List of Abstracts

STRAIN VARIATIONS IN THE LEISHMANIA DONOVANI COMPLEX DETECTED IN SELECTED PROTEIN-CODING SEQUENCES

E. Zemanová1, M. Jirku1, I.L. Mauricio2, M.A. Miles2, J. Lukeš1
1Institute of Parasitology and Faculty of Biology, Ceské Budejovice, Czech Republic;
2London School of Hygiene and Tropical Medicine, London, England

Human visceral leishmaniasis (VL) is caused by members of the Leishmania donovani complex, particularly L. donovani in the Old World and L. infantum (=L. chagasi) in both the Old and New Worlds. Leishmania archibaldi is a possible third member of the group. Currently, the “gold standard” for identification and classification of Leishmania spp. is multilocus isoenzyme electrophoresis, which relies on the analysis of 15 selected enzymes. Each zymodeme is represented by a unique combination of mobilities of these enzymes in a starch or polyacrylamide gel.
In order to study the genetic background of different enzymatic mobilities, we have amplified and directly sequenced PCR products of genes encoding isocitrate dehydrogenase (1,3 kb; involved in carbohydrate metabolism), malic enzyme (1,7 kb; involved in malate metabolism), mannose phosphate isomerase (1,3 kb; involved in carbohydrate metabolism) and glucose-6-phosphate dehydrogenase (1,7 kb; involved in pentose pathway). Sequences have been obtained from 25 different strains of the L. donovani complex from the Mediterranean region, India and East Africa.
Sequencing of about 170 kb of protein-coding regions from strains of the Leishmania donovani complex enabled : i/ identification of point mutations sometimes leading to changes in the charge of the protein; ii/ detection of a surprisingly high number of putative heterozygotes; iii/ construction of well-supported concateneted phylogenetic trees and phylogeny network. We propose differences in sequences that in several cases disagree with phenotypes determined by multilocus isoenzyme analysis are caused by posttranslational modification of the proteins. The documented genetic polymorphism correlates strongly with geographic origin of the L. donovani complex strains.

EQUINE LEISHMANIASIS IN PORTUGAL : FIRST REPORT

N. Rolão, A. João, J. Cristóvão, L. Campino
Instituto de Higiene e Medicina Tropical, Unidade Leishmanioses/CMDT, Universidade Nova de Lisboa, Portugal. e-mail: campino@ihmt.unl.pt

In order to check for the occurrence of equine leishmaniasis in Portugal, a serological screening was carried out in 13 horses living in a farm located in an endemic area (Metropolitan Region of Lisbon), where 3 cases of canine leishmaniasis were previously diagnosed. The horses were born in Portugal and had never travelled abroad. One of the horses presented a single irregular ulcerative skin lesion in the right metatarsus, which evolved from a small erosion within 2 months. Analysis by CIE revealed the presence of anti-Leishmania antibodies in the horse serum. Leishmania DNA was detected in the skin lesion by Real-time TaqManâ PCR (using a L. infantum probe), confirming the infection.
This study led to the finding of the first horse infected with Leishmania in Portugal. Equine leishmaniasis due to L. braziliensis is quite common in South and Central America. In Europe, equine leishmaniasis caused by L. infantum has been reported in Germany and Spain. In the present case the detection of anti-Leishmania antibodies may be indicative of a concomitant visceral involvement. The lesion described in the present report is different from lesions described in other equine leishmaniasis cases caused by L. infantum, suggesting that the pathogen is capable of producing a variety of cutaneous lesions in horse such as in canine and human hosts.
Our results strongly point out that equine infection with L. infantum need to be explored further in order to clarify the clinical evolution of the equine infections and the role of the horse as reservoir of the parasite in endemic areas.

Work supported by funding from the EC research project no. QLK2-CT-2001-01810, EU.

ISOENZYMATIC POLYMORPHISM OF LEISHMANIA INFANTUM IN PORTUGAL

L. Campino1, F. Pratlong3, P. Abranches1, J.- A. Rioux3, G. Santos-Gomes1, C. Pires2, S. Cortes1, O. Afonso2, J.P. Dedet3
1Instituto de Higiene e Medicina Tropical, Unidade de Leishmanioses/ CMDT, Universidade Nova de Lisboa, Portugal
2Instituto de Higiene e Medicina Tropical, Unidade de Entomologia Médica/UPMM
3Centre National de Référence des Leishmania, Université de Montpellier, France.
e-mail: campino@ihmt.unl.pt

Leishmaniasis in Portugal is a zoonosis caused by Leishmania infantum transmitted by P. ariasi and P. perniciosus, considered hypo-endemic as in other southern European countries. Although sporadic cases occur all over the country, three endemic foci have been identified: Alto Douro Region (ADR), Lisbon Metropolitan Region (LMR) and Algarve Region (AR). Since the beginning of the nineties, leishmaniasis as become increasingly important in the country due to the high number of diagnosed cases in patients infected with HIV. Recently, epidemiological surveys performed in ADR and LMR revealed an increase in canine prevalence.
This study reports to the isoenzymatic diversity of 210 Leishmania stocks isolated from: humans with cutaneous and visceral leishmaniasis, either immunocompetent children (13) or immunocompetent and immunocompromised adults (78), dogs (110), foxes (4) and vectors (5), from different regions of Portugal, from 1982 to 2004. Parasites were isolated in NNN medium and characterised by multilocus enzyme electrophoresis.
All strains isolated in dogs from all over the country, and in foxes from LMR, were identified as L. infantum MON-1. Strains isolated in P. ariasi from ADR were identified as MON-1 (2) and MON-24 (2), whereas the strain from P. perniciosus, AR, was MON-1 (1). As to the human stocks, 94.5 % (86/91) were identified as MON-1. Four strains isolated from Leishmania/HIV co-infection cases from LMR were zymodemes MON-80 (1), MON-29 (2) and MON-24 (1). Another MON-29 strain was isolated from an immunocompetent adult with cutaneous leishmaniasis from the AR. Zymodeme MON-1 was predominant from all the different geographic regions in Portugal, and in all hosts, being the only one identified in canids. So the life cycle of MON-1 is fully demonstrated in Portugal. The higher variability of zymodemes was observed in HIV-positive adults which represent the major group of patients from LMR. The low enzymatic polymorphism found in Portugal contrasts with the high diversity reported from other southern European foci namely in Spain.

Work supported partially by funding from the EC research project no. QLK2-CT-2001-01810, EU; CMDT and UPMM.

LEISHMANIA INFANTUM DIVERSITY IN PORTUGAL : APPLICATION OF KDNA AS A MOLECULAR MARKER

S. Cortes1, A. Almeida1, F. Pratlong2, J.P. Dedet2, L. Campino1
1Instituto de Higiene e Medicina Tropical, Unidade de Leishmanioses/ CMDT, Universidade Nova de Lisboa, Portugal
2Centre National de Référence des Leishmania, Université de Montpellier, France.
e-mail: campino@ihmt.unl.pt

Leishmania infantum is the main etiological agent of visceral and cutaneous leishmaniasis in Portugal and zymodeme MON-1 as been isolated in more than 95 % of the leishmaniasis cases. Previous studies on genetic characterisation and variability of Leishmania, namely L. infantum, with a range of molecular targets have demonstrated the potential of genotyping. In this work we applied kinetoplast DNA (kDNA) as a genetic marker and PCR - Restriction Length Fragment Polymorphism method (PCR-RFLP). Amplification of minicircle kDNA partial sequence by PCR and analysis by RFLP using nine endonucleases was performed in 112 samples (human, canine and vector) from several portuguese regions (Alto Douro, Lisbon Region, Alentejo and Algarve) and 16 from Mediterranean, East African and Brazilian foci.
Leishmania portuguese isolates were grouped into 14 different restriction profiles. A predominance of profile A (55/112) was observed, being this exclusive of the portuguese isolates. Both human and canine strains (79) belonging to zymodeme MON-1 were characterised in 11 profiles, whereas MON-1 vector isolates (3) were typed in 3 (B, C, L). The high genetic variability found in isolates from phlebotomines could be related with the fact that these samples belong to different vector species (P. perniciosus and P. ariasi).
PCR-RFLP method using kDNA as a marker permitted the evaluation of genetic diversity of L. infantum population isolated from different hosts in Portugal and also the application of direct genotyping of parasites in host tissues.

Work supported by funding from the EC research project no. QLK2-CT-2001-01810, EU.

PROTECTIVE IMMUNITY AND RESISTANCE TO RE-INOCULATION IN IRON-LOADED BALB/C MICE INOCULATED WITH A LOW NUMBER OF LEISHMANIA MAJOR METACYCLIC PROMASTIGOTES IS ASSOCIATED WITH NF-?? ACTIVATION

S. Bisti1, G.Milon2, K. Soteriadou1
1 Laboratory of Molecular Parasitology, Hellenic Pasteur Institute, Athens, Greece; sbisti@pasteur.gr 2 Institut Pasteur, Unite d’ Immunophysiologie et Parasitisme Intracellulaire, Paris, France

By inoculating a high number of L.major in the footpad, we previously established that systemic iron delivery in “susceptible” BALB/c mice is beneficial for the host. We report here, that the partial protective effect of iron delivery is also observed after the i.d. inoculation of a “high dose” in the BALB/c ear and is associated to a transient increase of oxidative burst. Interestingly, systemic iron delivery, in a “low dose” ear model, results in the complete absence of (a) ear lesion and (b) any culturable parasites from the ear, the DLN, the liver and the spleen. The iron-induced protective status is associated with a sustained, at late stages after parasite inoculation, increase in NADPH oxidase-mediated oxidative burst. The iron-induced protective status was reversed -i.e. development of lesions- in iron-loaded mice treated with the antioxidant diphenyleneiodonium chloride (DPI), two weeks after parasite inoculation. This correlated with significantly lower levels of oxidative burst at the dermal site of these mice compared to the non DPI-treated iron-loaded mice. Iron-loaded mice were resistant to the re-inoculation, at a distant site (right hind footpad), of 106 stationary phase promastigotes or to the re-inoculation of a ‘low dose’ of metacyclics at the contra-lateral ear. Interestingly, both the protective and the “resistant to re-inoculation” status were coupled to a) NF-?B activation in cells within the LN draining the sites of primary and secondary challenge of iron-loaded mice and b) high level of IFN? produced by CD4+ T-cells recovered from the DLNs. Once stimulated in vitro with anti-CD3, CD4+-T lymphocytes recovered at weeks 8 post second parasite inoculation from iron-loaded mice proliferated with higher rate (CFSE–labelling) than the similarly stimulated cells recovered from control mice. Our results strongly suggest that in iron-loaded mice the intracellular progeny of parasites remains very low. The generation of ROS and the NF-?? activation in iron-loaded cells are coupled to enhanced T-cell proliferative properties along with IFN-? production: these phenotypic parameters assess a sustained tightly regulated type 1 profile of the immune response.

EXPLOITATION OF HISTONE H1 AND K26 GENE POLYMORPHISM FOR GENOTYPING STRAINS BELONGING TO THE LEISHMANIA DONOVANI COMPLEX

C.Haralambous, D.Smirlis, K. Soteriadou
Molecular Parasitology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521, Athens, Greece. christos@pasteur.gr

Leishmania Histone H1 (LH1) and the K26 genes were evaluated for the genotyping of strains belonging to the L.donovani complex. Both proteins possess repeated amino acid motifs and show gene size variability. The K26 protein (also known as GBP) is a hydrophilic surface protein, which processes a highly variable central 14 amino acid repeat region. The repeat region of L.donovani and L.infantum (PKEDGH/RTQKNDGDG) is 64% identical with that of L.major. Since the L.major gene has considerable size polymorphism, we thought that this would also be the case for its homologue gene in L.donovani. PCR amplification of the K26 coding region revealed considerable size polymorphism within the L.donovani complex, ranging from 300b.p. to 1k.b. The latter reflects differences in the number of the repeats. A simple PCR-based method, based on size differences of the amplified K26 gene, easily detectable on agarose gels, was developed and used for genotyping strains belonging to the L.donovani complex. Results were correlated with the isoenzyme typing system. The Leishmania histone H1 (LH1) gene was also exploited as a genotyping marker. LH1 is a small, basic, lysine rich protein whose structure resembles the C-terminal domain of metazoan H1. It is encoded by two tandem genes which are identical or similar depending on the strain. Sequence analysis of LH1 in different Leishmania species and subspecies revealed considerable size polymorphism. The size polymorphism of this gene formed the basis for the development of a PCR-based method for genotyping strains belonging to the L.donovani complex. The LH1 sequences were also used for phylogenetic analysis. Since Leishmania LH1 represents one of the first eukaryotic histones on earth, its study would give us insights on the function and evolution of this gene superfamily.

This work was in part supported by EU grant QLK-CT-2001-01810

GENES ENCODING METABOLIC ENZYMES AS MARKERS FOR GENOTYPING THE LEISHMANIA DONOVANI COMPLEX

IL Mauricio1, R Silk1, M Baghaei1, F Pratlong2, J-P Dedet2, E Zemanova3, J Lukes3 & MA Miles1

1London School of Hygiene and Tropical Medicine, London, UK; 2Laboratoire de Parasitologie, Montpellier, France; 3Czech Academy of Sciences, Ceske Budovice, Czech Republic.
isabel.mauricio@lshtm.ac.uk

Multilocus enzyme electrophoresis (MLEE) has been the gold standard for the identifcation of Leishmania. Drawbacks include: detection of only amino acid polymorphisms that change protein mobility; different allozymes may have indistinguishable mobilities; the limited number of markers available, and bulk culture of parasites is required. However, an advantage is that thousands of strains have already been characterised by MLEE. Of the 15 enzymes used for MON-typing by MLEE, we have initially studied the genetic diversity of the genes encoding NH1, NH2, ASAT (GOT1), GPI and PGD, firstly to define the genetic basis of the allozyme phenotypes, and secondly, to replace MLEE with an equivalent or higher resolution DNA-based system, particularly for L. infantum. We amplified genes by PCR and the products were sequenced directly. In most cases a single amino acid polymorphism was shown to be responsible for differences in allozyme mobility. Some indistinguishable phenotypes were generated by different genetic polymorphisms; furthermore, some silent polymorphisms provided increased power of discrimination between isolates. Heterozygotes were unexpectedly abundant at several loci, and they suggested the presence of genetic exchange. The PCR-based enzyme genotyping described here will enable rapid assignment of strains to zymodemes, with resolution within a single zymodeme. The method is applicable for population genetics and further characterization of the heterozygotes will allow assessment of the extent of genetic exchange.

MOLECULAR EPIDEMIOLOGY OF VISCERAL LEISHMANIASIS : APPLICATION OF INNOVATIVE PCR-BASED ASSAYS FOR DIRECT PARASITE TYPING IN HOST TISSUES

K. W. Quispe-Tintaya1, T. Laurent1, S. Decuypere1, J. P. Dedet2, S. Rijal3, M. Hide4, A. L. Bañuls4, I. Mauricio5, M. Miles5, J. Alvar6, L. Campino7, J.C. Dujardin1.
1Laboratory of Molecular Parasitology, Instituut voor Tropische Geneeskunde ‘Prins Leopold’, 155 Nationalestraat, B-2000 Antwerpen, Belgium (wquispe@itg.be). 2Laboratoire de Parasitologie. U. Montpellier, France. 3BP Koirala Institute for Health Sciences, Dharan, Nepal. 4UMR CNRS/IRD 2724, Montpellier, France. 5LSHTM, London, UK. 6ISCIII, Madrid, Spain. 7IHMT, Lisboa, Portugal.

Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow a direct identification of species and intra-species groups of the etiological agents (L. donovani complex) in host tissues. We describe two PCR-based assays using (i) restriction fragment length polymorphism analysis of amplicons (PCR-RFLP), and (ii) fluorogenic hybridisation probes (FRET/MCA). Both assays targeted the gene locus of cysteine proteinase b (cpb), an important Leishmania antigen, and they were applied to the characterization of 23 reference strains of the L. donovani complex. The two assays distinguished the parasites according to their geographical origin. However, discrepancy was observed with the current taxonomy based on isoenzyme analysis and concerned specifically East African strains. This supports previous reports and strengthens the need for a systematic re-evaluation of the taxonomy of the L.donovani complex. The two assays were also applied directly in clinical samples (blood and bone marrow from Nepalese and Spanish VL patients, and from Portuguese VL dogs) and allowed a sensitive and specific typing. Our assays constitute direct, rapid and high-throughput alternatives for molecular diagnosis and epidemiology of VL and could be adapted to other Leishmania species.

Clinical Diagnosis of Cutaneous Leishmaniasis : Evaluation of Standardized Graded Microscopy and ITS1-PCR of Giemsa-stained Slides.

A. Al-Jawabreh 1, 2, W. Presber1, O. Hamarsheh1 and G. Schoenian 1
1 Institute of Microbiology and Hygiene, Charité University Medicine, Berlin, Germany
2 Leishmania research Unit, ICS- Jericho. Palestine.
islahjr@yahoo.com

Parasitological diagnosis of cutaneous leishmaniasis is a strict obligation before treatment. Direct microscopy of scrapings taken from margins of skin lesions is the most routinely used method for clinical diagnosis of leishmaniasis. In this study we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR. Three 5mmx5mm squares were marked on each of the 20 Giemsa-stained touch smears which represented 20 patients. Out of the 60 squares scanned for amastigotes using x100 oil- immersion light microscopy, 45 (75%) gave usable results. Fifteen (25%) squares could not be microscopically scanned, 12 due to thick stain and 3 due to poor stain. DNA from each square was extracted separately after microscopy and, subsequently, amplified by ITS1-PCR. Of the 23 microscopy-positive squares 20 (87%) were positive by PCR. Of the three negative, one failed to extract for DNA, the second showed only one amastigote in the entire square, while the third was normally graded as +1, but not amplified for unknown reasons. Of the 22 squares negative for microscopy, 18 (82%) were ITS1-PCR positive. In addition, all three poorly- stained squares were ITS1-PCR positive. Of the 12 darkly- stained squares, 11 were positive. In addition, a negative control group of 15 individuals from which DNA was extracted from 3 squares of Giemsa-stained slides, were tested. ITS1-PCR showed a sensitivity of 87% with positive predictive value of 100% and a specificity of 100% with negative predictive value of 85%.

Molecular epidemiology and population genetic studies of Leishmania major : A multilocus microsatellite typing approach.

A. Al-Jawabreh, G. Schönian,
1 Institute of Microbiology and Hygiene, Charité University of Medicine, Berlin,Germany
2 Leishmania research Unit, ICS- Jericho. Palestine.
islahjr@yahoo.com

