of TL. samples were seeded in biphasic medium (NNN/Schneider’s) plus 10% fetal bovine serum and incubated at 26–28 C. The culture was examined weekly for 30 days. For PCR, the samples were kept at 20 C until the use in the assays. All procedures performed in this study was approved and licensed by the Ethics Committee of Animal Users (CEUA-FIOCRUZ) under No. L23/06. For DNA isolation, the Illustra Tissue and Cells GenomicPrep Mini Spin kit (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, U.K.) was used according to the manufacturer’s instructions. The parasite DNA was detected by PCR amplification with the use of a pair of primers (B1: 5 0 -GGGGTTGGTGTAATATAGTGG-3 0 ; B2: 5 0 -CTAATTGTGCACGGGGAGG-3 0 ) that amplify the variable region of kDNA minicircles of species of the subgenus Viannia belonging to Leishmania braziliensis complex (De Bruijn and Barker, 1992). The PCR reaction was performed in a final volume of 25.0 ll containing 3.0 ll of sample DNA, Tris-HCl 10 mM buffer solution, MgCl2 1.5 mM, dNTP 0.2 mM, 10 pmol of each primer, and 2.5 U Taq DNA polymerase. Amplification cycles were started at 95 C for 3 min, followed by 33 cycles at 94 C for 30 sec, 60.5 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 10 min. The purified kDNA (5 ng/ll) of the reference strain L. (V.) braziliensis (MHOM/BR/1975/M2903) was used as a positive control in all PCR reactions and the negative control was composed of all the components, except DNA. To confirm the absence of inhibition factors, an internal control corresponding to a segment of the beta-globin gene was amplified in all samples (Saiki et al., 1985). The amplified products were analyzed by horizontal electrophoresis on 1.5% agarose gels and visualized under ultraviolet light and photographed in a gel documentation system (BioDoc.It Gel Documentation system, UVP an Analytik Jena Company Upland, California). The amplified products were transferred by passive transfer to nylon membranes, and molecular hybridization was performed according to the protocols previously described (Pacheco et al., 2000; Oliveira et al., 2005) with the use of the radioactively labeled kDNA of L. (V.) braziliensis (MHOM/BR/1975/M2903) reference strain as probe. The hybridization was performed under a high stringency (1.53 saline sodium citrate [SSC], 1% sodium dodecyl sulfate [SDS], and 0.5% skim milk at 60 C, overnight) condition. The membranes were washed 3 times in a solution containing 0.1X SSC and 0.5% SDS for 30 min at 60 C and exposed to X-ray film (Kodak X-OMAT, Sigma-Aldrich, St. Louis, Missouri) at 70 C overnight.
Leishmaniasis is a vector-borne protozoal disease affecting approximately 350 million people (World Health Organization [WHO], 2010). According to a recent review (Alvar et al., 2012) a total of 98 countries and 3 territories on 5 continents reported endemic leishmaniasis transmission. In the new world, Brazil has the highest incidence, with approximately 28,000 cases of ´ tegumentary leishmaniasis (TL) per year (Minist erio da Saude, ´ 2006). In this country, the cutaneous form is caused by at least 7 different species of Leishmania belonging to the subgenera Viannia and Leishmania, and Leishmania (Viannia) braziliensis is the prevalent species, affecting humans and domestic animals, mainly dogs (Marzochi and Marzochi, 1994). The role of the domestic dog in the transmission cycle of TL is still not completely understood, and this issue has been discussed for a long time (Reithinger and Davies, 1999; Padilla et al., 2002; Dantas-Torres, 2007, 2011). One reason for this debate is related to the frequent presence of infected dogs in all endemic areas of TL and the failure to detect L. (V.) braziliensis in other anatomical sites out of the cutaneous lesion (Madeira et al., 2005, 2006), suggesting that the sandflies may have a low probability of getting infected when feeding from this site (Silveira et al., 1989). At the diagnostic level, PCR with or without molecular hybridization has contributed to the diagnosis and to our understanding of the epidemiology of TL (Oliveira et al., 2005; Marco et al., 2012). Given the potential importance of the dogs in the transmission cycle of TL, our study aimed to perform a specific PCR assay associated with molecular hybridization to detect DNA of L. (V.) braziliensis in tissue samples obtained from naturally infected dogs from the municipality of Rio de Janeiro, Brazil. MATERIALS AND METHODS Nine naturally infected dogs were analyzed in this study. Infection in these animals was confirmed by identification of L. (V.) braziliensis isolated from cutaneous lesion fragments. These dogs were gathered in the municipality of Rio de Janeiro and were euthanized, because they showed positive serologic reactivity to Leishmania according to the Brazilian ´ Program for the Control of Leishmaniasis (Ministerio da Saude, ´ 2006). Cutaneous lesions and healthy skin tissue samples were obtained by biopsy procedure after euthanasia. For culture, the tissue fragments were kept in saline containing 1,000 U penicillin, 200 lg streptomycin, and 50 lg 5 0 -fluorocytocine per milliliter and stored at 4 C for 24 hr. Then the
