Infection, Genetics and Evolution 11 (2011) 557–563

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Infection, Genetics and Evolution

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PCR-RFLP of ribosomal internal transcribed spacers highlights inter and intra-species variation among Leishmania strains native to ,

Rosio Buitrago a, Elisa Cupolillo b, Brigitte Bastrenta c, Francois Le Pont a, Eddy Martinez d,e, Christian Barnabe´ c, Simone Fre´de´rique Brenie`re a,* a Institut de Recherche pour le De´veloppement (IRD), de´partement Socie´te´s et Sante´, UR 016, «Caracte´risation and Control of Populations of Vectors», Representation in Bolivia Av Hernando Siles N85290 Esq Calle 7 Obrajes, La Paz, Bolivia b Laborato´rio de Pesquisas em Leishmanioses, Departamento de Imunologia, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, 21045-900 RJ, Brazil c Institut de Recherche pour le De´veloppement (IRD), de´partement Socie´te´s et Sante´, UR 165, 911 Avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France d Instituto de Investigacio´n en Salud y Desarrollo (IINSAD), Unidad de Parasitologı´a, Medicina Tropical y Medio Ambiente, Calle Claudio Sanjinez s/n, Edificio IBBA, Complejo Hospitalario de Miraflores, La Paz, Bolivia e Ca´tedra de Parasitologı´a, Departamento de Patologı´a, Facultad de Medicina, Universidad Mayor de San Andre´s, Av. Saavedra 2246, La Paz, Bolivia

ARTICLE INFO ABSTRACT

Article history: Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the Received 23 September 2010 genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis Received in revised form 26 November 2010 species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Accepted 29 November 2010 Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus Available online 5 December 2010 chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE Keywords: and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis,21toL. (L.) Leishmania amazonensis,1toLeishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the Bolivia MLEE RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, RFLP-ITS rRNA genes were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), Pathology the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question. ß 2010 Published by Elsevier B.V.

1. Introduction In Bolivia leishmaniasis is endemic in six out of nine departments. At the present time, leishmaniasis shows a rising Leishmaniasis is a parasitosis caused by protozoan of the pattern in tropical and subtropical lowlands which are suitable for Leishmania genus, which is transmitted through the bites of insects agriculture and livestock (Le Pont et al., 1992). Previous which belongs to the genera Phlebotomus and Lutzomyia in the Old epidemiological studies based on isoenzyme electrophoresis and and New World respectively. This disease affects populations from molecular identification of parasites have described a complex 88 countries with an incidence of 2 million new cases each year distribution of different Leishmania species among the endemic worldwide, of which 1.5 million are cases of cutaneous leishmani- zone of the department of La Paz: Leishmania (Viannia) braziliensis asis and 500,000 cases of visceral leishmaniasis. According to (Desjeux et al., 1986a; Le Pont et al., 1992), Leishmania (Leishmania) estimates by the World Health Organization, the number of chagasi (Desjeux et al., 1986b; Le Pont et al., 1992), Leishmania infected people exceeds 12 millions (www.opsoms.org/spanish/ (Leishmania) amazonensis (Martı´nez et al., 1998), and Leishmania ad/dpc/cd/leishmaniasis.htm). (Viannia) lainsoni (Martinez et al., 2001; Bastrenta et al., 2002). The overlap of various Leishmania species in the region complicates the control of transmission because each species has a different * Corresponding author. Tel.: +591 2 278 2969; fax: +591 2 278 2944. epidemiological pattern. The clinical outcome of the disease E-mail address: [email protected] (S.F. Brenie`re). is likely to be the result of host–parasite interactions which

