Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1 Katrin Kuhls 1 *, Carmen Chicharro 2 , Carmen Can ˜ avate 2 , Sofia Cortes 3 , Lenea Campino 3 , Christos Haralambous 4 , Ketty Soteriadou 4 , Francine Pratlong 5 , Jean-Pierre Dedet 5 , Isabel Mauricio 6 , Michael Miles 6 , Matthias Schaar 1 , Sebastian Ochsenreither 1 , Oliver A. Radtke 1 , Gabriele Scho ¨ nian 1 1 Institut fu ¨ r Mikrobiologie und Hygiene, Charite ´ Universita ¨tsmedizin Berlin, Berlin, Germany, 2 WHO Collaborating Centre for Leishmaniasis, Servicio de Parasitologı ´a, Instituto de Salud Carlos III, Mahadahonda (Madrid), Spain, 3 Unidade de Leishmanioses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal, 4 Laboratory of Molecular Parasitology, Hellenic Pasteur Institute, Athens, Greece, 5 Laboratoire de Parasitologie and Centre National de Re ´fe ´rence des Leishmania, Universite ´ Montpellier 1 and CHU Montpellier, Montpellier, France, 6 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom Abstract Background: Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1. Methodology/Principal Findings: We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups—MON-1 and non-MON-1—could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands–Majorca/Ibiza; (3) mainland Portugal/ Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles. Conclusion: In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations. Citation: Kuhls K, Chicharro C, Can ˜ avate C, Cortes S, Campino L, et al. (2008) Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1. PLoS Negl Trop Dis 2(7): e261. doi:10.1371/journal.pntd.0000261 Editor: Charles Jaffe, Hebrew University, Israel Received November 30, 2007; Accepted June 12, 2008; Published July 9, 2008 Copyright: ß 2008 Kuhls et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research was supported by a grant (QLK2-CT-2001-01810) of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Katrin.Kuhls@charite.de Introduction Visceral leishmaniasis (VL) caused by Leishmania infantum (synonym L. chagasi, [1]) is a public-health problem in most countries bordering the Mediterranean, China and South America. Currently, the epidemiology of Mediterranean VL is changing. Increasing incidence [2] and a shift in the bulk of cases from children to adults [3,4], related to the emergence of HIV, has been reported. Since 1985 up to 80% of the cases have occurred in immunocompromised adults [5,6]. Leishmania/HIV co-infections have become increasingly frequent in Southern Europe, particularly in Spain, France, and Italy, with 25–70% of adult cases being related to HIV infection and up to 9% of AIDS cases developing VL [7,8]. In addition to the classical vector-based disease transmission, an anthroponotic cycle has emerged among intravenous drug users where syringes replace the sand fly vector [9–14]. Recently, L. infantum parasites have been found to have spread northward in continental Italy perhaps due to climatic changes [15–17]. Dogs are the main reservoir hosts for L. infantum, being part of the domestic (pet dogs) and peridomestic (stray dogs and wild canids) transmission cycles. The prevalence of canine leishmaniasis is high in all European Mediterranean countries [18–20]. The gold standard method for typing Leishmania is still Multilocus Enzyme Electrophoresis (MLEE, isoenzyme analysis). Most widely used is the Montpellier system (MON) which is based on the analysis of 15 enzymes [21]. Leishmania infantum is www.plosntds.org 1 July 2008 | Volume 2 | Issue 7 | e261