Leishmania major is a widely distributed parasite of desert and savannah rodents from the Old World with man being an incidental host. Isoenzyme typing showed the species L. major being epidemiologically quite uniform. Their hypervariability and ubiquitous occurrence made microsatellites, or simple sequence repeats (SSR), very useful markers for population genetic studies. Eleven pairs of PCR primers, annealing to the unique flanking regions, were designed to amplify microsatellite loci identified in the genome sequence of L. major on chromosomes 1, 3, 5, 21, 35. The microsatellite repeats are (CA)n, (AT)n, (GTG)n, and (GACA)n with varying length. A total of 105 strains isolated in different endemic regions of Central Asia, Middle East and Africa were analysed in this study. PCR products were separated and screened for length polymorphisms by polyacrylamide gel electrophoresis, fully-automated capillary electrophoresis and Metaphore agarose electrophoresis. Analysis of genetic distance revealed the existence of 6 discrete populations of L. major. This was confirmed by a Bayesian model-based clustering approach that assigned the individual strains to the same number of populations. All strains from Central Asia had highly similar genotypes suggesting recent emergence of this population. Considerable heterogeneity and further geographical subdivision was observed in strains isolated in the Middle East. The highest degree of variation was seen in African strains which would be in agreement with an African origin of the species. However, more strains of different regions in Africa have to be investigated to elucidate their population structures and the putative correlation to the reservoir hosts in different endemic foci. This study shows the apparent strength of microsatellites to discriminate between intra-species variations which, subsequently, aids in forming a global epidemiological picture of L. major.

CLINICAL DIAGNOSIS OF CUTANEOUS LEISHMANIASIS IN JERICHO : FILTER PAPERS AND UNSTAINED SMEARS AS POTENTIAL SAMPLING METHODS FOR ITS1-PCR

A. Al-Jawabreh1&2, W. Presber1 O. Hamarsheh1, and G. Schönian1
1 Institute of Microbiology and Hygiene, Charité University Medicine, Berlin, Germany
2 Leishmania Research Unit-ICS- Jericho, Palestine.
Islahjr@yahoo.com

The clinical diagnosis and strain genotyping of cutaneous leishmaniasis in endemic areas of mixed species is becoming a more vital and inevitable step before treatment. The use of skin scrapings spotted on filter papers and unstained tissue smears as direct clinical sampling methods for the diagnosis and typing of CL in Jericho was evaluated using a PCR assay amplifying region 1 of the internal transcribed spacer (ITS1) of the RNA operon. ITS1-PCR-RFLP using HaeIII and MnlI was used for the typing of isolates. In-vitro cultivation using NNN medium and direct smear microscopy of Giemsa-stained slides was compared. ITS1-PCR detected leishmanial DNA in 219 (52.4%) of the 418 filter papers, whereas DNA was detected in 96 (55.5%) of 173 unstained tissue smears. The rate of positivity is 53.3% when using both filter papers and unstained smear as potential sampling methods for ITS1-PCR. RFLP using unstained- smears, filter-papers and culture showed 178 (68%) L. major and 105 (36%) L. tropica and 12 (4%) were undetermined. The rate of positivity of ITS1-PCR was significantly higher than that obtained with cultivation (45%) and microscopy (42%) which rules out their use as ‘gold standard’ to evaluate new methods. The two sampling methods, filter papers and unstained smears, have comparable results and provide a secure tool in an optimized and well-controlled molecular-based assay for the direct clinical diagnosis and strain genotyping of cutaneous leishmaniasis in terms of long shelf-life, relative non-invasiveness compared to culture and histopathology, culture-independent, simplicity and time-saving.

MULTILOCUS MICROSATELLITE TYPING EMPHASIZES THE NEED FOR A REVISED TAXONOMY OF THE LEISHMANIA DONOVANI COMPLEX

K. Kuhls, S. Hertweck, M. Schaar, L. Keilonat, O. Radtke, G. Schönian

Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin Berlin, Dorotheenstr. 96, D-10117 Berlin
Katrin.Kuhls@charite.de

Conflicting results between the current isoenzyme based taxonomy of the Leishmania donovani complex and recent genotype studies including different DNA markers require population and evolutionary investigations of this parasite. For this purpose we have studied polymorhisms within 26 microsatellites for 63 strains of L. donovani, L. archibaldi and L. infantum. Seven populations generally corresponding with geographical origin could be identified using a Bayesian approach. This was confirmed by analysis of genetic distances. Leishmania infantum from the Mediterranean area and China (as well as its synonym L. chagasi from the New World) formed one of the two main clades of the phylogenetic tree. The second main clade included all L. donovani strains, as well as L. archibaldi and L. infantum from East Africa. Microsatellite analysis showed that L. donovani (MON-18), L. archibaldi (MON-82, 257, 258) and L. infantum (MON-30, 81, 267) from Sudan/Ethiopia are con-specific, placing all three species in one monophyletic clade. Consequently there is just a single species in that region, consisting of two discrete lineages (populations), which both include L. donovani, L. infantum and L. archibaldi strains. The L. donovani strains from India and Kenya are distinct from those from Sudan/Ethiopia, and form also two main populations. The Kenyan strains are most closely related to one of the two Indian populations. The relationship between the Indian and Kenyan strains seems to be closer as between the strains from Sudan and Kenya. Population structure analysis suggests an ancestral Sudanese population and the first separation of the Indian and Kenyan strains from this Sudanese population. We propose either to abandon the name „L. archibaldi“ and to apply L. donovani for all East African strains or to differentiate between the Indian/Kenyan strains as L. donovani and the Sudanese/Ethiopian strains as L. archibaldi as the supposed ancestral population.