1567-1348/$ – see front matter ß 2010 Published by Elsevier B.V. doi:10.1016/j.meegid.2010.11.019 558 R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563 depend on various factors such as the environment, host (Fig. 1), are listed in Table 1. Reference strains included: L. (L.) nutritional conditions, health and immunity status, as well as amazonensis (IFLA/BR/67/PH8), L. (L.) chagasi (MHOM/BR/74/ the genetic constitution of the host and the parasite. Thus, PP75), L. (V.) braziliensis (MHOM/BR/66/M2903), L. (L.) donovani genotyping and analysis of parasite genetic variability are (MHOM/IN/61/L13) and L. (V.) lainsoni (MHOM/BR/81/M6426), essential to identify the species and the intra-species variants, the last two were only used for PCR-RFLP analysis. All strains to clarify many aspects of these infections such as their were thawed and cultured on NNN biphasic medium enriched epidemiology and their relationships with to the clinical forms with foetal bovine serum (FBS) until they adapted. Then (Cupolillo et al., 2001). subcultures were performed at 26 8CinmonophasicSchneider In the present work, we analyzed together 35 autochthones medium supplemented with different concentrations of FBS. All Leishmahnia strains from 31 patients, 3 sandflies Lu. nuneztovari Leishmania strains included in this study were deposited at the anglesi, and 1 mammal (Conepatus chinga) from Bolivia; some of cryobank of the Leishmania collection from the Oswaldo Cruz them had been studied previously in a piecemeal fashion (Martinez Institute (‘‘Colec¸a˜odeLeishmania do Instituto Oswaldo Cruz – et al., 2001; Martı´nez et al., 1998, 1999, 2002; Bastrenta et al., CLIOC’’ – WDCM 731). 2002). We applied two genetic markers: the multi-locus enzyme electrophoresis (MLEE), which is still considered a gold standard to 2.2. Multilocus enzyme electrophoresis (MLEE) identify and classify Leishmania strains, and restriction fragment length polymorphism of the intergenic regions of rRNA genes Analysis was performed using agarose gel electrophoresis, (RFLP-ITS) according to Cupolillo et al. (1994, 1995) methods. This according to procedures previously described (Cupolillo et al., later genetic marker is highly variable and has already produced 1994). Fourteen enzymatic systems were analyzed: glucose-6- interesting results on within and between species variability of phosphate dehydrogenase (G6PDH, EC 1.1.1.49); phosphogluco- Leishmania, so it was applied for the first time with the current mutase (PGM, EC 2.7.5.1); malic enzyme (ME, EC 1.1.1.40); Bolivian strains. isocitrate dehydrogenase (IDH NADP+, IDH NAD+, EC 1.1.1.42); malate dehydrogenase (MDH, EC 1.1.1.37); 6-phosphogluconate 2. Materials and methods dehydrogenase (6PGDH, EC 1.1.1.44); glucose phosphate isomer- ase (GPI, EC 5.3.1.9); proline dipeptidase (PEPD, EC 3.4.13.9); 2.1. Origin of the strains nucleosidase (NH 1and NH 2, EC 3.1.3.5.); fumarate hydratase (FUM, EC 4.2.1.2); hexokinase (HK, EC 2.7.1.1); mannose phosphate Geographical and host origins, date of isolation of 35 studied isomerase (MPI, EC 5.3.1.8); and aconitate hydratase (ACON, EC Leishmania isolates, mainly from the department of La Paz 45.2.1.3).

Fig. 1. Map of the Bolivian localities where the 35 isolates of Leishmania have been isolated. R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563 559

Table 1 Origin of Bolivian strains isolated from patients, vectors and a mammal from the endemic zone of La Paz department and Leishmania reference strains.

Strains Location (community; Species Patients Type of lesion Zymodeme province) IOC/codea IOC/L codea WHO code Age (years) Sex

IOC/L2311 MHOM/BO/93/EQZ Carrasco; L. L. a. 5 F Diffuse Z100 IOC/L2314 MHOM/BO/95/ALA Cajuata; Inquisivif L. L. a. 39 M Cutaneous Z98 IOC/L2454 MHOM/BO/95/AF Cajuata; Inquisivif L. L. a. 65 M Cutaneous Z96 IOC/L2315 MHOM/BO/95/EN Cajuata; Inquisivif L. L. a. 63 M Cutaneous Z96 IOC/L2317 MHOM/BO/95/CJ5 Cajuata; Inquisivif L. L. a. 78 M Cutaneous Z95 IOC/L2306 MHOM/BO/95/CJ10 Cajuata; Inquisivif L. L. a. 42 M Cutaneous Z96 IOC/L2318b MHOM/BO/95/CJ11 Cajuata; Inquisivif L. L. a. 2 F Cutaneous Z99 IOC/L2307b MHOM/BO/95/CJ13 Cajuata; Inquisivif L. L. a. 50 M Cutaneous Z96 IOC/L2319b MHOM/BO/95/CJ15 Cajuata; Inquisivif L. L. a. 4 M Cutaneous Z96 IOC/L2320 MHOM/BO/95/CJ16 Cajuata; Inquisivif L. L. a. 53 M Cutaneous Z96 IOC/L2308 MHOM/BO/95/CJ17 Cajuata; Inquisivif L. L. a. 1 F Cutaneous Z96 IOC/L2321b MHOM/BO/95/CJ18 Cajuata; Inquisivif L. L. a. 15 M Cutaneous Z96 IOC/L2322b MHOM/BO/95/CJ21 Cajuata; Inquisivif L. L. a. 24 M Cutaneous Z96 – MHOM/BO/96/CJ27 Cajuata; Inquisivif L. L. a. 12 M Cutaneous nd IOC/L2323 MHOM/BO/96/CJ28 Cajuata; Inquisivif L. L. a. 57 M Cutaneous Z96 IOC/L2309 MHOM/BO/96/CJ34 Cajuata; Inquisivif L. L. a. 4 F Cutaneous Z96 IOC/L2324 MHOM/BO/96/CJ39 Cajuata; Inquisivif L. L. a. 32 M Cutaneous Z97 IOC/L2326 MHOM/BO/97/LP02 Quilla Quillani; Loayza L. L. a. 39 F Cutaneous Z96