RESULTS Parasite isolation in culture media was obtained in all lesion fragments processed, but healthy skin samples were 100% negative by this method. The PCR assays revealed the presence of L. (V.) braziliensis DNA in all lesions samples, showing diagnostic bands of 750 base pairs, confirming the previous results obtained by isoenzyme electrophoresis. In 3 dogs, the L. (V.) braziliensis DNA was also detected in healthy skins (Table I).
Received 9 May 2014; revised 30 July 2014; accepted 4 August 2014. DOI: 10.1645/14-567.1 91
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THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 1, FEBRUARY 2015
TABLE I. Culture and specific PCR results of cutaneous lesion and healthy skin fragments collected from 9 dogs. þ, positive results; , negative results. Culture Dog 1 2 3 4 5 6 7 8 9
PCR
Lesion
Healthy skin
Lesion
Healthy skin
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ
The specificity of the PCR amplified products was confirmed by hybridization with the use of radioactively labeled kDNA from the reference strain of L. (V.) braziliensis as probe. A great copy number of homologous sequences to the kDNA of L. (V.) braziliensis reference stain were detected in all amplified products (Fig. 1). All clinical samples had previously been submitted to inhibition tests, thereby avoiding false-negative results. PCR-negative samples showed no reactivity with the probe after confirmatory testing by molecular hybridization (not shown). DISCUSSION The role of domestic dog in the transmission cycle of TL has been widely discussed, particularly in Brazil, where this disease is quite prevalent in these animals (Marzochi and Marzochi, 1994). To our knowledge, this is the first study in Brazil that shows the presence of L. (V.) braziliensis DNA in healthy skin of naturally infected dogs. Dogs are reservoirs of L. (L.) chagasi (syn. L. infantum) and infections can be spread worldwide (Dantas-Torres, 2007). Euthanasia of seropositive dogs is 1 of the control measures used ´ for visceral leishmaniasis (VL) in Brazil (Ministerio da Saude, ´ 2006). However, the possibility of cross-reactions, mainly in areas of overlapping with other agents, is a common occurrence (Madeira et al., 2006). In this context, all dogs herein studied came from municipality of Rio de Janeiro where L. (V.) braziliensis and L. (L.) chagasi coexist. These dogs were euthanized for suspicion of VL supporting the need for accurate diagnoses in this region (Silva et al., 2011). The parasite’s isolation in culture media is considered the gold standard in the diagnosis of Leishmania sp. infection and in VL, healthy skin has also proven to be an excellent target for this approach in dogs (Madeira et al., 2009; Almeida et al., 2011). In this study, it was only possible to detect L. (V.) braziliensis by PCR, suggesting low parasite load on this site in comparison to cutaneous lesion samples. Nonetheless, this finding adds elements in the discussion about the role of dogs in the transmission cycle of TL. Furthermore, the detection of L. (V.) braziliensis DNA in all lesion fragments analyzed demonstrates reliability in the use of PCR as a tool in TL diagnostic. Although the role of the domestic dog in the transmission cycle of TL is unclear, some studies have suggested that this animal acts
FIGURE 1. (A) PCR products amplified with primers B1/B2 specific for the Leishmania braziliensis complex and visualized on ethidium-bromide– stained 1.5% agarose gel, showing the diagnostic bands (750 base pairs [bp]). M: 100-bp DNA ladder size marker; lane 1: cutaneous lesion sample from dog 1; lanes 2–4: healthy skin from dogs 1, 2, and 9; lane 5: negative control (no DNA); lane 6: positive control (purified kDNA from Leishmania (Vianna) braziliensis [MHOM/BR/1975/M2903]). (B) Southern blot hybridization of the amplified products with the use of kDNA from Leishmania (V.) braziliensis MHOM/BR/1975/M2903 as probe.
