Evolution of the Leishmania braziliensis species complex from amplified fragment length polymorphisms, and clinical implications Samwel Odiwuor a,c,d , Nicolas Veland b , Ilse Maes a , Jorge Arévalo b , Jean-Claude Dujardin a,d , Gert Van der Auwera a,⇑ a Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium b Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru c Center for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya d Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium article info Article history: Received 30 January 2012 Received in revised form 29 March 2012 Accepted 31 March 2012 Available online 10 April 2012 Keywords: Leishmania braziliensis Leishmania peruviana Heat-shock protein 70 Tegumentary leishmaniasis Peru AFLP abstract In order to get more insight into its evolution and geographical distribution, we investigated the Leish- mania (Viannia) braziliensis species complex using amplified fragment length polymorphisms and sequencing of a heat-shock protein 70 gene fragment. Previously, several assays had alluded to the high genetic diversity of the group, and single-locus assays typically identified two species, i.e. L. braziliensis and Leishmania peruviana, with occasional genetic signatures of both in the same strain. By analysis of 53 parasite isolates from Peru, and eight additional ones from other countries, we identified an atypical L. braziliensis cluster, and confirmed the origin of L. peruviana from the L. braziliensis cluster during the colonization of the western Andean coastal valleys. We discuss the clinical and taxonomical implications of our findings in relation to currently used species typing assays. Ó 2012 Elsevier B.V. All rights reserved. 1. Introduction Leishmania braziliensis (Vianna, 1911) and Leishmania peruviana (Velez, 1913) are two closely related species united in the L. brazili- ensis complex, the most important dermotropic Leishmania group in the Americas (Grimaldi et al., 1989). In Peru, they are differen- tially distributed within a zone where the Brazilian Amazon rain forest transitions into the Pacific coastal ecosystem through the Peruvian Andes (Lumbreras and Guerra, 1985), with local areas of sympatric occurrence (Dujardin et al., 1993b,c, 1995b; Nolder et al., 2007). The distribution is characterized by populations rela- tively separated within specific bio-geographical units demarcated by physical barriers such as rivers, deserts and mountains (Lamas, 1982). L. peruviana causes human cutaneous leishmaniasis (CL), locally referred to as ‘‘uta’’ (Lainson and Shaw, 1987; Lumbreras and Guerra, 1985), and has been isolated from both dogs and small peridomestic animals such as mice and opossums, suggesting it is a zoonosis (Llanos-Cuentas et al., 1999). The species is endemic in Peru, where it occurs at altitudes between 800 and 3000 m above sea level (a.s.l.) on the western slopes of the Andes and in the inter-Andean valleys in the lower (500–1500 m a.s.l.) and upper (1500–3500 m a.s.l.) montane forests. The departments of Piura and Ayacucho are the northern and southern limits respec- tively. L. braziliensis on the other hand has a much wider distribu- tion within Central and South America, both as a native anthropozoonosis (Grimaldi et al., 1989; Oddone et al., 2009) or as incidental cases imported by human migration (mier-David et al., 1993). Circulation is sylvatic, and it is encountered mainly on the eastern slopes of the Peruvian Andes, stretching into the Brazilian jungle lowlands at altitudes below 800 m a.s.l. (Lumbr- eras and Guerra, 1985). Contrary to L. peruviana, human CL caused by L. braziliensis is characteristically known for its ability to pro- gress to the severe mucocutaneous form also known as ‘‘espundia’’ or Amazonian leishmaniasis (Lainson and Shaw, 1987; Lumbreras and Guerra, 1985). The two species were originally separated by their differences in genomic and kinetoplast DNA buoyant density and by 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.03.028 Abbreviations: AFLP, amplified fragment length polymorphism; a.s.l, above sea level; CL, cutaneous leishmaniasis; cpb, cysteine proteinase B gene; hsp70, heat- shock protein 70 gene; ML, mucocutaneous leishmaniasis; MLEE, multilocus enzyme electrophoresis; MLST, multilocus sequence typing; mpi, mannose phos- phate isomerase gene; MPI, mannose phosphate isomerase; RAPD, random ampli- fied polymorphic DNA; RFLP, restriction fragment length polymorphism. ⇑ Corresponding author. Address: Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Tel.: +32 32476586; fax: +32 32476359. E-mail address: gvdauwera@itg.be (G. Van der Auwera). Infection, Genetics and Evolution 12 (2012) 1994–2002 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid