Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations

International Journal for Parasitology 39 (2009) 801–811

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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations Najla Chargui a, Ahmad Amro b,g, Najoua Haouas a, Gabriele Schönian b, Hamouda Babba a, Sonja Schmidt b, Christophe Ravel c, Michele Lefebvre c, Patrick Bastien c, Emna Chaker d, Karim Aoun e, Mohamed Zribi f, Katrin Kuhls b,* a

Laboratoire de Parasitologie-Mycologie à la Faculté de Pharmacie, 99-UR/08-05 Monastir, Tunisia Institute of Microbiology and Hygiene, Charité University Medicine Berlin, Germany French Reference Centre on Leishmaniasis, GEMI (UMR2724), University of Montpellier, Montpellier, France d Le laboratoire de Parasitologie-Mycologie La Rabta, Tunis, Tunisia e Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunisia f Laboratoire de Parasitologie, Centre d0 Hygiène de Sfax, Sfax, Tunisia g Al-Quds Nutrition and Health Research Institute, Faculty of Medicine, Al-Quds University, P.O. Box 20760, Abu-Deis, Palestine b c

a r t i c l e

i n f o

Article history: Received 30 September 2008 Received in revised form 17 November 2008 Accepted 20 November 2008

Keywords: Microsatellites Leishmania infantum Tunisia Hybrids Recombination Population genetics Molecular epidemiology

a b s t r a c t Twenty-seven strains of Leishmania infantum from north and central Tunisia belonging to the three main MON zymodemes (the MON-typing system is based on multilocus enzyme electrophoresis (MLEE) of 15 enzymes) found in this country (MON-1, MON-24 and MON-80) and representing different pathologies (visceral, cutaneous and canine leishmaniasis) have been studied to understand the genetic polymorphism within this species. Intraspecific variation could be detected in L. infantum by the use of 14 hypervariable microsatellite markers. In addition to microsatellite repeat length variation, a high degree of allelic heterozygosity has been observed among the strains investigated, suggestive of sexual recombination within L. infantum groups. The two major clusters found by using Bayesian statistics as well as distance analysis are consistent with the classification based on isoenzymes, dividing Tunisian L. infantum into MON-1 and MON-24/MON-80. Moreover, the existence of hybrid strains between the MON-1 and the non-MON-1 populations has been shown and verified by analysis of clones of one of these strains. Substructure analysis discriminated four groups of L. infantum. The major MON-1 cluster split into two groups, one comprising only Tunisian strains and the second both Tunisian and European strains. The major MON-24 cluster was subdivided into two groups with geographical and clinical feature correlations: a dermotropic group of strains mainly from the north, and a viscerotropic group of strains from the centre of Tunisia. The four viscerotropic hybrid strains all originated from central Tunisia and were typed by MLEE as MON-24 or MON-80. To our knowledge, this is the first report describing relationships between clinical picture and population substructure of L. infantum MON-24 based on genotype data, as well as the existence of hybrids between zymodemes MON-1 and MON-24/MON-80, and proving one of these hybrid strains by molecular analysis of the parent strain and its clones. Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Leishmaniasis is a spectrum of diseases ranging from self-curing cutaneous leishmaniasis (CL) to fatal visceral leishmaniasis (VL), caused by infection with different species of the protozoan parasite Leishmania. In Tunisia, leishmaniasis is prevalent in many areas and caused by three species of Leishmania: (i) Leishmania infantum causes VL or CL in the north and centre of the country, (ii) Leishmania major causes CL in the centre and the south and (iii) Leishmania killicki (syn. Leishmania tropica) causes CL in a micro-focus * Corresponding author. Tel.: +49 30 450524028; fax: +49 30 450524902. E-mail address: [email protected] (K. Kuhls).

in the south–east of Tunisia (Ben Ismail et al., 1986; Kallel et al., 2005; BenSaid et al., 2006; Haouas et al., 2007; Kallel et al., 2008a; Kallel et al., 2008b). The identification and classification of Tunisian Leishmania spp. isolates was mainly performed by the analysis of isoenzymes using multilocus enzyme electrophoresis (MLEE) (Rioux et al., 1990), the current standard typing method. Three MON zymodemes (based on this 15-enzyme system developed in Montpellier, France) of L. infantum have been identified and quantified in four studies in Tunisia: MON-1 (56–94% of the studied strains), MON-24 (3–38.5%) and MON-80 (0.5–13%) (Aoun et al., 2001; Belhadj et al., 2002; Haouas et al., 2007; Kallel et al., 2008b). The correlation between the clinical presentation of disease and the zymodemes is not clear-cut, as the predominating MON-1

0020-7519/$36.00 Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2008.11.016

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N. Chargui et al. / International Journal for Parasitology 39 (2009) 801–811