IOC/L2304 INUN/BO/95/CJA Cajuata; L. L. a. – Peripylariac Z95 IOC/L2305 INUN/BO/96/CJB Cajuata; Inquisivi L. L. a. – Peripylariac Z96 IOC/L2325 INUN/BO/96/CJC Cajuata; Inquisivi L. L. a. – Peripylariac Z96

IOC/L2316 MCON/BO/96/1948d Cajuata; Inquisivi L. V. b. – – Asintoma´tico Z27

IOC/L2312 MHOM/BO/96/MR Carrasco; Caranavi L. V. l. 11 F Cutaneous nd

IOC/L2452 MHOM/BO/97/LP06 Eslabon; Iturralde L. V. b. 22 F Cutaneous Z27 IOC/L2453 MHOM/BO/97/LP07 ; Nor yungas L. V. b. 6 M Cutaneous Z27 IOC/L2299 MHOM/BO/97/LP09 Coripata; Nor Yungas L. V. b. 2 F Cutaneous Z27 IOC/L2300 MHOM/BO/97/LP10 Carrasco; Caranavi L. V. b. 48 M Cutaneous Z27 IOC/L2327 MHOM/BO/97/LP015 San Buena Ventura; Iturralde L. V. b. 6 M Mucocutaneous Z27 IOC/L2328 MHOM/BO/97/LP16 ; Iturralde L. V. b. 47 M Cutaneous Z27 IOC/L2302 MHOM/BO/98/LP26 Wara; Loayza L. V. b. 50 F Cutaneous Z27 IOC/L2303 MHOM/BO/98/LP31 Charcas, Sud Yungas L. V. b. Mucocutaneous Z27 MHOM/BO/98/CHU14 ; Sud Yungas L. V. b. Cutaneous Z27 MHOM/BO/99/LP45 Milluguaya; Nor Yungas L. V. b. 6 F Cutaneous nd MHOM/BO/99/LP46 Rurrenabaque; Balliviane L. V. b. 23 M Cutaneous nd

IOC/L2301 MHOM/BO/97/LP17 Taipiplaya; Caranavi L. L. c. 2a 11 m M Visceral Z01

a All strains are stored at the Leishmania Bank at the Instituto Oswaldo Cruz (Brasil). b These stocks were not analyzed by PCR-RFLP. c Location of the parasites in the proven vector Lutzomya nuneztovari anglesi. d Conepatus chinga (skunk). e Rurrenabaque belongs to Beni department, nd, not done. f These strains were isolated during an outbreak.

2.3. RFLP-ITS analysis amplification step of 30 cycles: 94 8C for 1 min, 55 8C for 1 min, and 72 8C for 2 min. PCR products were analyzed by electrophoresis on Total DNA was extracted from 50 mg of pellets of parasites 1.5% agarose gels in 0.5 TAE buffer and visualised by ethidium diluted in 400 ml of TE buffer (10 mM Tris–Cl pH 7.4; 1 mM EDTA bromide staining. Then PCR products were ethanol precipitated pH 8) and incubated overnight at 56 8C with proteinase K (10 mg/ and digested overnight with 6 restriction enzymes: Eco RI (37 8C), 100 mg of pellet) and SDS 1% final. After consecutive phenol and HhaI (37 8C), AluI (37 8C), RsaI (37 8C), TaqI (65 8C), and HaeIII chloroform:isoamylalcohol (24:1) extractions and ethanol precip- (37 8C) at a concentration of 0.01 U/ml. The electrophoretical itation, the DNA was dissolved in sterile water (50 ml/75 mg separation of the fragments was performed in a 12% acrylamide gel pellet). Previously described primers used in PCR to amplify the with the Gene Gel Excel 12.5/24 Kit (Pharmacy Biotech, San Intergenic Region of rRNA genes were IR1 = 50GCT-GTA-GGT-GAA- Francisco, USA) in the Genephor apparatus. CCT-GCA-GCA-GCT-GGA-TCA-TT30 and IR2 = 50GCG-GGT-AGT- CCT-GCC-AAACA-CTC-AGG-TCT-G30 which were obtained from 2.4. Phenetic analysis conserved sequences of the short subunit 30 end (ITS1) and of the large subunit 50 end (ITS2) of rRNA genes. They amplify two ITSs For each stock, the presence or absence of a particular band (internal transcribed spacers noncoding region) and include the was coded by 1 or 0 respectively. Genetic distances between gene that codes for the smaller sub unit 5.8S rRNA (Cupolillo et al., pairs of stocks were based on either matching index, of which 1995). Amplification reactions were performed in a volume of the formula is (00 + 11)/(00 + 01 + 11) or Jaccard’s index 100 ml containing 100 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM (11/(11 + 01)), where (00) is the number of bands which are