as an accidental host (Padilla et al., 2002; Dantas-Torres, 2011). Indeed, little is known regarding the tissue tropism of L. (V.) braziliensis in naturally infected dogs. Our group has been focused on this issue and results recently obtained showed that infection in dogs can be manifested by dissemination and tissue tropism of genetically distinct L. (V.) braziliensis populations (Oliveira et al., 2013). This finding is important and contributes to a better understanding of the epidemiology of canine tegumentary leishmaniasis. The finding of L. (V.) braziliensis DNA in healthy skin suggests a much larger role than thought for the domestic dog, in view of their large presence in an urban environment. This assumption may be closer to reality, because in previous investigations we have reported the persistence and viability of L. braziliensis in human scars many years after cure of the cutaneous lesion (Schubach et al., 1998). In this scenario, the possibility of sandflies becoming infected when fed outside of the lesion, must then be considered. In this way our results suggest the possibility of more active participation of the domestic dog in the transmission cycle of TL. The PCR, with the use of target kDNA minicircles, combined with molecular hybridization as a confirmatory method, proved to be a very useful tool for detection of L. (V.) braziliensis DNA in healthy skin of naturally infected dogs. The sequencing of the amplicon would be an alternative, but the purified kDNA of L. (V.) braziliensis, used as a probe in high-stringency hybridization conditions, has been demonstrated not to cross-react with members of the Leishmania subgenus. Finally, clarifying the real importance of dogs in the transmission cycle of TL is of great importance to public health, so that effective measures to control the disease can be defined and specific protocols for handling these infected animals can be established. ACKNOWLEDGMENTS This work received financial support from Conselho Nacional de ´ Desenvolvimento Cient ´ıfico e Tecnologico (CNPq) and Instituto Kinder do Brasil (IKB). R. S. Pacheco and M. F. Madeira are the recipients of fellowships from CNPq.
DE OLIVEIRA ET AL.—L. BRAZILIENSIS IN HEALTHY SKIN IN CANINE TEGUMENTARY LEISHMANIASIS
LITERATURE CITED ALMEIDA, A. B. F., V. R. SOUSA, E. C. BOA-SORTE, F. B. FIGUEIREDO, D. A. DE PAULA, M. F. PIMENTEL, V. DUTRA, AND M. F. MADEIRA. 2011. Use of parasitological culture to detect Leishmania (Leishmania) chagasi in naturally infected dogs. Vector-Borne and Zoonotic Diseases 11: 1555–1560. ALVAR, J., I. D. VE´ LEZ, C. BERN, M. P. DESJEUX, J. CANO, J. JANNIN, M. DEN BOER, and WHO LEISHMANIASIS CONTROL TEAM. 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671. DANTAS-TORRES, F. 2007. The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Veterinary Parasitology 149: 139– 146. ———. 2011. Dogs as reservoirs for Leishmania braziliensis. Emerging Infectious Diseases 17: 326–327. DE BRUIJN, M. H. L., AND D. C. BARKER. 1992. Diagnosis of New World leishmaniasis: Specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Tropica 52: 45–58. MADEIRA, M. F., F. B. FIGUEIREDO, A. G. PINTO, L. D. NASCIMENTO, M. FURTADO, E. MOUTA-CONFORT, C. C. DE PAULA, A. BOGIO, M. C. GOMES, A. M. BESSA, ET AL. 2009. Parasitological diagnosis of canine visceral leishmaniasis: Is intact skin a good target? Research in Veterinary Science 87: 260–262. ———, A. O. SCHUBACH, T. M. SCHUBACH, S. A. PEREIRA, F. B. FIGUEIREDO, C. BAPTISTA, C. A. LEAL, C. X. MELO, E. M. CONFORT, AND M. C. MARZOCHI. 