zymodeme, mostly associated with VL cases, was also found to cause CL (Aoun et al., 2000; Kallel et al., 2005; Kallel et al., 2008b). Moreover, the dermotropic MON-24 zymodeme (Aoun et al., 2000) was occasionally isolated from VL cases (Gramiccia et al., 1991; Belhadj et al., 2000; Aoun et al., 2001; Belhadj et al., 2002; Kallel et al., 2008b), whereas zymodeme MON-80 was linked with both VL and CL (Aoun et al., 1999; Aoun et al., 2001; Belhadj et al., 2002; Haouas et al., 2007; Kallel et al., 2008b). VL in Tunisia is considered to be zoonotic with canines being the reservoir host, however only MON-1 has to date been found in dogs in that country (Aoun et al., 2003; Bouratbine et al., 2005). The reservoir host/s of MON-24 and MON-80 have not yet been elucidated. Strains of MON-24 have been isolated from dogs only twice, in Morocco and Algeria (Benikhlef et al., 2004; Haralambous et al., 2007), suggesting the dog was rather an accidental host. MLEE typing has some disadvantages, as it needs bulk cultivation of parasites and specialised laboratories, post-translational modifications may influence the mobility of the protein, synonymous nucleotide substitutions may not be observed and different allozymes can have coincident mobilities. The discriminatory power is limited, which is most apparent and problematic in the case of Mediterranean L. infantum where the majority of strains, approximately 70%, belong to a single zymodeme, MON-1 (Gallego et al., 2001; Chicharro et al., 2003; Gramiccia, 2003; Martin-Sanchez et al., 2004; Pratlong et al., 2004; Campino et al., 2006; Kallel et al., 2008b). This zymodeme also constitutes between 56% and 94% of all strains in Tunisia. Of the many molecular typing methods developed during the last decade (Schonian et al., 2008) kinetoplast–DNA restriction fragment length polymorphism analysis (kDNA-RFLP) and Multilocus Microsatellite Typing (MLMT) were shown to be most discriminatory overall and able to further discriminate at the intrazymodeme level, as shown for strains of MON-1 (Morales et al., 2001; Botilde et al., 2006). However, kDNA-RFLP has the disadvantages of poor reproducibility and complex banding patterns which makes comparisons between different laboratories very difficult. Microsatellites are tandemly repeated stretches of short nucleotide motifs of 1–6 bp ubiquitously distributed in the genomes of eukaryotic organisms. Because of their high mutation rate it is necessary to develop species-specific marker sets. Several sets of such markers have been developed for the Leishmania donovani complex (Bulle et al., 2002; Jamjoom et al., 2002; Jamjoom et al., 2004; Ochsenreither et al., 2006; Kuhls et al., 2007; Montoya et al., 2007; Kuhls et al., 2008) and proven to be the most suitable molecular markers for population genetics and epidemiological studies of L. infantum at an intra-zymodeme level. The aim of the present study was to apply a MLMT approach based on size variations in 14 microsatellite markers (Ochsenreither et al., 2006; Kuhls et al., 2007; Kuhls et al., 2008) for population studies of L. infantum in Tunisia, including 13 strains of MON24, 14 strains of MON-1 and a single strain of MON-80, isolated from different parts of the country. Questions as to the degree of polymorphism within L. infantum and its zymodemes, the existence and geographical distribution of particular L. infantum populations, the existence of gene flow between those, and correlations to the clinical pictures were the focus of this work. 2. Materials and methods 2.1. Parasite strains, MLEE and DNA extraction Twenty-seven strains of L. infantum maintained in the laboratory in Monastir (Tunisia) were sent by the following laboratories (all in Tunisia): the Institut Pasteur de Tunis (10 strains), the Laboratoire de Parasitologie Mycologie La Rabta, Tunis (nine strains), the Laboratoire de Parasitologie Mycologie Fatouma Bourguiba

Monastir (four strains), the Laboratoire de Microbiologie du Centre d’Hygiene de Sfax (two strains), and two strains were isolated in the Monastir laboratory from dogs. Parasite promastigotes were maintained in Novy–McNeal–Nicolle medium. All human strains were isolated from immuno-competent patients, mainly from children aged between 1 month and 6 years, but also from adults (Table 1). In addition, five L. infantum strains were used as references. The reference strains of the zymodemes MON-1 and MON24, MHOM/FR/1978/LEM75 (MON-1) and MHOM/DZ/1982/LIPA59 (MON-24), representing two of the three zymodemes of L. infantum present in Tunisia, were obtained from the French National Centre on Leishmaniasis, Montpellier, France. Three L. infantum MON-1 strains from the collection of the Institute of Microbiology and Hygiene, Charité University Medicine Berlin, Germany, were used as references for microsatellite analysis: INF1-MHOM/TN/1980/IPT1; INF41-MHOM/ES/1993/PM1; and INF40-MHOM/FR/1995/LPN114. All the strains were typed by MLEE as described previously (Rioux et al., 1990) (Table 1). DNA was extracted from cultured parasites following the protocol of (Schonian et al., 1996). 2.2. Cloning of parasites Single cells of strain MHOM/TN/2005/PLV36MO were visualised, mechanically isolated using a Leitz DMIL micromanipulator (Wetzlar, Germany) and then cultivated on Novy–MacNeal–Nicolle (NNN) medium as reported previously (Alekseev and Saf’ianova, 1977). 2.3. Analysis of microsatellites Fourteen microsatellite markers (Table 2) have been used for the study of genetic polymorphism within Tunisian L. infantum strains, using PCR conditions as described elsewhere (Ochsenreither et al., 2006; Kuhls et al., 2007). Screening of length variations of the amplified markers was performed by electrophoresis in 4% MetaPhor agarose gels (BioWhittaker Molecular Applications) as described by Ochsenreither et al. (2006), which were prepared according to the manufacturer’s instructions. Four strains with known numbers of repeats including the cloned and sequenced strain INF41 (MHOM/ES/1993/PM1) were used as size references for comparison. The separation took 5–7 h at 140 V in a standard horizontal electrophoresis system. Strains with PCR products of different sizes or showing double bands were re-amplified using fluorescence-conjugated forward primers (Proligo, France) and subjected to automated fragment analysis on the capillary sequencer by using the fragment analysis tool (CEQ 8000; Beckman Coulter) (Schwenkenbecher et al., 2006). This technique permits the exact determination of the molecular size, and thus the number of repeats, for the amplified microsatellite markers. The multilocus genotype data, consisting of the number of repeats in each microsatellite marker for each strain, were analysed by two different approaches: the Bayesian model-based clustering method using the software STRUCTURE (Pritchard et al., 2000) and a distance-based method. The first approach has been shown to be suitable for inferring population structure from microsatellite data in various organisms including Leishmania (Pritchard et al., 2000; Rosenberg et al., 2001; Rosenberg et al., 2002; Evanno et al., 2005; Schwenkenbecher et al., 2006; Kuhls et al., 2007; AlJawabreh et al., 2008; Kuhls et al., 2008). The complete set of individual strains was divided into K sub-populations, with K ranging from 1 to 8. Isolates were assigned probabilistically to clusters or, in the case of admixed genotypes, jointly to more than one cluster, with the membership coefficients of all sub-populations adding up to 1. The following parameters have been used: burn-in period 20,000 iterations and 200,000 Markov Chain Monte Carlo iterations. The most probable number of populations was determined by comparing the log-likelihood values for K 1–8 and calculation of DK

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N. Chargui et al. / International Journal for Parasitology 39 (2009) 801–811 Table 1 Designation and characteristics of the Leishmania infantum strains used in this study including reference strains. Strain no.