MgCl2, 125 mM of each deoxynucleotide triphosphate, 0.2 mMof absent in the two stocks, (11) is the number of bands present in each primer, 1.5 U of Taq DNA polymerase (Pharmacia Biotech, San the two stocks and (01) the number of bands present in one stock Francisco, USA) and 50 ng of DNA template. The tubes were placed and absent in the other. Distances were computed by PHYL- on a Biometra, Trio thermoblock (Go¨ttingen, Germany) for an TOOLS, which is a freeware utilities package that works with the 560 R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563

PHYLIP package (Felsenstein, J. 2002. PHYLIP (Phylogeny Infer- restriction enzymes (EcoRI, HhaI, AluI, RsaI, TaqI and HaeIII) and ence Package) version 3.6a3, distributed by the author, Depart- the fragments separated by high resolution electrophoresis in ment of Genome Sciences, University of Washington, Seattle). acrylamide gel. Then, for each strain, the presence (1) or absence UPGMA trees were built by the program NEIGHBOR from the (0) of bands was scored. A total of 206 generated fragments were PHYLIP package, while bootstrap trees were computed by retained ranging from 26 for AluIto43forTaqIrestriction SEQBOOT, MIX and CONSENSE programs successively. MIX is a enzymes. The number of bands per strain ranged between 42 and general parsimony program which carries out the Wagner and 79 (average 64.3 9.07). Nine bands were common to all strains Camin-Sokal parsimony methods in mixture. The bootstrap (4.4%) while 42 bands were present in only one of the strains method (Felsenstein, 1985) was used to test the robustness (singleton, 20.4%). Indeed, each strain presented a unique restric- of nodes within the trees. Both kinds of trees (UPGMA, tion pattern. UPGMA trees constructed with either Jaccard’s or Bootstrap) were plotted by the freeware NJPLOT (Perrie` re and Matching’s distances depicted 4 clusters composed of stocks Gouy, 1996). belonging to L. (V.) braziliensis, L. (L.) chagasi, L. (L.) amazonensis and L. (V.) lainsoni. Corresponding nodes were supported by high 3. Results bootstrap values (99; see Fig. 2) except for the L. (V.) braziliensis group (70.8). When bands corresponding to singletons are removed 3.1. MLEE variability from the analysis, the general structure is unchanged, as well as the bootstrap values (77.4 for L. (V.) braziliensis group). It is worth Among the 14 enzyme systems tested, the NH inferred the noting that the bootstrap value of the L. (V.) braziliensis group activity of two different enzymatic loci and therefore 15 enzyme without MHOM/BO/97/LP16 and MCAR/BO/96/1948 stocks pre- loci were resolved. However, the activity of the second loci of NH sented a significant value (99.8). We also investigated the current was missing (null allele) in some isolates characterized as L. (L.) sample of stocks by identical analysis of RFLP-ITS patterns for each amazonensis. The phenotype composition of the different enzyme separately. A similar topology to that of the joined data was zymodemes is given in Table 2. All loci proved to be polymorphic. only obtained for the Taq-I restriction enzyme which depicted a It is worth noting that for 18 isolates the patterns observed for total of 43 bands (largest number of bands among the restriction FUM, GPI, MDH, MPI, PGM, and 6PGDH loci were all characteristic enzyme systems used); however, the bootstrap value was lower for of heterozygotes (see below). According to MLEE patterns and L. (L.) amazonensis (66.1%) but similar for the other groups (76.8% for clustering analysis based on the construction of dendrogram L. (V.) braziliensis). (Fig. 2), 3 Leishmania species are clearly identified among the 31 Bolivian stocks analyzed by this genetic marker: 10 isolates 4. Discussion presented a MLEE pattern identical to L. (V.) braziliensis (ICO/ Z27), 1 isolate MHOM/BO/97/LP17, had a pattern identical to L. Among the 35 stocks analyzed in this work, by either MLEE and/ (L.) chagasi (ICO/Z01), and the others (n = 20) were significantly or PCR-RFLP ITS-rRNA, 12 were characterized as L. (V.) braziliensis, grouped with a bootstrap value of 99% in a cluster that included 21 as L. (L.) amazonensis,1asL. (V.) lainsoni and 1 as L. (L.) chagasi.In the L. (L.) amazonensis (IOC/Z07) reference strain. In this group, 6 a general way the two markers are in agreement to discriminate different zymodemes were identified among the Bolivian stocks, the species but the second marker highlights an additional and all were distinct from the L. (L.) amazonensis reference strain variability among L. (V.) braziliensis and L. (L.) amazonensis.In by at least one locus; the number of stocks belonging to a single fact, the polymorphism of ITS sequences identifies each stock in a zymodeme ranges from 1 to 13. Remarkably, the zymodemes finger printing way, confirming the ability of this technique to numbered IOC/Z95, IOC/Z96 and IOC/Z99 (Table 1), which discriminate between closely related strains. represent 85% of the stocks belonging to the L. (L.) amazonensis MLEE analysis does not show any variability among L. (V.) showed a heterozygous pattern in three loci, GPI, PGM and braziliensis. All stocks were identical to the reference stock isolated 6PGDH which are new patterns heterozygous in L. (L.) in 1966 which originated from a Brazilian patient. The absence of amazonensis. isoenzyme variability of L. (V.) braziliensis stocks in the studied area differs from the data obtained in other endemic regions. Saravia 3.2. RFLP-ITS polymorphisms of rRNA genes et al. (1998) describe 6 enzymatic variants of L. (V.) braziliensis isolated from patients in Colombia, similarly Cupolillo et al. (1994, PCR products of ITS rRNA were of different molecular weights 2001, 2003) identified up to 8 variants in Brazilian Amazonia. As according to the Leishmania subgenus, 1 kb for L. (V.) braziliensis expected, the analysis of the sequence polymorphism of the and L. (V.) lainsoni and 1.2 kb for L. (L.) amazonensis and L. (L.) ITSrRNA detected additional variability among this group of stocks chagasi. PCR products of each strain were digested with the 6 (Cupolillo et al., 2003). Indeed, the current analysis shows that two