2006. Post mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil. Veterinary Parasitology 138: 366–370. ———, ———, ———, C. M. SERRA, S. A. PEREIRA, F. B. FIGUEIREDO, E. M. CONFORT, L. P. QUINTELLA, AND M. C. MARZOCHI. 2005. Is Leishmania (Viannia) braziliensis preferentially restricted to the cutaneous lesions of naturally infected dogs? Parasitology Research 97: 73–76. MARCO, J. D., P. A. BARROSO, T. MIMORI, F. M. LOCATELLI, A. TOMATANI, M. C. MORA, S. P. CAIAL, J. R. NASSER, L. A. PARADA, T. TANIGUCHI, ET AL. 2012. Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina. BMC Infectious Diseases 12: 1–8. MARZOCHI, M. C. A., AND K. B. F. MARZOCHI. 1994. Tegumentary and visceral leishmaniasis in Brazil: Emerging anthropozoonosis and possibilities for their control. Cadernos de Saude ´ Publica ´ 10: 359–375. ´ ´ DA SAUDE . 2006. Manual de Vigilancia ˆ e Controle da MINISTERIO ´ ´ Leishmaniose Visceral. Serie A. Normas e Manuais Tecnicos. ´ Ministerio da Saude, ´ Brasilia, DF, Brasil, 120 p.
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OLIVEIRA, F. S., C. PIRMEZ, M. Q. PIRES, R. P. BRAZIL, AND R. S. PACHECO. 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. Veterinary Parasitology 129: 219– 227. OLIVEIRA, G. M., M. F. MADEIRA, F. S. OLIVEIRA, M. Q. PIRES, AND R. S. PACHECO. 2013. Canine cutaneous leishmaniasis: Dissemination and tissue tropism of genetically distinct Leishmania (Viannia) braziliensis populations. Veterinary Medicine International 3013: 1–4. PACHECO, R. S., O. FERNANDES, G. SALINAS, I. SEGURA, H. MOMEN, W. DEGRAVE, N. G. SARAVIA, AND D. A. CAMPBELL. 2000. Intraspecific heterogeneity in the mini-xon gene localization of Leishmania (V.) panamensis and Leishmania (V.) guyanensis from Colombia. Journal of Parasitology 86: 1250–1253. PADILLA, A. M., J. D. MARCO, P. DIOSQUE, M. A. SEGURA, M. C. MORA, M. M. FERNA´NDEZ, E. L. MALCHIODI, AND M. A. BASOMBRIO. 2002. Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina. Veterinary Parasitology 110: 1–10. REITHINGER, R., AND C. R. DAVIES. 1999. Is the domestic dog (Canis familiaris) a reservoir host of American cutaneous leishmaniasis? A critical review of the current evidence. American Journal of Tropical Medicine and Hygiene 61: 530–541. SAIKI, R. K., S. SCHARF, F. FALOONA, K. B. MULLIS, G. T. HORN, H. A. ERLICH, AND N. ARNHEIM. 1985. Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 20: 1350–1354. SCHUBACH, A., M. C. MARZOCHI, T. CUZZI-MAYA, A. V. OLIVEIRA, M. L. ARAUJO, A. L. OLIVEIRA, R. S. PACHECO, H. MOMEN, F. CONCEICAOSILVA, S. G. COUTINHO, ET AL. 1998. Cutaneous scars in American tegumentary leishmaniasis patients: A site of Leishmania (Viannia) braziliensis persistence and viability eleven years after antimonial therapy and clinical cure. American Journal of Tropical Medicine and Hygiene 58: 824–827. SILVA, D. A., M. F. MADEIRA, A. C. TEIXEIRA, C. M. SOUZA, AND F. B. FIGUEIREDO. 2011. Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Veterinary Parasitology 179: 257–261. SILVEIRA, F. T., R. LAINSON, J. J. SHAW, E. A. ISHIKAWA, A. A. SOUZA, AND R. R. BRAGA. 1989. Sensitivity of the culture of circulating leukocytes in the detection of Leishmania in the peripheral blood of patients with tegumentary leishmaniasis. Revista da Sociedade Brasileira de Medicina Tropical 22: 143–146. WHO (WORLD HEALTH ORGANIZATION). 2010. Technical Report Series 949. Report of a meeting of the WHO Expert Committee on the control of leishmaniasis, Geneva, Switzerland, 186 p.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