Clinical picture

MON

WHO code

Region

Age of the patient

Population

1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 LEM75 LEM417 INF1 INF41 INF40

VL VL VL VL VL VL VL VL VL VL VL VL VL VL VL VL CanL CanL CanL CanL CL CL CL CL CL CL CL VL CL VL VL/HIV+ VL

1 24 24 24 80 24 24 24 24 1 1 1 1 1 1 1 1 1 1 1 1 24 24 24 24 24 24 1 24 1 1 1

MHOM/TN/2001/Tus167 MHOM/TN/2002/Tus227 MHOM/TN/2002/PLV14 MHOM/TN/2005/PLV11 MHOM/TN/2005/PLV36MO MHOM/TN/2005/PLV15 MHOM/TN/2005/PLV28 MHOM/TN/2002/PLV26 MHOM/TN/2005/PLV13 MHOM/TN/2002/27M MHOM/TN/2002/246M MHOM/TN/2002/20S MHOM/TN/2002/22MO MHOM/TN/2002/21S MHOM/TN/2002/Tus221 MHOM/TN/2002/Tum222 MCAN/TN/2002/LCnJ20S MCAN/TN/2002/GGCH1/02 MCAN/TN/2002/FCH2/02 MCAN/TN/2002/LCnJ20G MHOM/TN/2004/LC78 MHOM/TN/2002/LC148 MHOM/TN/2004/LC64 MHOM/TN/2002/LC95 MHOM/TN/2002/SFC89 MHOM/TN/2005/SFC51 MHOM/TN/2004/TLC3 MHOM/FR/1978/LEM75b MHOM/DZ/1982/LIPA59b MHOM/TN/1980/IPT1a MHOM/ES/1993/PM1 MHOM/FR/1995/LPN114

Monastir CTN) Monastir (CTN) Kairouan (CTN) Kairouan (CTN) Kairouan (CTN) Kairouan (CTN) Kairouan (CTN) Kairouan (CTN) Kairouan (CTN) Tunis (NTN) Tunis (NTN) Beja (NTN) Seliana (NTN) Béja (NTN) Monastir (CTN) Monastir (CTN) Tunis (NTN) Monastir (CTN) Monastir (CTN) Tunis (NTN) Tunis (NTN) Bizerte (NTN) Tunis (NTN) Béja (NTN) Sfax (CTN) Sfax (CTN) Siliana (NTN) France Algeria Tunisia Spain France

4 years 3 years 2 years 9 months 1 year 1 year 2 years 1 month 6 years 4 years 8 months 9 months 2 years 13 months 3 years 3 years 2 years 4 years 4 years 2 years 30 years 19 years 6 years 15 years 54 years 50 years 10 years nd nd nd nd nd

1 3 1/3 3 1/3 3 3 1/3 1/3 1 1 1 1 1 1 1 1 1 1/2 1 2 4 4 4 4 4 4 2 4 1 2 2

CL, cutaneous leishmaniasis; VL, Visceral leishmaniasis; CanL, canine leishmaniasis; NTN, North Tunisia; CTN, Central Tunisia; MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990); WHO, World Health Organisation. a WHO reference strain of L. infantum. b Reference strain of the zymodeme; nd, not defined.

(Evanno et al., 2005), which is based on the rate of change in the log probability of data between successive K values. Ancestral source populations were identified by decreasing the number of K (Pritchard et al., 2000; Rosenberg et al., 2001; Falush et al., 2003; Parker et al., 2004). Genetic distances (DAS – proportion of shared alleles (Bowcock et al., 1994)) were calculated using MSA software (Dieringer and Schlotterer, 2002), and a Neighbor Joining (NJ) tree based on these distances has been constructed using POPULATIONS 1.2.28 (http:// www.legs.cnrs-gif.fr/bioinfo/populations) and MEGA 3.1 (Kumar et al., 2004) programmes. Microsatellite markers as well as populations were analysed with respect to diversity of alleles (A), expected (gene diversity) and observed heterozygosity (He and Ho, respectively) and the inbreeding coefficient FIS applying GDA software (http:// www.hydrodictyon.eeb.uconn.edu/people/plewis/software.php). Genetic differentiation and gene flow was assessed by F-statistics calculating the FST (theta) values (Weir and Cockerham, 1984) with the corresponding p-values (confidence test) using MSA software. Recombination has been evaluated through Neighbor Joining networks (NeighborNet) obtained by SplitsTree (Huson, 1998; Bryant and Moulton, 2004) and by the population membership coefficients obtained using STRUCTURE. 3. Results 3.1. Genetic diversity of Tunisian L. infantum The initial MLEE analysis of the collected Tunisian L. infantum strains typed 13 of the 27 strains as MON-1, 13 as MON-24 and a single strain as MON-80 (Table 1).

Fourteen microsatellite markers that were shown to be polymorphic within strains of L. infantum, and even within strains of zymodeme MON-1 (Ochsenreither et al., 2006; Kuhls et al., 2008), were tested for variation of repeat numbers within the Tunisian L. infantum strains. All markers were polymorphic showing between three and nine different alleles per marker for the 28 Tunisian strains tested (IPT1 included). The most variable markers were CS20 and Lm4TA (nine and seven alleles, respectively), and the least polymorphic were Li46-67, Li71-5/2, LIST7031 and LIST7039 (three alleles). Within the Tunisian MON-1 strains nine markers were polymorphic with two to four alleles per locus, and five were monomorphic. The degree of polymorphism was significantly higher within the Tunisian MON-24 strains, where all markers were polymorphic, showing two to eight different alleles per marker (Table 2). Gene diversity was hence much higher in MON-24 (mean 0.662) than in MON-1 (mean 0.251) strains (Tables 2 and 3). For all loci either single or two different alleles were observed. Three or more peaks, suggestive of aneuploidy or mixed heterozygous strains, did not occur. Double fragments were much more frequent in MON-24 than in MON-1 strains, occurring in 4–14 (mean 8.7) and zero to two markers (mean 0.92), respectively. This was also shown by the observed heterozygosity values in Tables 2 and 3, the mean of which was 0.071 for MON-1 strains and 0.545 for MON-24 strains. Inbreeding coefficients suggested clonal propagation in MON1 (mean 0.723), in contrast to MON-24 (mean 0.185) (Tables 2 and 3). Thirteen alleles were found to be specific for MON-1 strains and 42 for MON-24 strains, but there are also a few overlapping alleles that were less frequent in both zymodemes (Tables 2 and 3).

