Mixed infection with Leishmania (Viannia) braziliensis and ...

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 from Rio de Janeiro, Brazil M.F. Madeira a,∗, A. Schubach b, T.M.P. Schubach b, R.S. Pacheco c, F.S. Oliveira c, S.A. Pereira b, F.B. Figueiredo b,d, C. Baptista a, M.C.A. Marzochi a,e a

Serviço de Parasitologia, Instituto de Pesquisa Cl´ınica Evandro Chagas — FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil b Serviço de Zoonoses, Instituto de Pesquisa Cl´ınica Evandro Chagas — FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil c Departamento de Bioqu´ımica e Biologia Molecular, Instituto Oswaldo Cruz — FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil d õ em Pesquisa Cl´ınica em Doenças Infecciosas, Instituto de Pesquisa Cl´ınica Evandro Curso de P´ os-Graduaça Chagas — FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil e Secretaria Municipal de Sa´ ude 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 2005 Available online 27 October 2005

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

∗

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

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

1. Introduction Leishmaniasis is caused by different species of the genus Leishmania, which mainly occur in tropical and subtropical countries (WHO, 1990). The geographic expansion of the disease is mainly caused by population displacement associated with environmental changes (Marzochi and Marzochi, 1994). The overlap of endemic areas can lead to changes in classical epidemiological patterns, with the occurrence of mixed infections caused by distinct Leishmania species (Martinez et 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 transmission of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis and visceral leishmaniasis (VL) caused by Leishmania (Leishmania) chagasi has been observed in various regions of the Municipality of Rio de Janeiro. The simultaneous occurrence of canine cases with both diseases in the same geographic area impairs the diagnosis and implementation of control measures (Minist´ erio da Sa´ ude, 2000, 2003). We report here an autochthonous case of mixed CL and VL in a male dog (Canis familiaris) from an endemic area in the city of Rio de Janeiro, Brazil.

2. Methods The animal was referred to the Zoonosis Service, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, with indication for euthanasia according to the recommendations of the Brazilian Program for the Control of Leishmaniasis (Minist´ erio da Sa´ ude, 2003) after the detection of IgG Leishmania antibodies (titres 1:160) by an IFAT. The study was approved by the Ethics Committee on Animal Experimentation of the Oswaldo Cruz Foundation (CEUA/FIOCRUZ; programme No. P0195-03). The dog showed a cutaneous ulcer on the internal surface of the ear but no other alterations, and was active and in good general condition. The dog was killed by a thiopental overdose and fragments of the cutaneous lesion, popliteal lymph node, spleen and blood mononuclear cells were collected and inoculated separately into appropriate tubes containing biphasic medium (NNN and Schneider’s Drosophila medium) supplemented with 10% fetal calf serum. The tubes were incubated at 26—28 ◦ C. Promastigote forms isolated from the blood mononuclear cell layer and from fragments of the popliteal lymph node and cutaneous lesion were characterised by isoenzyme electrophoresis as described by Cupolillo et al. (1994) and Pacheco et al. (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-phosphogluconate dehydrogenase (6PGDH). The enzyme profiles of isolates were compared with those of the following reference strains: 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, PCR analysis was carried out on biopsy fragments of the cutaneous lesion using primers specific for the L. braziliensis complex: B1: 5 -GGGGTTGGTGTAATATAGTGG-3 and B2:

443 5 -CTAATTGTGCACGGGGAGG-3 (De Bruijn and Barker, 1992). For spleen and popliteal lymph node fragments, generic primers that amplify the conserved region of Leishmania kDNA minicircle molecules were used: A: 5 (G/C)(G/C)(C/G)CC(A/C)CTAT(A/T)TTACACAACCCC-3 and B: 5 -GGGGAGGGGCGTTCTGCGAA-3 (Degrave et al., 1994). All amplified PCR products were visualised on agarose gels followed 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 popliteal lymph node fragments were identified as L. (L.) chagasi and one isolate from the cutaneous lesion was identified as L. (V.) braziliensis by isoenzyme electrophoresis (Figure 1). Spleen fragments were negative in culture. PCR in combination with molecular hybridisation was used to investigate the presence of L. (V.) braziliensis DNA in the cutaneous lesion biopsy and of L. (L.) chagasi in spleen and lymph node fragments. No mixed infection with L. (V.) braziliensis and L. (L.) chagasi was detected in the cutaneous lesion, spleen or lymph node after hybridisation of the PCR products. This finding is important because it suggests that: first, the two different species present a specific tropism even if they occur as a co-infection; and second, that it is essential to perform several samplings in dogs with cutaneous lesions. Figures 2 and 3 show the PCR/hybridisation results, which confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. Despite the differences between the clinical manifestations of CL and VL (Marzochi et al., 1985; Silva et al., 2001), the diagnosis of VL and the consequent elimination of dogs from endemic areas are based on positive serological tests, irrespective of general condition or the presence of signs of VL (Minist´ erio da Sa´ ude, 2003). In areas where CL and VL overlap, as is the case for some suburban areas of the Municipality of Rio de Janeiro, the aetiological diagnosis of infected animals is difficult. The confirmation of a mixed

Figure 1 Isoenzyme profiles (glucose phosphate isomerase) of strains isolated from the dog’s cutaneous lesion (lane 2), blood mononuclear cell layer (lane 3) and popliteal lymph node (lane 4). Reference strains of Leishmania (Viannia) braziliensis (lane 1), L. (L.) chagasi (lane 5) and L. (L.) amazonensis (lane 6).

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M.F. Madeira et al. a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ ogico (CNPq), Brazil, and PAPESIII/FIOCRUZ.

References

Figure 2 (A) PCR products amplified with primers B1/B2 specific for the Leishmania braziliensis complex and visualised on ethidium bromide-stained 1.5% agarose gel, showing the diagnostic band (750 bp). M: 100 bp DNA ladder size marker; lane 1: cutaneous lesion of the dog; lane 2: negative control (no DNA); lane 3: positive control (kDNA from L. (V.) braziliensis MHOM/BR/75/M2903). (B) Southern blot hybridisation of the amplified products using kDNA from L. (V.) braziliensis MHOM/BR/75/M2903 as probe.

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

infection in a dog supports the importance of incorporation of biochemical and molecular methods for the correct identification of circulating aetiological agents, and the consequent re-assessment of control and epidemiological surveillance measures. Conflicts of interest statement The authors have no conflict of interest concerning the work reported in this paper.

Acknowledgements We wish to thank Cristianni A. Leal and Cintia Xavier de Melo (Parasitology Service, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation — FIOCRUZ) for technical assistance. (Cristianni A. Leal was the recipient of a technical support fellowship from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ ogico (CNPq), Brazil.) õ de Apoio This work was partially supported by the Fundac ¸a

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