J. Parasitol., 101(1), 2015, pp. 91–93 Ó American Society of Parasitologists 2015
PCR ASSOCIATED WITH MOLECULAR HYBRIDIZATION DETECTS LEISHMANIA (VIANNIA) BRAZILIENSIS IN HEALTHY SKIN IN CANINE TEGUMENTARY LEISHMANIASIS Guilherme Marx de Oliveira, Maria de Fatima Madeira, Fernanda S. Oliveira, and Raquel S. Pacheco Laborat´ orio de Epidemiologia e Sistema´tica Molecular, Instituto Oswaldo Cruz, Funda¸c˜ao Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, 21040-360. Correspondence should be sent to: [email protected]
ABSTRACT:
Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis` a-vis the importance of the domestic dogs in endemic areas of TL. samples were seeded in biphasic medium (NNN/Schneider’s) plus 10% fetal bovine serum and incubated at 26–28 C. The culture was examined weekly for 30 days. For PCR, the samples were kept at 20 C until the use in the assays. All procedures performed in this study was approved and licensed by the Ethics Committee of Animal Users (CEUA-FIOCRUZ) under No. L23/06. For DNA isolation, the Illustra Tissue and Cells GenomicPrep Mini Spin kit (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, U.K.) was used according to the manufacturer’s instructions. The parasite DNA was detected by PCR amplification with the use of a pair of primers (B1: 5 0 -GGGGTTGGTGTAATATAGTGG-3 0 ; B2: 5 0 -CTAATTGTGCACGGGGAGG-3 0 ) that amplify the variable region of kDNA minicircles of species of the subgenus Viannia belonging to Leishmania braziliensis complex (De Bruijn and Barker, 1992). The PCR reaction was performed in a final volume of 25.0 ll containing 3.0 ll of sample DNA, Tris-HCl 10 mM buffer solution, MgCl2 1.5 mM, dNTP 0.2 mM, 10 pmol of each primer, and 2.5 U Taq DNA polymerase. Amplification cycles were started at 95 C for 3 min, followed by 33 cycles at 94 C for 30 sec, 60.5 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 10 min. The purified kDNA (5 ng/ll) of the reference strain L. (V.) braziliensis (MHOM/BR/1975/M2903) was used as a positive control in all PCR reactions and the negative control was composed of all the components, except DNA. To confirm the absence of inhibition factors, an internal control corresponding to a segment of the beta-globin gene was amplified in all samples (Saiki et al., 1985). The amplified products were analyzed by horizontal electrophoresis on 1.5% agarose gels and visualized under ultraviolet light and photographed in a gel documentation system (BioDoc.It Gel Documentation system, UVP an Analytik Jena Company Upland, California). The amplified products were transferred by passive transfer to nylon membranes, and molecular hybridization was performed according to the protocols previously described (Pacheco et al., 2000; Oliveira et al., 2005) with the use of the radioactively labeled kDNA of L. (V.) braziliensis (MHOM/BR/1975/M2903) reference strain as probe. The hybridization was performed under a high stringency (1.53 saline sodium citrate [SSC], 1% sodium dodecyl sulfate [SDS], and 0.5% skim milk at 60 C, overnight) condition. The membranes were washed 3 times in a solution containing 0.1X SSC and 0.5% SDS for 30 min at 60 C and exposed to X-ray film (Kodak X-OMAT, Sigma-Aldrich, St. Louis, Missouri) at 70 C overnight.