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Table 2 Characteristics of the 14 microsatellite markers used for population analysis of Tunisian Leishmania infantum strains. Marker

Population

Repeat numbers

Fragment sizes [bp]

n

A

He

Ho

FIS

Lm2TG

MON-1 MON-24

TG 13, 19, 24, 25 TG 13, 18, 24

118, 130, 140, 142 118, 128, 140

14 (17) 11

4 (4) 3

0.616 (0.683) 0.437

0.071 (0.059) 0.545

0.888 (0.916) 0.263

TubCA

MON-1 MON-24

CA 9, (13) CA 9, 12, 13, 14, 16

80, (88) 80, 86, 88, 90, 94

13a (16) 11

1 (2) 5

0.000 (0.121) 0.684

0.000 (0.000) 0.454

0.000 (1.000) 0.346

Lm4TA

MON-1 MON-24

TA (10), 12, 13, 14, 15 TA 9, 10, 11, 12, 14

(75), 79, 81, 83, 85 73, 75, 77, 79, 83

14 (17) 10b

4 (5) 5

0.727 (0.772) 0.747

0.071 (0.059) 0.700

0.905 (0.926) 0.067

Li41-56

MON-1 MON-24

CA 10 CA 7, 9, 10, 11, 12, 13

90 84, 88, 90, 92, 94, 96

14 (17) 11

1 (1) 6

0.000 (0.000) 0.788

0.000 (0.000) 0.636

0.000 (0.000) 0.200

Li46-67

MON-1 MON-24

CA 9 CA 6, 7

80 74, 76

14 (17) 11

1 (1) 2

0.000 (0.000) 0.485

0.000 (0.000) 0.000

0.000 (0.000) 1.000

Li22-35

MON-1 MON-24

CA (11, 12), 13, 14, 15 CA 5, 10, 12, 13, 14

(90, 92), 94, 96, 98 78, 88, 92, 94, 96

14 (17) 11

3 (5) 5

0.362 (0.547) 0.736

0.071 (0.059) 0.636

0.809 (0.895) 0.141

Li23-41

MON-1 MON-24

GT 14, 15, 16, 17 GT 9, 16, 17, 18, 21

81, 83, 85, 87 71, 85, 87, 89, 95

14 (17) 11

4 (4) 5

0.325 (0.512) 0.801

0.214 (0.176) 0.727

0.350 (0.662) 0.096

Li45-24

MON-1 MON-24

CA 5, (14), 15, 16 CA 8, 14, 16, 17

85, (103), 105, 107 91, 103, 107, 109

14 (17) 10c

3 (4) 4

0.204 (0.437) 0.610

0.071 (0.059) 0.200

0.658 (0.869) 0.684

Li71-33

MON-1 MON-24

TG 11 TG 10, 11, 12, 13, 15, 16

105 103, 105, 107, 109, 113, 115

14 (17) 11

1 (1) 6

0.000 (0.000) 0.719

0.000 (0.000) 0.545

0.000 (0.000) 0.250

Li71-5/2

MON-1 MON-24

CA 8, 9, 10 CA 8, 10

108, 110, 112 108, 112

14 (17) 11

3 (3) 2

0.140 (0.219) 0.312

0.071 (0.059) 0.364

0.500 (0.738) 0.176

Li71-7

MON-1 MON-24

CA 11, 12, 13 CA 8, 9, 10, 11, 13

96, 98, 100 90, 92, 94, 96, 100

14 (17) 11

3 (3) 5

0.321 (0.426) 0.810

0.214 (0.176) 1.000

0.340 (0.593) 0.250

CS20

MON-1 MON-24

TG 18, 19 TG 7, 9, 10, 11, 19, 20, 21, 22

83, 85 61, 65, 67, 69, 85, 87, 89, 91

14 (17) 11

2 (2) 8

0.423 (0.499) 0.840

0.143 (0.118) 0.909

0.671 (0.770) 0.087

LIST7031

MON-1 MON-24

CA 10, 11, (12) CA 8, 10, 11

109, 111, (113) 105, 109, 111

14 (17) 11

2 (3) 3

0.389 (0.526) 0.671

0.071 (0.059) 0.636

0.822 (0.891) 0.054

LIST7039

MON-1 MON-24

CA 15 CA 15, 16, 17

207 207, 209, 211

14 (17) 11

1 (1) 3

0.000 (0.000) 0.636

0.000 (0.000) 0.273

0.000 (0.000) 0.583

Overall

MON-1

13.93 (16.93)

2.36 (2.79)

0.251 (0.339)

0.071 (0.059)

0.723 (0.831)

Overall

MON-24

10.86

4.43

0.662

0.545

0.185

a

MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990). Hybrid MON-1/MON-24-80 strains (3, 5, 8, 9) have been excluded. In brackets are given the respective values with the three European strains (LEM75, INF40, INF41) included. The reference strain LEM417 from Algeria is included in the MON24 population. Predominating alleles are marked as bold numbers. Population-specific alleles are underlined. n, sample size; A, number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; FIS, inbreeding coefficient. Missing data: a, strain No. 14; b, LEM417; c, strain No. 24.

Table 3 Characterisation of the Tunisian Leishmania infantum populations found using STRUCTURE (K = 2 and K = 4) analysis. Population

Number of strains

p

MNA

He

Ho

FIS

NAu

K=2 MON-1 MON-24 Hybrids MON-1/MON-24

14 (17)a 11 (15)b 4

0.643 (0.714)a 1.000 (1.000)b 1.000

2.357 (2.786)a 4.429 (5.000)b 2.571

0.251 (0.339)a 0.662 (0.718)b 0.622

0.071 (0.059)a 0.545 (0.634)b 0.875

0.723 (0.831)a 0.185 (0.120)b 0.508

13/7/(7 b)(15/9/(9 b)) a 42/25/(44 b)(38/23/(40 b)) 2

K=4 Pop1 MON-1 TN Pop2 MON-1 TN + EU Pop3 MON-24 VL Pop4 MON-24 CL Hybrids MON-1/MON-24

12 5 4 (8)b 7 4

0.571 0.714 0.643 (1.000)b 0.929 1.000

1.857 2.286 2.000 (3.000)b 3.286 2.571

0.170 0.365 0.360 (0.580)b 0.568 0.622

0.036 0.114 0.375 (0.625)b 0.646 0.875

0.798 0.712 0.050 (0.084)b 0.153 0.508

5/2/(4b) 4/4/(4b) 11/2 (13b) 19/19/(19b) 2

P, proportion of polymorphic loci; MNA, mean number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; FIS, inbreeding coefficient; NAu, number of unique alleles. MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990); TN, Tunisia; EU, Europe; VL, visceral leishmaniasis; CL, cutaneous leishmaniasis. Hybrid MON-1/MON-24-80 strains (3, 5, 8, 9) have been treated either as a separate population or as member of population 3. The Algerian reference strain LEM417 is part of population 4. The hybrid strain of MON-1 populations 1 and 2 (strain 20) is included in population 2, as inferred by STRUCTURE. Underlined numbers for NAu are the respective values if hybrid strains are included in the comparison as separate population. a In brackets are given the respective values with the three European strains (LEM75, INF40, INF41) included. b Hybrid MON-1/MON-24-80 strains (3, 5, 8, 9) included in population 3 (italic values in brackets).