Table 2 Multilocus genotypes of the 9 zymodemes identified with 15 polymorphic loci among Leishmania isolates native of Bolivia.

Zymodeme Species Number of Acon Fum G6pd Gpi Hk Idh Nadp Idh Nad Mdh Me Mpi Nh I Nh 2 Pepd Pgm 6Pgdh IOC/code studied stocks

IOC/Z07a L. L. a. 0 3 1, 4 2 2 3 3 3 3 2 2, 3 1 Absent 3 1 5 IOC/Z100 L. L. a. 1 3 1, 4 2 2 3 3 3 3 2 2, 3 1 Absent 5 1 5 IOC/Z97 L. L. a. 1 3 1, 4 2 2 3 3 3 3 2 2, 3 1 Absent 6 1 5 IOC/Z98 L. L. a. 1 3 1, 4 2 1, 2 3 3 3 3 2 2, 3 1 2 2 1 3, 4, 5 IOC/Z95 L. L. a. 2 3 1, 4 2 1, 2 3 3 3 3 2 2, 3 1 2 3 1, 3 3, 4, 5 IOC/Z96 L. L. a. 14 3 1,4 2 1,2 3 3 3 3 2 2,3 1 2 5 1,3 3,4,5 IOC/Z99 L. L. a. 1 3 1, 4 2 1, 2 3 3 3 3 2 2, 3 1 2 4 1, 3 3, 4, 5 IOC/Z27a L. V. b. 10 1 2, 3 3 3 1 1 2 1 1 1 3 3 5 2 1 IOC/Z01a L. L. c. 1 2 1 1 4 2 2 1 2, 3 2 1, 2 2 1 1 1 2

a These zymodemes correspond to references strains: L. L. amazonensis (IFLA/BR/67/PH8) is IOC/Z07, L. V. braziliensis (MHOM/BR/66/M2903) is IOC/Z27 and L. L. chagasi (MHOM/BR/74/PP75) is IOC/Z01. R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563 561

