Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 442—445

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Mixed infection with Leishmania (Viannia)braziliensis and Leishmania (Leishmania)chagasi in a naturally infected dog fromRio de Janeiro, Brazil

M.F. Madeiraa,∗, A. Schubachb, T.M.P. Schubachb, R.S. Pachecoc,F.S. Oliveirac, S.A. Pereirab, F.B. Figueiredob,d,

a a,e

C. Baptista , M.C.A. Marzochi

a Servico de Parasitologia, Instituto de Pesquisa Clınica Evandro Chagas — FIOCRUZ, Av. Brasil 4365,21045-900 Rio de Janeiro, RJ, Brazilb Servico de Zoonoses, Instituto de Pesquisa Clınica Evandro Chagas — FIOCRUZ, Av. Brasil 4365,21045-900 Rio de Janeiro, RJ, Brazilc Departamento de Bioquımica e Biologia Molecular, Instituto Oswaldo Cruz — FIOCRUZ, Av. Brasil 4365,21045-900 Rio de Janeiro, RJ, Brazild Curso de Pos-Graduacao em Pesquisa Clınica em Doencas Infecciosas, Instituto de Pesquisa Clınica EvandroChagas — FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazile Secretaria Municipal de Saude da Cidade do Rio de Janeiro, Rua Afonso Cavalcanti 455, 20211-901 Rio de Janeiro, RJ, Brazil

Received 3 May 2005; received in revised form 26 July 2005; accepted 26 July 2005Available online 27 October 2005

KEYWORDSLeishmania (Viannia)braziliensis;Leishmania(Leishmania) chagasi;Mixed infection;Isoenzymes;PCR;Brazil

Summary We report here the first case of co-infection with Leishmania (Viannia) braziliensisand Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil.Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesionas L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysisusing specific primers followed by molecular hybridisation for direct Leishmania species identi-fication in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneouslesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This reportemphasises the importance of identification of Leishmania species infecting seropositive dogsin endemic areas, and the consequent re-assessment of control and epidemiological surveillancemeasures for the control of leishmaniasis, as is the case in Brazil.© 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rightsreserved.

∗ Corresponding author. Tel.: +55 21 3865 9594; fax: +55 21 3865 9541.E-mail address: fmadeira@ipec.fiocruz.br (M.F. Madeira).

0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.trstmh.2005.07.011

Mixed infection Leishmania in a dog 443

1. Introduction

Leishmaniasis is caused by different species of the genusLeishmania, which mainly occur in tropical and subtropicalcountries (WHO, 1990). The geographic expansion of the dis-ease is mainly caused by population displacement associatedwith environmental changes (Marzochi and Marzochi, 1994).The overlap of endemic areas can lead to changes in classi-cal epidemiological patterns, with the occurrence of mixedinfections caused by distinct Leishmania species (Martinezet al., 2002; Oliveira-Neto et al., 1986; Silveira et al., 1984)or co-infection with Trypanosoma cruzi (Bastrenta et al.,2003; Chiaramonte et al., 1996). Overlapping transmissionof cutaneous leishmaniasis (CL) caused by Leishmania (Vian-nia) braziliensis and visceral leishmaniasis (VL) caused byLeishmania (Leishmania) chagasi has been observed in vari-ous regions of the Municipality of Rio de Janeiro. The simul-taneous occurrence of canine cases with both diseases inthe same geographic area impairs the diagnosis and imple-mentation of control measures (Ministerio da Saude, 2000,2003).

We report here an autochthonous case of mixed CL andVL in a male dog (Canis familiaris) from an endemic area inthe city of Rio de Janeiro, Brazil.

2. Methods

5′-CTAATTGTGCACGGGGAGG-3′ (De Bruijn and Barker,1992). For spleen and popliteal lymph node fragments,generic primers that amplify the conserved region ofLeishmania kDNA minicircle molecules were used: A: 5′-(G/C)(G/C)(C/G)CC(A/C)CTAT(A/T)TTACACAACCCC-3′ andB: 5′-GGGGAGGGGCGTTCTGCGAA-3′ (Degrave et al., 1994).All amplified PCR products were visualised on agarose gelsfollowed by molecular hybridisation with a specific probe,as previously described (Oliveira et al., 2005; Silva et al.,2002).

3. Results and discussion

Two isolates from the mononuclear cell layer and popliteallymph node fragments were identified as L. (L.) chagasi andone isolate from the cutaneous lesion was identified as L.(V.) braziliensis by isoenzyme electrophoresis (Figure 1).Spleen fragments were negative in culture. PCR in com-bination with molecular hybridisation was used to investi-gate the presence of L. (V.) braziliensis DNA in the cuta-neous lesion biopsy and of L. (L.) chagasi in spleen andlymph node fragments. No mixed infection with L. (V.)braziliensis and L. (L.) chagasi was detected in the cuta-neous lesion, spleen or lymph node after hybridisation ofthe PCR products. This finding is important because it sug-gests that: first, the two different species present a spe-cific tropism even if they occur as a co-infection; andsiPoLf