Leishmaniasis is a vector-borne protozoal disease affecting approximately 350 million people (World Health Organization [WHO], 2010). According to a recent review (Alvar et al., 2012) a total of 98 countries and 3 territories on 5 continents reported endemic leishmaniasis transmission. In the new world, Brazil has the highest incidence, with approximately 28,000 cases of ´ tegumentary leishmaniasis (TL) per year (Minist erio da Saude, ´ 2006). In this country, the cutaneous form is caused by at least 7 different species of Leishmania belonging to the subgenera Viannia and Leishmania, and Leishmania (Viannia) braziliensis is the prevalent species, affecting humans and domestic animals, mainly dogs (Marzochi and Marzochi, 1994). The role of the domestic dog in the transmission cycle of TL is still not completely understood, and this issue has been discussed for a long time (Reithinger and Davies, 1999; Padilla et al., 2002; Dantas-Torres, 2007, 2011). One reason for this debate is related to the frequent presence of infected dogs in all endemic areas of TL and the failure to detect L. (V.) braziliensis in other anatomical sites out of the cutaneous lesion (Madeira et al., 2005, 2006), suggesting that the sandflies may have a low probability of getting infected when feeding from this site (Silveira et al., 1989). At the diagnostic level, PCR with or without molecular hybridization has contributed to the diagnosis and to our understanding of the epidemiology of TL (Oliveira et al., 2005; Marco et al., 2012). Given the potential importance of the dogs in the transmission cycle of TL, our study aimed to perform a specific PCR assay associated with molecular hybridization to detect DNA of L. (V.) braziliensis in tissue samples obtained from naturally infected dogs from the municipality of Rio de Janeiro, Brazil. MATERIALS AND METHODS Nine naturally infected dogs were analyzed in this study. Infection in these animals was confirmed by identification of L. (V.) braziliensis isolated from cutaneous lesion fragments. These dogs were gathered in the municipality of Rio de Janeiro and were euthanized, because they showed positive serologic reactivity to Leishmania according to the Brazilian ´ Program for the Control of Leishmaniasis (Ministerio da Saude, ´ 2006). Cutaneous lesions and healthy skin tissue samples were obtained by biopsy procedure after euthanasia. For culture, the tissue fragments were kept in saline containing 1,000 U penicillin, 200 lg streptomycin, and 50 lg 5 0 -fluorocytocine per milliliter and stored at 4 C for 24 hr. Then the
RESULTS Parasite isolation in culture media was obtained in all lesion fragments processed, but healthy skin samples were 100% negative by this method. The PCR assays revealed the presence of L. (V.) braziliensis DNA in all lesions samples, showing diagnostic bands of 750 base pairs, confirming the previous results obtained by isoenzyme electrophoresis. In 3 dogs, the L. (V.) braziliensis DNA was also detected in healthy skins (Table I).
Received 9 May 2014; revised 30 July 2014; accepted 4 August 2014. DOI: 10.1645/14-567.1 91
92
THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 1, FEBRUARY 2015
TABLE I. Culture and specific PCR results of cutaneous lesion and healthy skin fragments collected from 9 dogs. þ, positive results; , negative results. Culture Dog 1 2 3 4 5 6 7 8 9
PCR
Lesion
Healthy skin
Lesion
Healthy skin
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ
The specificity of the PCR amplified products was confirmed by hybridization with the use of radioactively labeled kDNA from the reference strain of L. (V.) braziliensis as probe. A great copy number of homologous sequences to the kDNA of L. (V.) braziliensis reference stain were detected in all amplified products (Fig. 1). All clinical samples had previously been submitted to inhibition tests, thereby avoiding false-negative results. PCR-negative samples showed no reactivity with the probe after confirmatory testing by molecular hybridization (not shown). DISCUSSION The role of domestic dog in the transmission cycle of TL has been widely discussed, particularly in Brazil, where this disease is quite prevalent in these animals (Marzochi and Marzochi, 1994). To our knowledge, this is the first study in Brazil that shows the presence of L. (V.) braziliensis DNA in healthy skin of naturally infected dogs. Dogs are reservoirs