DIFFERENTIATION OF LEISHMANIA INFANTUM MON-1 STRAINS BY MULTILOCUS MICROSATELLITE TYPING - APPLICATION FOR EPIDEMIOLOGICAL AND POPULATION STUDIES

K. Kuhls1, S. Hertweck1, M. Schaar1, O. Radtke1, C. Chicharro2, S. Cortes3, H. Haralambous4, G. Schönian

1Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin Berlin, Germany; 2Instituto de Salud Carlos III, Madrid, Spain; 3Instituto de Higiene e Medicina Tropica, Lisbon, Portugal; 4Hellenic Pasteur Institute, Athens, Greece

Katrin.Kuhls@charite.de

Leishmania infantum is a causative agent of visceral and cutaneous leishmaniasis. This species is endemic in the Mediterranean region and South America. An important problem in these countries is the high rate of HIV/L. infantum co-infections. Differentiation of L. infantum strains is based on MLEE; however, the discriminatory power of this method is restricted. MON-1 is the predominant and ubiquitous zymodeme in the endemic regions and it has the highest co-infection rate with HIV. In order to answer many epidemiological questions it is essential to discriminate strains of MON-1. We have developed a set of 15 microsatellite markers which can be used for differentiation of L. infantum strains. In this study we included 147 strains of L. infantum mainly from Spain, Portugal, Greece and some from France and 5 L. chagasi strains from the New World. 114 of these strains were MON-1. The microsatellite markers have the potential to discriminate MON-1 strains from L. infantum strains of other zymodemes. Moreover, nine of them are enough polymorphic to differentiate within MON-1 strains. Relationships according to their geographic origin could be detected, e.g. MON-1 strains from Greece could be separated from strains from Spain and Portugal. Among the Iberian MON-1 strains we could detect specific clades for Portuguese and for Spanish strains, as well as mixed clades. Within the Spanish strains that were collected in Madrid, Ibiza and Mallorca a clear clade consisting of all strains from Ibiza could be detected. There was no correlation between leishmanial genotypes and host specificity, clinical manifestation (VL or CL) and occurrence of HIV/leishmania co-infection. The potential of these markers for distinguishing between relapses and re-infections will be discussed.

MULTILOCUS MICROSATELLITE TYPING REVEALS GENETIC HOMOGENEITY OF STRAINS OF LEISHMANIA DONOVANI ISOLATED IN BIHAR STATE, INDIA

K. Kuhls1, C. Schweynoch1, S. Sundar2 and G. Schönian1
1Institute of Microbiology and Hygiene, Charité University Medicine, Berlin, Germany
2Banaras Hindu University, Varanasi, India
gabriele.schoenian@charite.de

In India, more than 100,000 cases of visceral leishmaniasis occur every year and the state of Bihar accounts for more than 90% of these cases. Over 60% of the cases in highly endemic areas of Bihar are refractory to traditional antimony therapy. A panel of 25 microsatellite markers that were proven to be informative for L. donovani were used to type 24 strains isolated from VL patients in Bihar between 2002 and 2003. Five older Indian strains including the WHO reference strain DD8 (MON-2) were included for comparison. Multilocus microsatellite typing (MLMT) identified two groups of Indian L. donovani strains. All strains from Bihar including the DD8 strain isolated in 1980 and two strains isolated in 1996 and 2000, respectively, formed a very homogenous clade. This clade was, however, genetically distinct from a second group that was formed by two other strains from India, including one MON-38 strain, and strains isolated in Kenya. The majority of the strains from Bihar (18) showed completely identical microsatellite patterns. The remaining strains presented different alleles with only 2 markers. One may speculate that refractoriness to antimonial drugs in Bihar is correlated with a particular parasite genotype. To test the hypothesis that the spread of therapy resistance is due to the expansion of a particular clone, more strains from Bihar but also from other endemic area in the Indian subcontinent have to be analysed.

MULTILOCUS MICROSATELLITE TYPING OF LEISHMANIA TROPICA SUGGESTS RECENT CLONAL EXPANSION IN THE MIDDLE EAST

J.M. Schwenkenbecher1, L.F. Schnur2 and G. Schönian1
1Institute of Microbiology and Hygiene, Charité University Medicine, Berlin, Germany
2 Dept of Parasitology, Hebrew University - Hadassah Medical School, Jerusalem, Israel gabriele.schoenian@charite.de