ttfioVMi

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The animal was referred to the Zoonosis Service, EvandroChagas Clinical Research Institute, Oswaldo Cruz Foun-dation, with indication for euthanasia according to therecommendations of the Brazilian Program for the Controlof Leishmaniasis (Ministerio da Saude, 2003) after thedetection of IgG Leishmania antibodies (titres 1:160) byan IFAT. The study was approved by the Ethics Committeeon Animal Experimentation of the Oswaldo Cruz Founda-tion (CEUA/FIOCRUZ; programme No. P0195-03). The dogshowed a cutaneous ulcer on the internal surface of the earbut no other alterations, and was active and in good generalcondition. The dog was killed by a thiopental overdoseand fragments of the cutaneous lesion, popliteal lymphnode, spleen and blood mononuclear cells were collectedand inoculated separately into appropriate tubes con-taining biphasic medium (NNN and Schneider’s Drosophilamedium) supplemented with 10% fetal calf serum. Thetubes were incubated at 26—28 ◦C. Promastigote formsisolated from the blood mononuclear cell layer and fromfragments of the popliteal lymph node and cutaneouslesion were characterised by isoenzyme electrophoresisas described by Cupolillo et al. (1994) and Pacheco etal. (1998). A total of five enzyme systems were analysed:nucleosidase (NH1 and NH2), glucose phosphate isomerase(GPI), glucose-6-phosphate dehydrogenase (G6PDH),phosphoglucomutase (PGM) and 6-phosphogluconatedehydrogenase (6PGDH). The enzyme profiles of isolateswere compared with those of the following referencestrains: L. (V.) braziliensis (MHOM/BR/75/M2903), L. (L.)chagasi (MHOM/BR/74/PP75) and L. (L.) amazonensis(IFLA/BR/67/PH8). To confirm the isoenzyme results, PCRanalysis was carried out on biopsy fragments of the cuta-neous lesion using primers specific for the L. braziliensiscomplex: B1: 5′-GGGGTTGGTGTAATATAGTGG-3′ and B2:

econd, that it is essential to perform several samplingsn dogs with cutaneous lesions. Figures 2 and 3 show theCR/hybridisation results, which confirmed the presencef L. (V.) braziliensis DNA in the cutaneous lesion and of. (L.) chagasi DNA in spleen and popliteal lymph noderagments.

Despite the differences between the clinical manifesta-ions of CL and VL (Marzochi et al., 1985; Silva et al., 2001),he diagnosis of VL and the consequent elimination of dogsrom endemic areas are based on positive serological tests,rrespective of general condition or the presence of signsf VL (Ministerio da Saude, 2003). In areas where CL andL overlap, as is the case for some suburban areas of theunicipality of Rio de Janeiro, the aetiological diagnosis of

nfected animals is difficult. The confirmation of a mixed

igure 1 Isoenzyme profiles (glucose phosphate isomerase)f strains isolated from the dog’s cutaneous lesion (lane 2),lood mononuclear cell layer (lane 3) and popliteal lymph nodelane 4). Reference strains of Leishmania (Viannia) brazilien-is (lane 1), L. (L.) chagasi (lane 5) and L. (L.) amazonensislane 6).

444 M.F. Madeira et al.

Figure 2 (A) PCR products amplified with primers B1/B2 spe-cific for the Leishmania braziliensis complex and visualised onethidium bromide-stained 1.5% agarose gel, showing the diag-nostic band (750 bp). M: 100 bp DNA ladder size marker; lane1: cutaneous lesion of the dog; lane 2: negative control (noDNA); lane 3: positive control (kDNA from L. (V.) brazilien-sis MHOM/BR/75/M2903). (B) Southern blot hybridisation ofthe amplified products using kDNA from L. (V.) braziliensisMHOM/BR/75/M2903 as probe.

Figure 3 (A) PCR products amplified with generic primersA/B and visualised on ethidium bromide-stained 2% agarosegel, showing the diagnostic band (120 bp). M: 100 bp DNA lad-der size marker; lanes 1 and 2: spleen and popliteal lymphnode; lane 3: positive control (kDNA from Leishmania (L.) cha-gasi MHOM/BR/74/PP75); lane 4: negative control (no DNA). (B)Southern blot hybridisation of the amplified products using kDNAfrom L. (L.) chagasi MHOM/BR/74/PP75 as probe.

infection in a dog supports the importance of incorporationof biochemical and molecular methods for the correctidentification of circulating aetiological agents, and theconsequent re-assessment of control and epidemiologicalsurveillance measures.

Conflicts of interest statementThe authors have no conflict of interest concerning the workreported in this paper.

Acknowledgements

We wish to thank Cristianni A. Leal and Cintia Xavier deMelo (Parasitology Service, Evandro Chagas Clinical ResearchInstitute, Oswaldo Cruz Foundation — FIOCRUZ) for tech-nical assistance. (Cristianni A. Leal was the recipient ofa technical support fellowship from Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq), Brazil.)This work was partially supported by the Fundacao de Apoio

a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq), Brazil, and PAPESIII/FIOCRUZ.