of L. (L.) chagasi (syn. L. infantum) and infections can be spread worldwide (Dantas-Torres, 2007). Euthanasia of seropositive dogs is 1 of the control measures used ´ for visceral leishmaniasis (VL) in Brazil (Ministerio da Saude, ´ 2006). However, the possibility of cross-reactions, mainly in areas of overlapping with other agents, is a common occurrence (Madeira et al., 2006). In this context, all dogs herein studied came from municipality of Rio de Janeiro where L. (V.) braziliensis and L. (L.) chagasi coexist. These dogs were euthanized for suspicion of VL supporting the need for accurate diagnoses in this region (Silva et al., 2011). The parasite’s isolation in culture media is considered the gold standard in the diagnosis of Leishmania sp. infection and in VL, healthy skin has also proven to be an excellent target for this approach in dogs (Madeira et al., 2009; Almeida et al., 2011). In this study, it was only possible to detect L. (V.) braziliensis by PCR, suggesting low parasite load on this site in comparison to cutaneous lesion samples. Nonetheless, this finding adds elements in the discussion about the role of dogs in the transmission cycle of TL. Furthermore, the detection of L. (V.) braziliensis DNA in all lesion fragments analyzed demonstrates reliability in the use of PCR as a tool in TL diagnostic. Although the role of the domestic dog in the transmission cycle of TL is unclear, some studies have suggested that this animal acts
FIGURE 1. (A) PCR products amplified with primers B1/B2 specific for the Leishmania braziliensis complex and visualized on ethidium-bromide– stained 1.5% agarose gel, showing the diagnostic bands (750 base pairs [bp]). M: 100-bp DNA ladder size marker; lane 1: cutaneous lesion sample from dog 1; lanes 2–4: healthy skin from dogs 1, 2, and 9; lane 5: negative control (no DNA); lane 6: positive control (purified kDNA from Leishmania (Vianna) braziliensis [MHOM/BR/1975/M2903]). (B) Southern blot hybridization of the amplified products with the use of kDNA from Leishmania (V.) braziliensis MHOM/BR/1975/M2903 as probe.
as an accidental host (Padilla et al., 2002; Dantas-Torres, 2011). Indeed, little is known regarding the tissue tropism of L. (V.) braziliensis in naturally infected dogs. Our group has been focused on this issue and results recently obtained showed that infection in dogs can be manifested by dissemination and tissue tropism of genetically distinct L. (V.) braziliensis populations (Oliveira et al., 2013). This finding is important and contributes to a better understanding of the epidemiology of canine tegumentary leishmaniasis. The finding of L. (V.) braziliensis DNA in healthy skin suggests a much larger role than thought for the domestic dog, in view of their large presence in an urban environment. This assumption may be closer to reality, because in previous investigations we have reported the persistence and viability of L. braziliensis in human scars many years after cure of the cutaneous lesion (Schubach et al., 1998). In this scenario, the possibility of sandflies becoming infected when fed outside of the lesion, must then be considered. In this way our results suggest the possibility of more active participation of the domestic dog in the transmission cycle of TL. The PCR, with the use of target kDNA minicircles, combined with molecular hybridization as a confirmatory method, proved to be a very useful tool for detection of L. (V.) braziliensis DNA in healthy skin of naturally infected dogs. The sequencing of the amplicon would be an alternative, but the purified kDNA of L. (V.) braziliensis, used as a probe in high-stringency hybridization conditions, has been demonstrated not to cross-react with members of the Leishmania subgenus. Finally, clarifying the real importance of dogs in the transmission cycle of TL is of great importance to public health, so that effective measures to control the disease can be defined and specific protocols for handling these infected animals can be established. ACKNOWLEDGMENTS This work received financial support from Conselho Nacional de ´ Desenvolvimento Cient ´ıfico e Tecnologico (CNPq) and Instituto Kinder do Brasil (IKB). R. S. Pacheco and M. F. Madeira are the recipients of fellowships from CNPq.
DE OLIVEIRA ET AL.—L. BRAZILIENSIS IN HEALTHY SKIN IN CANINE TEGUMENTARY LEISHMANIASIS
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