The resurgence, dispersal and clinical diversity of Leishmania tropica justify its epidemiological and evolutionary investigation. Isoenzyme analysis and different molecular biological methods have revealed extensive genetic variation within this species with no correlation to either geographical origin or pathogenesis. Multilocus microsatellite typing (MLMT) was based on variations in 21 microsatellite loci of 117 strains collected in different African and Asian locations. MLMT revealed a clear genetic structure. Ten genetic clusters were identified using a Bayesian approach and by analysing genetic distances that correlated predominantly with geographical origin. The ‘Asian’ population of strains was very heterogeneous with interrelated genotypes spread over a large territory. The other populations formed homogeneous clusters with distinct genetic differentiation. Analysis of linkage disequilibrium favoured a predominantly clonal propagation for all populations. In the Middle East, strains belonged to different populations. Strains isolated before 1990 and a few recently isolated strains grouped with the ‘Asian’ strains. Two more populations that were genetically separate from each other and the ‘Asian’ clade were comprised of strains isolated in that area recently. It appears that new genotypes have spread rapidly into an area with an ‘established’ population. Overlapping, genetically distinct populations of L. tropica were also identified in Morocco that are probably related to anthroponotic and zoonotic transmission cycles. MLMT revealed a clear population structure within the species L. tropica, suggesting links between epidemiological systems and the genetic composition of this species.

EVIDENCE OF THE PRESENCE OF CIS-REGULATING ELEMENTS IN THE 3’ UTR’S OF HISTONE GENES AND OF PROTEIN(S) INTERACTING WITH THIS ELEMENT

C. Haralambous, D. Smirlis, K. Soteriadou
Laboratory of Molecular Parasitology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521, Athens, Greece. christos@pasteur.gr

The mechanisms regulating cell-cycle dependent gene expression represent an unexplored aspect of gene regulation in trypanosomatids. In the protozoan Crithidia fasciculata an octamer consensus sequence (A/CATAGAAG/A) is present in the 5’ UTR of several DNA replication genes (TOP2, RPA1, DHFR-TS, Histone-like kinetoplast associated protein 3). This element was shown to be necessary for the cycling of these genes and was therefore termed cycling element. Two protein complexes (CSBP and CSBPII) that bind specifically to the motif were also described. Homologues of the proteins forming the two CSBP complexes also exist in Leishmania and Trypanosoma. These proteins show high sequence similarity to the C.fasciculata proteins. We report here the presence of a similar cycling element in all the histone genes of Leishmania and Trypanosoma. Gel retardation assays using RNA probes possessing the cycling element sequence revealed the presence of Leishmania proteins specifically interacting with the cis-regulating element. The regulation of histone synthesis in Leishmania occurs exclusively at the translational level. Therefore, it could be speculated that the cycling element could be involved in histone mRNA translational accessibility in the different phases of the cell cycle. Further experiments are required for the understanding of the cycling element’s interaction with the protein complexes and the mechanism that regulates histone translation.

MUTATIONAL ANALYSIS OF T. BRUCEI AND L. MAJOR HYPOXANTHINE TRANSPORTERS

I. Papageorgiou1.2, K. P. Soteriadou2, H. P. de Koning3 and G. Diallinas1
1Department of Botany, Faculty of Biology,University of Athens, Athens, Greece, 2Laboratory of
Molecular Parasitology, Hellenic Pasteur Institute, Athens, Greece ioannis@pasteur.gr, 3Institute
of Biomedical and Life Sciences, Division of Infection and Immunity,University of Glasgow, Glasgow

In the last few years major progress has been made in the identification of the genes encoding nucleobase and nucleoside transporters of parasitic protozoa. Purine transporters of protozoan parasites have attracted much attention because of their role in the salvage of essential nutrients and drug uptake. The high affinity nucleobase transporter (TbNBT1), from T. brucei shows homology with a gene from L. major, identified using BLAST analysis from the Leishmania Genome database. The cloning and biochemical characterization of the respective gene in Leishmania, which is in progress, will help us understand the transport of hypoxanthine in the parasite. Despite the importance of nucleobase and nucleoside transporters in cellular nutrition, little is known about the aminoacid residues involved in binding of their substrates. In the present study we used random mutagenesis with hydroxylamine and site-directed mutagenesis of the TbNBT1 to further elucidate the role of specific aminoacid residues in the transport process. By expression of TbNBT1 in a S. cerevisiae strain lacking an endogenous purine transporter, we checked for mutants conferring resistance to 8-azaguanine. The biochemical characterization of the mutants showed that the generated mutations resulted in the loss of function of the gene. Since the mutated residues are conserved in the two protozoan parasites the respective mutations will be generated also in the putative Leishmania transporter. This will reveal the transmembrane segments that are responsible for the formation of the substrate permeation pathway. Since the purine metabolic pathways of Leishmania have been studied in detail and most of the key enzymes have been characterized, the mutational analysis of these transporters will allow the design of specific inhibitors of pharmacological interest.