MLMT combining the results for the 14 markers differentiated 23 genotypes for the 28 Tunisian strains (IPT1 included), as seen in Fig. 3. Nine of the 14 MON-1 strains had different genotypes and six MON-1 strains were identical. All 14 MON-24 strains had unique genotypes, as well as the single MON-80 strain.

3.2. Population structure and characteristics of Tunisian L. infantum – differentiation and gene flow To infer the population structure of Tunisian L. infantum the MLMT data of the tested strains were processed using STRUCTURE

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software, which applies a Bayesian model-based analysis algorithm. Three European MON-1 strains and the reference strain for MON-24, LEM417 from Algeria, have been included in this analysis. Based on the analysis of allele frequencies four populations have been identified in the data set (Fig. 1A). FST values varied between 0.335 and 0.627 (Table 4 – values in brackets), confirming that these four populations are genetically differentiated. Ancestral populations were identified by decreasing the number of K (Fig. 1B). The first split (K = 2) indicates two major populations, one comprising all MON-1 (Tunisian and European), the other all MON-24 strains and the single MON-80 strain (FST = 0.358, p = 0.0001). Five strains (INF40, 3, 5, 8, 9) showed significant shared membership in both populations. At K = 3 the MON-1 strains were further subdivided into MON-1 strains from Tunisia (population 1) and MON-1 strains from Europe (population 2), however the latter contained two Tunisian strains (22 and 20). Strain 20 showed shared 1:1 membership in both MON-1 popula-

A

tions. Also strains 10, 11 and 13 showed significant portions of membership from the other MON-1 population, indicating gene flow between these two populations. At K = 4 two MON-24 populations (populations 3 and 4) were identified, one of which comprised all strains from VL (population 3), the other all strains from CL (population 4). Four strains, three typed as MON-24 (3, 8, 9) and one as MON-80 (5), had shared membership between MON-24 population 3 and the MON-1 population 1 (Table 5). These four strains had heterozygous alleles in 10–14 markers, one of which was characteristic for MON-1 and the other for MON-24 (Table 6). This suggested that these strains might represent either mixed MON-1 and MON-24/MON-80 strains or real MON-1/MON-24–80 hybrids. Fig. 2 shows that there is a significant amount of gene flow between the European and the Tunisian MON-1 populations, as well as between the Tunisian MON-1 and the combined MON-24 populations. Interestingly, when looking separately at the two MON-24 populations, MON-24 CL (popula-

DeltaK

300 200 100 0 1

2

3

4

5

6

7

8

9 10

K

B

populations 1 + 2

populations 3 + 4

MON-1

MON-24

1.00

0.60 0.40 0.20

VL+CanL Tunisia+Europe

hybrids MON-80

K=2

0.80

VL+CL Tunisia+Algeria

0.00 1 10 12 16 17 18 21 11 13 14 19 LEM7522 INF1INF4120 INF402 26 28 7 24 27 23 4

population 1

6 25 L417 3

populations 3 + 4

8

9 5

population 2

1.00

0.60 0.40 0.20

MON-1

MON-24

VL+CanL Tunisia

hybrids MON-80

K=3

0.80

VL+CL Tunisia+Algeria

MON-1 VL+CanL Europe+ Tunisia

0.00 1 12 16 17 18 21 19 INF1 14 13 10 11 2 7 26 27 28 24 23 4

6 25 L417 3

9 5

8 22LEM75 INF41 INF4020

gene flow MON-1 EU and TN population 1

population 3

population 4

population 2

1.00

0.60 0.40 0.20

MON-1

MON-24

VL+CanL Tunisia

hybrids

VL Tunisia 2

6 4

3 9

MON-1

VL+CanL CL Tunisia+Algeria Europe+ Tunisia

5

8 26 24 28 23 27 25 L417 22LEM75 INF41 INF40 20

0.00 1 12 16 17 18 21 19 INF1 14 13 10 11 7

MON-24

MON-80

K=4

0.80

gene flow MON-1 EU and TN Fig. 1. Estimated population structure for Leishmania infantum from Tunisia as inferred by STRUCTURE software based on the data for 14 microsatellite loci. (A) The derived graph for DK shows a peak at K = 4, indicating the existence of four populations in the dataset. (B) Barplots for K = 2 till K = 4 with each strain being represented by a single vertical line divided into K colours, where K is the number of populations assumed. Each colour represents one population, and the length of the coloured segment shows the strain’s estimated proportion of membership in that population. VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; CanL, canine leishmaniasis; MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990); EU, Europe; TN, Tunisia.

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Table 4 FST values and respective p-values (upper and lower triangle, respectively) of the four Leishmania infantum populations inferred on the basis of 14 microsatellite markers by Bayesian model-based analysis with STRUCTURE software. Populationa

No. of strains

MON-1 TN

MON-1 EU + TN

MON-24 VL

MON-24 CL

Hybrids

Pop1 Pop2 Pop3 Pop4 Hybrids

12 5 4 (8) 7 4 (0)

0 0.0001 0.0001 (0.0001) 0.0001 0.0037

0.514 0 0.0112 (0.0001) 0.0001 0.0158

0.750 (0.537) 0.564 (0.371) 0 0.0033 (0.0001) 0.0115

0.627 0.443 0.423 (0.335) 0 0.0004

0.442 0.289 0.287 0.355 0

According to STRUCTURE K = 4 the hybrids have the highest membership coefficient in population 3 (Table 5). TN, Tunisia; EU, Europe; CL, cutaneous leishmaniasis; VL, visceral leishmaniasis; MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990). a Populations as assumed by STRUCTURE K = 4. Hybrid strains (3, 5, 8, 9) have been either treated as separate populations or included in population 3 (values in brackets).