EQZ, IOC/Z100 IOC/Z96 989 CJ39, IOC/Z97 CJA, IOC/Z95 * CJ15, IOC/Z96 IOC/Z95 AF, IOC/Z96 CJ18, IOC/Z96 CJ11, IOC/Z99 IOC/Z99 CJ17, IOC/Z96 CJ13, IOC/Z96 CJ16, IOC/Z96 IOC/Z98 CJ5, IOC/Z95 CJC, IOC/Z96 * 999 CJ10, IOC/Z96 IOC/Z100 CJB, IOC/Z96 * L. L. amazonensis PH8, IOC/Z07 CJ28, IOC/Z96 IOC/Z07 EN, IOC/Z96 LP002, IOC/Z96 ALA, IOC/Z98 CJ21, IOC/Z96 IOC/Z97 CJ34, IOC/Z96 CJ27 M6426, IOC/Z15 1000 L. V. lainsoni MR, IOC/Z15 IOC/Z01 998 L13 (a) L. L. donovani LP17, IOC/Z01 L. L. chagasi 1948, IOC/Z27 ** LP16, IOC/Z27 986 LP007, IOC/Z27 LP15, IOC/Z27 997 M2903, IOC/Z27 LP010, IOC/Z27 LP45 LP31, IOC/Z27 CHU14, IOC/Z27

LP46 L. V. braziliensis IOC/Z27 LP006, IOC/Z27 LP26, IOC/Z27 LP009, IOC/Z27

0.02 0.1

PCR-RFLP of ITS rRNA MLEE

Fig. 2. UPGMA trees built from genetic distances (matching index), obtained by PCR-RFLP of ITS rRNA genes (left) and MLEE (right), showing relationships between 35 Bolivian Leishmania isolates studied here, including reference species strains (underlined). The values at the main nodes of the tree are the bootstrap values over 1000 replicates. (a) L13 (MHOM/IN/61/L13) strain was only analyzed using PCR-RFLP; the MR strain and the reference L. (V.) lainsoni strain (M6426) were not included in the MLEE analysis in the current work; *strains from vectors; strain from the only mammal.

stocks are not significantly gathered with the L. (V.) braziliensis diversity of the Bolivian stocks infecting human remains reference. The first stock was isolated from a small carnivore, complex among L. (V.) braziliensis species and it is necessary a skunk (C. chinga), in Cajuata (Province Inquisivi) and the to study with precision the species status of strains within the second one from a cutaneous lesion of a patient in Ixiamas braziliensis complex. This diversity should be related to that (Province Iturralde) which is a distance of 500 km from Cajuata. which exists in a sylvatic cycle where many mammalian hosts The other stocks were significantly grouped to L. (V.) braziliensis are involved in the transmission cycle. However, parasite (bootstrap value of 99.8%). This result shows that the genetic diversity in sylvatic cycles is scarcely documented in Bolivia. 562 R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563