LEISHMANIA HISTONE H1 REGULATES PARASITE INFECTIVITY AND CELL-CYCLE PROGRESSION

D.Smirlis, E..Xyngi, S. Bisti, M. Thiakaki, K. Soteriadou
Laboratory of Molecular Parasitology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521,
Athens, Greece. penny@pasteur.gr

Leishmania histone H1 (LH1) is a low molecular weight, lysine-rich protein which is expressed in greater abundance in stationary phase parasites and regulates Leishmania infectivity in vitro. The role of the LH1 was subsequently studied in vivo using transgenic promastigotes that over- and under- express LH1. ?f great interest is the observation that the modulated levels of LH1 in L. major transgenic parasites affect also parasite infectivity in vivo. No lesions were observed in mice inoculated with L. major parasites that over-express LH1. Unexpected was the finding that transgenic parasites expressing lower amounts of LH1 were also found to be less infective in Balb/c mice. These results strongly suggest that the level of expression of this protein affects parasite infectivity. The differences in infectivity maybe correlated with differences in their cell-cycle progression status. By FACS analysis we have shown that synchronized with hydroxyurea parasites over-expressing LH1 had a delayed cell-cycle progression. The mammalian histone H10 homolog delays cell-cycle progression in mammalian cells. Since cell-cycle control elements in mammalian cells are better studied than in parasitic protozoa we have investigated whether LH1 may also affect cell-cycle progression of mammalian cells. For this purpose we have expressed LH1 in mammalian cells. LH1 was also found to delay cell-cycle progression both in COS7 and NIH 3T3 cells. The latter strengthens the phenotype observed in Leishmania and provide evidence that critical functions of histone H1 molecules are conserved throughout evolution. The reduction of parasite infectivity maybe attributed to the cell-cycle differences observed in transgenic parasites with modulated levels of LH1.
This work was in part supported by EU grant QLK-CT-2001-01810

DETECTION OF THREE LEISHMANIA INFANTUM - L. MAJOR HYBRID STRAINS FROM THREE HIV INFECTED PATIENTS IN PORTUGAL

1C. Ravel, 1F. Pratlong, 2L. Campino, 2S. Cortes, 1P. Lami, 1G. Serres, 1J.P. Dedet,
(1Laboratoire de Parasitologie, Centre National de Référence des Leishmania and WHO Collaborating Center on Leishmaniasis, Centre Hospitalier Universitaire, Montpellier, France and 2Instituto de Higiene e Medicina Tropical, Unidade de Leishmanioses/ CMDT, Universidade Nova de Lisboa, Portugal)

Isoenzymatic and molecular analysis of three Leishmania strains from Portuguese HIV patients showed complex features, with hybrid patterns in all the enzymatic systems, some of them being multibands. Typic hybrid patterns are also demonstrated by the sequence of RNA pol II large sub-unit gene. Both techniques are compatible with genetic hybrids between L. infantum and L. major.
These hybrids seems identical by the detection means used.
These three strains corresponded to cutaneous, visceral and viscero-cutaneous leishmaniasis cases, occurring in three endovenous drug addicts, who never travelled outside Portugal in 2 cases, or was resident in Portugal for over two years.
This observation highlights the possibility of genetic exchanges between two distinct species, one mainly viscerotropic (L. infantum), and the other strictly dermotropic (L. major). The occurrence and stability of such hybrid strains with so divergent Leishmania species is an exceptional finding.

Work supported by funding from the EC research project no. QLK2-CT-2001-01810, EU.

COMPARISON OF ISOENZYMATIC AND MOLECULAR IDENTIFICATION OF OLD AND NEW WORLD ISOLATES OF LEISHMANIA.

C. Ravel, F. Pratlong, J. Puechberty, Y. Balard, P. Lami, G. Serres and J.P. Dedet
(Laboratoire de Parasitologie, Centre National de Référence des Leishmania and WHO Collaborating Center on Leishmaniasis, Centre Hospitalier Universitaire, Montpellier, France)

In the past two years, molecular typing was carried out on 242 human Leishmania isolates from the Old and New Worlds, collected for diagnostic purposes at the Centre National de Référence des Leishmania of Montpellier (France). Among them, 26 were contaminated by bacteria or fungi and 34 were culture-negative. The 182 remaining strains were typed by both molecular and isoenzymatic techniques to evaluate the correlation between the two approaches.
The molecular typing was based on the systematic sequencing of a part of the RNA pol II large sub-unit gene. The isoenzymatic approach was based on 15 enzymatic systems.
Among the 242 isolates analysed, 218 were from cutaneous lesions and 24 from visceral leishmaniasis. 159 (66%) and 83 (34%) were from the Old World and the New World, respectively. 16 different Leishmania taxa were identified: L. aethiopica, L. archibaldi, L. donovani, L. infantum, L. killicki, L. major and L. tropica from the Old World; and, L. braziliensis, L. guyanensis, L. lainsoni, L. mexicana, L. naiffi, L. panamensis, L. peruviana, L. shawi and L. sp. from Martinique Island from the New World.
For the 182 strains analysed by both molecular and isoenzymatic techniques the correlation was 100%. The molecular approach was validated by this large scale comparison with the isoenzymatic technique, which is currently the gold standard for identification. Advantages of the molecular approach include a short response time and its ability to be carried out on primary samples, as well as on negative or contaminated cultures.
Acknowledgments : This work received financial support from the French Ministry of Health, from CNRS, and from EC research project no. QLK2-CT-2001-01810.