Table 5 Population membership coefficients of hybrid MON-1/MON-24-80 strains and strains with other mixed ancestries. Strain

Population 1 MON-1 TN

Population 2 MON-1 TN + EU

Population 3 MON-24 VL

Population 4 MON-24 CL

3 5 8 9 20 10 11 13

0.380 0.400 0.427 0.401 0.457 0.776 0.694 0.812

0.035 0.029 0.006 0.026 0.504 0.219 0.302 0.183

0.582 0.568 0.565 0.570 0.006 0.002 0.002 0.003

0.003 0.003 0.002 0.003 0.032 0.002 0.002 0.002

Population as inferred for K = 4 using STRUCTURE. MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990); TN, Tunisia; EU, Europe; VL, visceral leishmaniasis; CL, cutaneous leishmaniasis. Bold numbers indicate the two populations with the highest membership coefficients for the respective strain.

tion 4) is almost completely isolated without significant gene flow to any of the other populations with the exception of traces of gene flow for the Algerian reference strain LEM417 and strain 25 (as shown in Fig. 1B). The Neighbor Joining tree derived from distance-based analysis of the MLMT data (Fig. 3) identified two main clusters comprising MON-1 and MON-24, respectively, and an intermediate group including the putative MON-1/MON-24-80 hybrids. The MON-1 cluster is subdivided into one subcluster comprising all but one Tunisian strains, and another comprising all European MON-1 strains and a single MON-1 strain (22) from Tunisia. Strain 20 identified by STRUCTURE as a hybrid between the MON-1 populations 1 and 2 has an intermediate position between the two MON-1 subclusters. Similarly, the MON-24 cluster is split into a CL and a VL subcluster. The nodes separating the major clusters (with the exception of population 4 – MON-24 CL) were, however, not supported by significant bootstrap values. This may be due to the extremely high similarity of these strains, a certain amount of homoplasy and the existence of strains with mixed or intermediate genotypes. Bootstrap values >50% were obtained only for nodes between some subgroups inside the major clusters. To asses the degree of recombination from the distance data we inferred a Neighbour Joining network of the strains by SplitsTree (Fig. 4). A reticulate pattern was seen mainly between populations 1 and 3 (MON-1 Tunisia and MON-24 VL), with the hybrids showing intermediate positions between these populations and also within population 4 (MON-24 CL). Population MON-24 CL (population 4, MNA = 3.3, He = 0.568) was found to be most diverse, whereas Tunisian MON-1 population 1 (MNA = 1.9, He = 0.17) was least diverse although it included the highest number of strains (Table 3). Observed heterozygosity (Ho = 0.646) was also highest in MON-24 CL and lowest (Ho = 0.036) in MON-1 Tunisia. Inbreeding coefficients were highest

in the two MON-1 populations, indicating clonal propagation. Strain 20, which showed membership coefficients of 0.457 and 0.504 for the two MON-1 populations 1 and 2, respectively, has been included in population 1 in these calculations. There was no significant change in the values of descriptive analysis or for the FST values, if this strain was moved to population 1 or even excluded (data not shown). Apart from the correlation between genotype and clinical picture observed we also detected some correlation to the geographical origin of the strains (Fig. 5). All but one strain (No. 2: Tus227) belonging to population 3 (MON-24 VL) as well as all the detected MON-1/MON-24-80 hybrids (population 1/3) have been isolated in the Kairouan district. 3.3. Recombination in Tunisian L. infantum – analysis of a cloned hybrid strain In order to test whether the putative MON-1/MON-24-80 hybrids are real hybrids, strain 5 (PLV36) was cloned and six of the clones obtained (PLV36cl01–PLV36cl06) were analysed by MLMT. All clones showed MLMT profiles identical to that of the parent strain (Table 6). Moreover, the comparison with strains of the parent populations 1 (MON-1) and 3 (MON-24) showed the presence of both population-specific alleles in the hybrid strains and the clones (Table 6), indicating that PLV36 is a real hybrid of population 1 (MON-1) and population 3 (MON-24). In further analyses we tested the influence of including these four hybrid strains in population 3 (since they have the highest membership coefficient for this population as inferred by STRUCTURE) or treating them as a separate population (Tables 3 and 4). The inclusion of the hybrids in population 3 led to an increase in the He and Ho values for this population by 61% and 66%, respectively. As a separate population they showed the highest values for He and Ho, and the lowest number of unique alleles. A remarkable influence was observed also on the number of population-specific alleles depending on the inclusion or exclusion of these strains in the descriptive analyses (Table 3). The FST values for the two main populations MON-1 and MON-24 (K = 2) are 0.453 and 0.358, with the four hybrid strains excluded and included in population MON-24, respectively. Table 4 shows the three variants of treatment of the four hybrid strains: (i) excluded hybrid strains, (ii) hybrid strains included in population 3 and (iii) as separate population. As expected the inclusion of the hybrid strains in population 3 led to a decrease of the respective FST values. The hybrids as a separate population showed the lowest FST values compared with the other four populations. 4. Discussion In this study a microsatellite based approach has been applied, to our knowledge for the first time, to investigate the population structure of L. infantum in Tunisia. Considerable polymorphism

Table 6 Multilocus microsatellite typing (MLMT) profiles of the hybrids, the respective cloned strains and of strains of parent populations 1 (MON-1)*** and 3 (MON-24). Lab code

Clinical picture

MON

Population

Region

Lm2TG

TubCA

Lm4TA

Li41-56

Li46-67

Li22-35

Li23-41

Li45-24

Li71-33

Li71-5/2

Li71-7

CS20

LIST7031

LIST7039

3 8 9 5 5cl01 5cl02 5cl03 5cl04 5cl05 5cl06 2 4 6 7 1 10 11 12 13 14 16 17 18 19 21 22

PLV14 PLV26 PLV13 PLV36 PLV36cl01 PLV36cl02 PLV36cl03 PLV36cl04 PLV36cl05 PLV36cl06 TUS227 PLV11 PLV15 PLV28 Tus167 27S 246M 20S 22MO 21S Tus 221 Tum 222 LCnJ20S GGCH1 LCnJ20G LC78

VL VL VL VL na na na na na na VL VL VL VL VL VL VL VL VL VL VL VL CanL CanL CanL CL

24 24 24 80 nd nd nd nd nd nd 24 24 24 24 1 1 1 1 1 1 1 1 1 1 1 1

Hybrid 1/3 Hybrid 1/3 Hybrid 1/3 Hybrid 1/3 Clone1 Clone2 Clone 3 Clone 4 Clone 5 Clone 6 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1

Kairouan Kairouan Kairouan Kairouan

18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 + 24 18 18 + 24 18 18 19 24 24 19 24 19 19 19 19 19 19 25

9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 9 + 16 16 16 16 16 9 9 9 9 9 nd 9 9 9 9 9 9

9 + 14 9 + 14 9 + 14 10 + 14 10 + 14 10 + 14 10 + 14 10 + 14 10 + 14 10 + 14 9 + 10 9 10 9 15 12 12 15 14 13 15 15 15 14 15 13