According to the origin of current Bolivian patients infected by L. Acknowledgements (V.) braziliensis, this isoenzyme variant presents a large geographical distribution from the sub-Andean valleys of This work received financial support from the IRD (Institut de Yungas (1700 m) to the lower Amazonian lands in Alto Beni Recherche pour le De´veloppement, France). E.C. is a fellow (250 m) being an old focus of leishmaniasis (Le Pont et al., 1992). researcher from the national Council for Scientific and Technologi- Moreover, this variant involved cutaneous forms as well as cal Development (CNPq, Brazil) and Carlos Chagas Filho Research mucosal forms but we did not find a relationship between Foundation of the state of Rio de Janeiro (FAPERJ, Brazil). ITSrRNA variability and clinical forms. Contrary to what was found among L. (V.) braziliensis,MLEE References methods detected several zymodemes among the stocks belonging to L. (L.) amazonensis and the ITSrRNA analysis Bastrenta, B., Buitrago, R., Vargas, F., Le Pont, F., Torrez, M., Flores, M., Mita, N., conferred additional variability, each stock presenting a single Brenie`re, S.F., 2002. First evidence of transmission of Leishmania (Viannia) lainsoni in a Sub Andean region of Bolivia. Acta Trop. 83, 249–253. pattern. So current data and previous analyses showed L. (L.) Bonfante-Garrido, R., Melendez, E., Barroeta, S., de Alejos, M.A., Momen, H., Cupo- amazonensis is a very diverse group, as indicated by a study of lillo, E., McMahon-Pratt, D., Grimaldi Jr., G., 1992. Cutaneous leishmaniasis in strains of L. (L.) amazonensis isolates from patients from North western Venezuela caused by infection with Leishmania venezuelensis and L. braziliensis variants. Trans. R. Soc. Trop. Med. Hyg. 86, 141–148. east Brazil (De Oliveira et al., 2007). However, according to the Cupolillo, E., Grimaldi Jr., G., Momen, H., 1994. A general classification of New clustering analysis, neither of the two markers detects a sub World Leishmania using numerical zymotaxonomy. Am. J. Trop. Med. Hyg. 50, group of stocks within L. amazonensis. Despite the genetic 296–311. diversity of these stocks, all except one (MHOM/BO/93/EQZ), Cupolillo, E., Grimaldi Jr., G., Momen, H., Beverley, S.M., 1995. Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution produced only cutaneous lesions in patients. In fact, an absence of Leishmania. Mol. Biochem. Parasitol. 73, 145–155. of correlation between Leishmania genotype and clinical mani- Cupolillo, E., Aguiar, F., Brahim, L.R.N., Naiff, M.F., Pereira, L.O.R., Oliveira-Neto, M.P., festation has been previously reported by several authors using Falqueto, A., Grimaldi Jr., G., 2001. Recent advances in taxonomy of the New World leishmanial parasites. Med. Microbiol. Immunol. 190, 57–60. different molecular markers (Cupolillo et al., 2003; Garcia et al., Cupolillo, E., Brahim, L.R., Toaldo, C.B., de Oliveira-Neto, M.P., de Brito, M.E., 2005; Guerbouj et al., 2001; Schonian et al., 2000). The specific Falqueto, A., de Farias Naiff, M., Grimaldi Jr., G., 2003. Genetic polymorphism case is the infection with MHOM/BO/93/EQZ of the young girl and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil. J. Clin. Microbiol. 41, 3126–3132. who developed an extremely serious diffuse leishmaniasis from Delgado, O., Cupolillo, E., Bonfante-Garrido, R., Silva, S., Belford, E., Grimaldi Jr., G., which she did not survive (Martı´nez et al., 2002). An explanation Momen, H., 1997. Cutaneous leishmanaisis in Venezuela caused by infection for the severe clinical manifestation in this patient could be with a new hybrid Leishmania (Viannia) braziliensis and L. (V.) guyanensis. Mem. Inst. Oswaldo Cruz 92, 581–582. the co-infection with L. (L.) chagasi and L. (L.) amazonensis De Oliveira, J.P., Fernandes, F., Cruz, A., Trombela, V., Monteiro, E., Camargo, A., which was highlighted after her death. As reported by the Barral, A., de Oliveira, C., 2007. Genetic diversity of Leishmania amazonensis authors, L. (L.) chagasi is known to depress the immune response strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR- based analyses and molecular karyotyping. Kinetoplastid. Biol. Dis. 6 (5). and in this context, a prior L. (L.) chagasi infection followed by a Desjeux, P., Le Pont, F., Mollinedo, S., Tibayrenc, M., 1986a.In: Les Leishmania de secondary infection with L. (L.) amazonensis could have provoked Bolivie. I. Leishmania braziliensis Vianna, 1911 dans les De´partements de la Paz diffuse cutaneous leishmaniasis in the patient (Martı´nez et al., et du Beni. Premiers isolements de souches d’origine humaine. Caracte´risation 2002). It is thus still of interest to be searching for genetic enzymatique, Coll. Int. CNRS/INSERM, 1984, IMEEE, Montpellier, pp. 401–410. Desjeux, P., Le Pont, F., Mollinedo, S., Tibayrenc, M., 1986b.In: Les Leishmania de markers that can be associated genetic variability with clinical Bolivie. I. Leishmania chagasi Cunha et Chagas, 1937. Premiers isolements dans manifestations. les ‘‘Yungas’’ du de´partement de La Paz. Comparaison isoenzymatique de The majority of L. (L.) amazonensis stocks was isolated during an souches de