References

Bastrenta, B., Mita, N., Buitrago, R., Vargas, F., Flores, M.,Machame, M., Yacsik, N., Torres, M., Le Pont, F., Breniere,F., 2003. Human mixed infections of Leishmania spp. andLeishmania—Trypanosoma cruzi in a sub Andean Bolivian area:identification by polymerase chain reaction/hybridization andisoenzyme. Mem. Inst. Oswaldo Cruz 98, 255—264.

Chiaramonte, M.G., Zwirner, N.W., Caropresi, S.L., Taranto, N.J.,Malchiodi, E.L., 1996. Trypanosoma cruzi and Leishmania spp.human mixed infection. Am. J. Trop. Med. Hyg. 54, 271—273.

Cupolillo, E., Grimaldi Jr, G., Momen, H., 1994. A general classifica-tion of New World Leishmania using numerical zymotaxonomy.Am. J. Trop. Med. Hyg. 50, 296—311.

De Bruijn, M.H.L., Barker, D.C., 1992. Diagnosis of New Worldleishmaniasis: specific detection of species of the Leishmaniabraziliensis complex by amplification of kinetoplast DNA. ActaTrop. 52, 45—58.

Degrave, W., Fernandes, O., Campbell, D., Bozza, M., Lopes, U.,1994. Use of molecular probes and PCR for detection and typ-ing of Leishmania: a mini review. Mem. Inst. Oswaldo Cruz 89,463—469.

Martinez, E., Mollinedo, S., Torrez, M., Munoz, M., Banuls, A.L.,Le Pont, F., 2002. Co-infection by Leishmania amazonensis and

M

M

M

M

O

O

P

S

L. infantum/L. chagasi in a case of diffuse cutaneous leish-maniasis in Bolivia. Trans. R. Soc. Trop. Med. Hyg. 96, 529—532.

arzochi, M.C.A., Marzochi, K.B.F., 1994. Tegumentary and vis-ceral leishmaniases in Brazil —– emerging anthropozoonosis andpossibilities for their control. Cad. Saude Publica 10, 359—375.

arzochi, M.C.A., Coutinho, S.G., Souza, W.J., Toledo, L.M.,Grimaldi, G., Momen, H., Pacheco, R.S., Sabroza, P.C., Souza,M.A., Rangel, F.B., Tramontano, N.C., 1985. Canine visceralleishmaniasis in Rio de Janeiro, Brazil. Clinical, parasitological,therapeutical and epidemiological findings (1977—1983). Mem.Inst. Oswaldo Cruz 80, 349—357.

inisterio da Saude, 2000. Manual de controle da leishmaniose tegu-mentar americana. Ministerio da Saude, Fundacao Nacional deSaude, Brasılia.

inisterio da Saude, 2003. Manual de vigilancia e controle da leish-maniose visceral. Ministerio da Saude, Secretaria de Vigilanciaem Saude, Departamento de Vigilancia Epidemiologica, Brasılia,Serie A. Normas e Manuais Tecnicos.

liveira, F.S., Pirmez, C., Pires, M.Q., Brazil, R.P., Pacheco, R.S.,2005. PCR-based diagnosis for detection of Leishmania in skinand blood of rodents from an endemic area of cutaneousand visceral leishmaniasis in Brazil. Vet. Parasitol. 129, 219—227.

liveira-Neto, M.P., Marzochi, M.C.A., Grimaldi Jr, G., Pacheco,R.S., Toledo, L.M., Momen, H., 1986. Concurrent human infec-tion with Leishmania donovani and Leishmania braziliensisbraziliensis. Ann. Trop. Med. Parasitol. 80, 587—592.

acheco, R.S., Marzochi, M.C.A., Pires, M.Q., Brito, C.M.M.,Madeira, M.F., Barbosa-Santos, E.G.O., 1998. Parasite genotyp-ically related to a monoxenous trypanosomatid of dog’s fleacausing opportunistic infection in an HIV positive patient. Mem.Inst. Oswaldo Cruz 93, 531—537.

ilva, E.S., Gontijo, C.M.F., Pacheco, R.S., Fiuza, V.O.P., Brazil, R.P.,2001. Visceral leishmaniasis in the metropolitan region of BeloHorizonte, state of Minas Gerais, Brazil. Mem. Inst. Oswaldo Cruz96, 285—291.

Mixed infection Leishmania in a dog 445

Silva, E.S., Pacheco, R.S., Gontijo, C.M.F., Carvalho, I.R., Brazil,R.P., 2002. Visceral leishmaniasis caused by Leishmania (Viannia)braziliensis in a patient infected with human immunodeficiencyvirus. Rev. Inst. Med. Trop. Sao Paulo 44, 145—149.

Silveira, F.T., Lainson, R., Shaw, J.J., Ribeiro, R.S.M., 1984.Leishmaniose cutanea na Amazonia. Registro do primeiro caso

humano de infeccao mista, determinado por duas especies dis-tintas de Leishmanias: Leishmania braziliensis e Leishmaniamexicana amazonensis. Rev. Inst. Med. Trop. Sao Paulo 26,272—275.

WHO, 1990. Control of Leishmaniasis. World Health Organization,Geneva, Technical Report Series No. 793.