9 + 10 9 10 10 10 10 10 10 10 10 9 9 9 9 10 10 10 10 10 10 10 10 10 10 10 10

6+9 6+9 6+9 6+9 6+9 6+9 6+9 6+9 6+9 6+9 6 6 6 6 9 9 9 9 9 9 9 9 9 9 9 9

12 + 15 12 + 15 10 + 15 10 + 15 10 + 15 10 + 15 10 + 15 10 + 15 10 + 15 10 + 15 12 10 + 13 10 + 12 14 15 15 13 15 15 15 15 15 15 15 15 13

15 + 20 15 + 20 16 + 21 16 + 21 16 + 21 16 + 21 16 + 21 16 + 21 16 + 21 16 + 21 21 18 + 21 21 18 + 21 15 15 15 + 16 15 15 15 15 15 15 15 15 17

8 + 16 16 8 + 16 8 + 16 8 + 16 8 + 16 8 + 16 8 + 16 8 + 16 8 + 16 8 8 8 8 16 16 16 16 16 16 16 16 16 5 + 16 16 15

11 + 12 11 + 12 11 + 16 11 + 16 11 + 16 11 + 16 11 + 16 11 + 16 11 + 16 11 + 16 13 + 16 12 + 16 12 + 16 12 + 16 11 11 11 11 11 11 11 11 11 11 11 11

8+9 9 8+9 8+9 8+9 8+9 8+9 8+9 8+9 8+9 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9

10 + 12 9 + 10 10 + 12 10 + 12 10 + 12 10 + 12 10 + 12 10 + 12 10 + 12 10 + 12 9 + 10 9 + 10 9 + 10 9 + 10 12 12 12 12 11 + 12 11 + 12 12 12 12 12 12 13

18 + 20 19 + 20 19 + 20 19 + 20 19 + 20 19 + 20 19 + 20 19 + 20 19 + 20 19 + 20 21 + 22 20 + 22 19 + 20 20 + 22 19 18 18 + 19 19 18 19 19 19 19 19 19 18

8 + 11 8 + 11 11 11 11 11 11 11 11 11 11 8 + 11 8 + 11 8 + 11 10 11 10 10 11 10 10 10 10 10 10 11

15 + 18 18 15 + 17 15 + 17 15 + 17 15 + 17 15 + 17 15 + 17 15 + 17 15 + 17 17 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15

Monastir Kairouan Kairouan Kairouan Monastir Tunis Tunis Beja Seliana Béja Monastir Monastir Tunis Monastir Tunis Tunis

N. Chargui et al. / International Journal for Parasitology 39 (2009) 801–811

StrainNo.

Nd, not defined; na, not applicable; MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990). Populations as defined by STRUCTURE analysis. Repeat numbers characteristic for population 3 (MON-24) found also in the hybrids are marked in bold, repeat numbers characteristic for population 1 (MON-1) present also in the hybrids are marked in italics. For marker Li41-56 most hybrid strains are monoallelic, showing only the specific MON-1 allele. Markers LIST7031 and LIST7039 have overlapping repeat numbers for the two populations 1 and 3.

807

808

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MON-24 Tunisia

MON-1 Tunisia

MON-1 Europe+Tunisia

Fig. 2. Illustration of gene flow between populations as inferred by Bayesian model-based analysis with STRUCTURE software. Each data point corresponds to a single strain whose proportion of ancestry from each of the three sources is represented by its proximity to the corresponding corner of the triangle. Strains with nearly identical genotype have overlapping positions. The upper corner combines the two MON-24 populations inferred. MON, zymodeme system based on multilocus enzyme electrophoresis (MLEE) of a 15 enzyme system developed by Rioux et al. (1990).

was detected within the strains studied, since all but six of those had different genotypes. In Tunisia only three different L. infantum zymodemes are circulating and this study has investigated representative numbers of isolates of the most common zymodemes MON-1 and MON-24 collected from different endemic microfoci, as well as one strain of the less frequent zymodeme MON-80. For comparison World Health Organisation (WHO) reference strains for the respective zymodemes were included. Analysis of MLMT patterns revealed a main split between the MON-1 and the MON-24 strains, in concordance with zymodeme classification. The MON-24 group was characterised by a much higher degree of genetic diversity than the MON-1 group, reflected by a higher number of alleles, higher values for He and Ho and a significantly higher number of private alleles, as shown by descriptive analysis. This is in agreement with previous observations from Europe, where MON-1 was also much less heterogeneous than the nonMON-1 group (Kuhls et al., 2008). However, here we could directly compare strains of two different zymodemes, in contrast to the European study where different zymodemes were included in the non-MON-1 group. More importantly, we could identify two different MON-1 as well as two MON-24 populations in Tunisia. All four populations were genetically distinct as shown by the FST values and by the presence of population-specific alleles. Interestingly, all but two

MON CP 1 CanL VL 1 1 CanL 54 VL 1 VL 1 VL 1 61 VL 1 14 1 CanL 19 R VL 1 IPT1 VL 1 10 VL 1 13 VL 1 11 CL 1 20 VL 1 PM1 VL LPN114 1 VL 1 81 22 VL 1 LEM75Z VL 24 mixed MON-1/MON-24-80 24 VL genotypes (including confirmed 24 VL hybrid - strain 5) 80 VL 78 24 VL 4 24 VL 7 24 VL 6 24 VL 2 24 CL 25 24 CL 27 24 CL LEM417Z 24 CL 23 24 CL 28 24 CL 24 24 CL 26 18 21 17 16 1 12

74

3

90

8 100

9 5

80 59 61

0.1

origin NTN NTN CTN CTN CTN NTN NTN CTN TN NTN NTN NTN NTN ES FR CTN FR CTN CTN CTN CTN CTN CTN CTN CTN CTN CTN DZ NTN NTN CTN NTN

Structure K=4

1

1+2

2

1+3

3

4

Fig. 3. Neighbour Joining tree (mid-point rooted) inferred from the DAS distances calculated for the microsatellite data of the 27 Tunisian strains of Leishmania infantum, the World Health Organisation (WHO) reference strains of L. infantum MON-1 IPT1 (also from Tunisia) and MON-24 LEM417 (from Algeria), and for three representative MON-1 strains from Europe. Bootstrap values >50% are shown. The four populations (1–4) inferred by Bayesian model-based analysis with STRUCTURE software are indicated by the bars on the right-hand side. The group representing the mixed MON-1/MON-24-80 (population 1/3) genotype as well as the single strain of mixed populations 1 and 2 genotype are indicated. R, WHO reference strain; Z, reference strain of the zymodeme; CP, clinical picture; NTN, North Tunisia; CTN, Central Tunisia; TN, Tunisia; ES, Spain; FR, France; DZ, Algeria.