l’homme, du chien et du phle´botome Lutzomyia longipalpis, Coll. Int. CNRS/INSERM, 1984, IMEEE, Montpellier, pp. 411–419. outbreak in an area which was not considered previously as an Dujardin, J.C., Ban˜uls, A.L., Llanos-Cuentas, A., De Doncker, S., Jackquet, D., Le Ray, D., endemic zone of leishmaniasis (Martı´nez et al., 1998). The Are´valo, J., Tibayrenc, M., 1993. Putative Leishmania hybrids in the Eastern heterozygous character of the large majority of the transmitted Andean Valley of Huanuco. Peru. Acta Trop. 59, 293–307. Evans, D.A., Kennedy, W.P.K., Elbihari, S., Chapman, C.J., Smith, V., Peter, W., 1987. parasites (zymodemes IOC/Z95, IOC/Z96, IOC/Z99), evidenced here Hybrid formation within the genus Leishmania. Parasitology 29, 165–173. with three additional heterozygous loci, could be the result of an Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the hybridization event between different strains; so the hybrid nature bootstrap. Evolution 39, 783–791. Garcia, A.L., Kindt, A., Quispe-Tintaya, K.W., Bermudez, H., Llanos, A., Arevalo, J., of these isolates could be linked to the high rate of human Ban˜uls, A.L., De Doncker, S., Le Ray, D., Dujardin, J.C., 2005. American tegumen- transmission because of better fitness of these parasites with tary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleo- respect to its host. However, among the current sample of L. (L.) morphism. Infect. Genet. Evol. 5, 109–116. Guerbouj, S., Guizani, I., Speybroeck, N., Le Ray, D., Dujardin, J.C., 2001. Genomic amazonensis stocks, we did not observe isoenzyme patterns polymorphism of Leishmania infantum: a relationship with clinical corresponding to the hypothetical parents. Other studies have pleomorphism? Infect. Genet. Evol. 1, 49–59. already reported hybrids between species of Leishmania in the Old Guevara, P., Alonso, G., Franco da Silveira, J., de Mello, M., Vicente Scorza, J., An˜ez, N., ´ World (Evans et al., 1987; Kelly et al., 1991) as well as in the New Ramırez, L., 1992. Identification of New World Leishmania using ribosomal gene spacer probes. Mol. Biochem. Parasitol. 56, 15–26. World where hybrids between species of the braziliensis complex Kelly, J.M., Law, J.M., Chapman, C.J., Van Eys, G.J., Evans, D.A., 1991. Evidence of were reported (Bonfante-Garrido et al., 1992; Delgado et al., 1997; genetic recombination in Leishmania. Mol. Biochem. Parasitol. 46, 253–263. Dujardin et al., 1993; Tojal da Silva et al., 2006). Here, putative Le Pont, F., Desjeux, P., Torrez, M., Fournet, A., Mouchet, J., 1992. Leishmanioses et phle´botomes en Bolivie. ORSTOM E´ ditions 116. parents remain to be researched and could be investigated with Martı´nez, E., Le Pont, F., Torrez, M., Tellerı´a, J., Vargas, F., Mun˜oz, M., De Donker, S., priority on wild cycles. Dujardin, J.C., Dujardin, J.P., 1998. A new focus cutaneous leishmaniasis due to In a general way, this study agrees with the complexity of the Leishmania amazonensis in Sub Andean region of Bolivia. Acta Trop. 71, 97–106. Martı´nez, E., Le Pont, F., Torrez, M., Tellerı´a, J., Vargas, F., Dujardin, J.C., Dujardin, J.P., leishmaniasis in Bolivia with the coexistence of several 1999. Lutzomyia nuneztovari anglesi (Le Pont & Desjeux, 1984) as a vector of Leishmania species, probably related to environmental changes Leishmania amazonensis in a Sub Andean leishmaniose focus of Bolivia. Am. J. and human colonization, which can explain the increase in Trop. Med. Hyg. 61, 846–849. Martinez, E., Le Pont, F., Mollinedo, S., Cupolillo, E., 2001. A first case of cutaneous declared cases of leishmaniasis in the sub-Andean region. leishmaniasis due to Leishmania (Viannia) lainsoni in Bolivia. Trans. R. Soc. Trop. Overall, the study confirms that the sequence variability of Med. Hyg. 95, 375–377. the ITS rRNA gene is relevant for our purpose (Guevara et al., Martı´nez, E., Mollinedo, S., Torrez, M., Ban˜uls, A.L., Le Pont, F., 2002. Co-infection by Leishmania amazonensis and L. infantun/L. chagasi in a case of difuse cutaneous 1992; Cupolillo et al., 1995) and this marker may be suggested leishmaniasis in Bolivia. Trans. R. Soc. Trop. Med. Hyg. 96, 529–532. as a means to explore the genetic clustering within Leishmania Perrie`re, G., Gouy, M., 1996. WWW-Query: an on-line retrieval system for biological species. sequence banks. Biochimie 78, 364–369. R. Buitrago et al. / Infection, Genetics and Evolution 11 (2011) 557–563 563

Saravia, N.G., Segura, I., Holguin, A., Santrich, C., Valderrama, L., Ocampo, C., 1998. Leishmania aethiopica does not correlate with clinical variations of cutaneous Epidemiologic, genetic, and clinical associations among phenotypically dis- leishmaniasis. Mol. Biochem. Parasitol. 5, 239–248. tinct populations of Leishmania (Viannia) in Colombia. Am. J. Trop. Med. Hyg. Tojal da Silva, A.C., Cupolillo, E., Volpini, A.C., Almeida, R., Romero, G.A., 2006. 59, 86–94. Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Schonian, G., Akuffo, H., Lewin, S., Maasho, K., Nylen, S., Pratlong, F., Eisenberger, Acre, Brazil. Trop. Med. Int. Health 11, 1388–1398. C.L., Schnur, L.F., Presber, W., 2000. Genetic variability within the species