N. Chargui et al. / International Journal for Parasitology 39 (2009) 801–811

809

Fig. 4. Network inferred with SplitsTree software using the DAS distances for the 14 microsatellite markers for all strains of Leishmania infantum studied. The respective populations 1-4 are indicated as well as the hybrids. For abbreviations, see Fig. 1.

studied Tunisian MON-1 strains (including the WHO reference strain IPT1 from Tunisia), exclusively originating from the humid northern and central (coastal) regions of the country, formed a unique population distinct from the European MON-1 population. Human and canine MON-1 strains presented identical MLMT profiles, emphasising the key role of dogs as reservoirs. Only two Tunisian strains isolated in Monastir and Tunis grouped together with the European MON-1 strains in population 2. One of those (strain 20) was identified as a putative hybrid between the two MON-1 populations by STRUCTURE and is the only Tunisian MON-1 strain isolated from a CL case. The occurrence of North African genotypes together with Southern European ones could be explained by frequent migration and travel of people and a long common history in the Mediterranean area. A similar observation has been made in a recent study of Algerian L. infantum (Seridi et al., 2008). A second important observation was that two MON-24 populations could be differentiated in Tunisia. One of those, population 4, included parasites isolated from CL and the other, population 3, from VL cases. Thus, for the first time a clear relationship could be demonstrated between clinical presentation of leishmaniasis and parasite genotype. Whether this is a general trait of these populations or a spurious association caused by sampling bias has to be tested with more strains. In addition, we could see a correlation with the age of the patients in populations 3 and 4. In the VL group all patients were less than 3 years old, whereas in the CL group the majority of the patients were adults. This is in agreement with previous reports that VL caused by zymodeme MON-24 in immunocompetent patients affects only children (Aoun et al., 2001; Benikhlef et al., 2001; Kallel et al., 2008b). All but one strain of population 3 as well as the population 1/3 hybrids (MON-1/MON-2480 VL) originated from the Kairouan district (all studied strains from Kairouan belonged to these populations). This area has been described as a newly emerged VL focus in central Tunisia with 91% of cases occurring in children up to 5 years old (Ben Salah et al., 2000). The reasons of the unusual increase in VL incidence

in the 1990s are still not understood. Whether MON-1/MON-24– 80 hybrids are indeed restricted to the Kairouan focus also needs clarification. The MON-24 CL group to which also the MON-24 reference strain from Algeria (LEM417) belongs is the most polymorphic L. infantum population. Strains of this population were collected from the northern humid parts but also from central arid and semiarid parts of Tunisia. In Tunisia three Phlebotomus species have been described to transmit L. infantum, depending on bioclimatic conditions (Ghrab et al., 2006). Phlebotomus perfiliewi was found in the CL foci of humid bioclimate, Phlebotomus perniciosus in VL and CL regions of semiarid and arid bioclimates, and Phlebotomus longicuspis in a VL focus of Saharan bioclimate. Whether there is a correlation with the different L. infantum populations detected remains be elucidated. The most striking finding of this study was evidence for the existence of MON-1/MON-24-80 hybrids. The occurrence of hybrid strains in L. infantum has been previously suggested in a MLMT study of Algerian L. infantum strains (Seridi et al., 2008), however these putative hybrids have not been tested by analysing cloned parasites. Hybrids seem to occur quite frequently, as in total 12 MLMT profiles (14.6% of the 82 strains studied) suggestive for hybrid genotypes have been identified through the studies in Algeria (55 strains studied, 14.5% of those showed a hybrid genotype) and Tunisia (27 strains studied, 14.8% had a hybrid genotype). Interestingly, all but one of the strains from the two countries typed as MON-80 (one from Tunisia and four from Algeria) exhibited hybrid MLMT patterns. For the Tunisian MON-80 strain (PLV36) four bands have been observed by isoenzyme analysis for NP1, which is the single discriminating enzyme for MON-1, MON-24 and MON-80. This strain was cloned and proven as a hybrid. The occurrence of gene flow and recombination is increasingly reported in Leishmania spp. Although the population structure is predominantly clonal (Tibayrenc et al., 1990; Bastien et al., 1992; Ayala, 1993; Tibayrenc, 1993; Tibayrenc et al., 1993; Tibayrenc and Ayala, 1999; Kuhls et al., 2008), the frequency of recombination

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of this species in Tunisia and gene flow between those, evidence of the existence of MON-1/MON-24-80 hybrids, correlations between genotype, geographical origin and clinical presentation, have been added to the understanding of the epidemiology of Mediterranean VL and CL caused by L. infantum. Acknowledgements We thank Omar Hamarsheh (Department of Biological Sciences, Al-Quds University, Abu-Deis, The Palestinian Authority) for support in performing the molecular analyses, and Francine Pratlong and Jean-Pierre Dedet (Centre National de Référence des Leishmania, Montpellier, France) for providing reference strains. Najla Chargui was supported by a grant from the Agence Universitaire de la Francophonie (AUF). References

Fig. 5. Map of Tunisia and geographical distribution of the Tunisian Leishmania infantum strains in this study. The four populations identified by Bayesian modelbased analysis with STRUCTURE software and by the Neighbour Joining tree are indicated by different colours of the symbols, as specified in the figure. The numbers in the symbols are the strain numbers as given in Table 1. For abbreviations, see Fig. 1.

events has been underestimated to date (Mauricio et al., 2006; Kuhls et al., 2008). Several inter-species hybrids have been described (Belli et al., 1994; Banuls et al., 1997; Ravel et al., 2006; Nolder et al., 2007), however the occurrence of recombination at the intra-species level between or within zymodemes is difficult to prove and requires highly discriminative markers. Gene flow between MON-1 and non-MON-1 populations and even between MON-1 sub-populations could be demonstrated only by MLMT (Kuhls et al., 2008; Seridi et al., 2008). In addition to the identification of MON-1/MON-24-80 hybrids, this study also detected gene flow between the two MON-1 populations, as shown for strain 20. Now new questions have to be addressed, such as (i) what is the mechanism of recombination in Leishmania spp., (ii) does recombination occur in the mammalian host or in the vector, (iii) do hybrids have selective advantage over parent strains? A recently described L. infantum/L. major hybrid was found to have a higher transmission potential in relation to the respective vectors (Volf et al., 2007). The present study demonstrated the usefulness of MLMT for population studies at the strain and intra-zymodeme levels for L. infantum. New aspects such as the occurrence of sub-populations

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