Najoua Haouas

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

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.

First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae) in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous leishmaniasis in Tunisia

BackgroundLeishmania killicki was originally described in 1980 in southeast Tunisia. It was also recently reported in Lybia and Algeria. Nevertheless, neither vector nor reservoirs of this parasite are known. The identification of the vector and the animal reservoir host of L. killicki is critical for the establishment of an efficient control strategy.Findingsblood, popliteal lymph node, spleen, bone marrow, liver and skin were collected from 50 rodents in 2009 in south western Tunisia. Samples were smeared onto glass slides, cultured on NNN medium and tested by polymerase chain reaction for Leishmania detection. Parasites were detected by PCR from 10 Psammomys obesus and from two Ctenodactylus gundi. Parasite identification was performed simultaneously by internal transcribed spacer 1 PCR-RFLP and by PCR sequencing. Both Leishmania major and Leishmania killicki were identified from infected Psammomys and Ctenodactylus gundi respectively.ConclusionThis is the first report of Leishmania killicki identified from Ctenodactylus gundi in Tunisia. This result supports the assumption that C. gundi is a potential reservoir for Leishmania killicki.

Leishmaniases in Maghreb: An endemic neglected disease

Maghreb is known to be one of the most endemic areas of leishmaniases where both visceral and cutaneous forms are reported. Cutaneous leishmaniasis (CL) is older and has a higher prevalence than visceral one (VL). It is caused by four taxa (Leishmania (L.) major, L. infantum, L. tropica and L. killicki) which are responsible for a large clinical spectrum of lesions. Most transmission cycles of these taxa are known and many phlebotomine sandflies vectors and reservoir hosts are identified. The zoonotic transmission is well established for L. major. However, for L. infantum and L. killicki it needs more investigations to be proven. Regarding L. tropica, studies suggest it to be of both zoonotic and anthroponotic types. The isoenzymatic characterization of these four taxa showed a large enzymatic polymorphism varying from two zymodemes for L. major to 10 zymodemes for L. tropica. Cutaneous leishmaniasis is widely distributed and covers all bioclimatic stages with the coexistence of more than one taxon in the same foci. Visceral leishmaniasis is the second form of leishmaniases in Maghreb. Only L. infantum is known to cause this disease. The transmission cycle of this parasite is zoonotic but still not well known. The isoenzymatic identification of L. infantum causing VL showed the presence of six zymodemes. Geographically, VL is distributed in all bioclimatic stages of Maghreb countries. Despite all the previous studies realized on leishmaniases in Maghreb, they are still considered as neglected diseases because of the rarity or the absence of efficient control strategies.

Transmission of Visceral Leishmaniasis in a Previously Non-Endemic Region of Tunisia: Detection of Leishmania Dna in Phlebotomus perniciosus

ABSTRACT: Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non-endemic region. Sixty-three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.

Twenty-four new human cases of cutaneous leishmaniasis due to Leishmania killicki in Metlaoui, southwestern Tunisia. Probable role of Phlebotomus sergenti in the transmission

Metlaoui district in the South-west of Tunisia is a classical focus of cutaneous leishmaniasis (CL) due to Leishmania major. Since 2005, a single case of CL due to L. killicki has been reported. We report twenty four human cases due to this parasite, affecting men and women from 2 to 70 years old. Leishmania killicki have been typed using molecular techniques: polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing. Four strains from patients have been successfully cultured on NNN medium and isoenzymatically typed as L. killicki MON-8. Our results strongly suggests that Metlaoui is a new L. killicki focus with a stable transmission cycle. Sand flies fauna in the same focus was also studied. 1400 Phlebotomine sand flies (785 males/615 females) have been caught during an entomological survey. Leishmania major DNA has been found in one P. papatasi female, the most abundant species, whereas L. killicki DNA has been found in one Phlebotomus sergenti female emphasizing the probable role of this species as vector of this zoonotic parasite.

Development of a polymerase chain reaction-restriction fragment length polymorphism assay for Leishmania major/Leishmania killicki/Leishmania infantum discrimination from clinical samples, application in a Tunisian focus.

Topoisomerase II gene of Leishmania genus was used to develop a molecular tool for detection and species differentiation of Leishmania from clinical samples. Identification was achieved by a polymerase chain reaction followed by digestion with 2 restriction endonucleases BstU1 and Taq1. Despite the relatively low sensitivity, it is able to differentiate between 3 complexes responsible for cutaneous leishmaniasis.

Blood meal analysis of culicoides (Diptera: ceratopogonidae) in central Tunisia.

To evaluate the host preferences of Culicoides species (Diptera: Ceratopogonidae) in Central Tunisia, we identified the source of blood meals of field collected specimens by sequencing of the cytochrome b (cyt b) mitochondrial locus and Prepronociceptine single copy nuclear gene. The study includes the most common and abundant livestock associated species of biting midges in Tunisia: C. imicola, C. jumineri, C. newsteadi, C. paolae, C. cataneii, C. circumscriptus, C. kingi, C. pseudojumineri, C. submaritimus, C. langeroni, C. jumineri var and some unidentified C. species. Analysis of cyt b PCR products from 182 field collected blood-engorged females’ midges revealed that 92% of them fed solely on mammalian species, 1.6% on birds, 2.4% on insects and 0.8% on reptiles. The blast results identified the blood origin of biting midges to the species level with exact or nearly exact matches (≥98%). The results confirm the presence of several Culicoides species, including proven vectors in Central Tunisia. Blood meal analyses show that these species will indeed feed on bigger mammals, thereby highlighting the risk that these viruses will be able to spread in Tunisia.

First detection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Culicoides spp. (Diptera: Ceratopogonidae).

BackgroundCulicoides (Diptera: Ceratopogonidae) species are known to be the vectors of Bluetongue virus and African Horses Sickness virus (AHSV) in different areas of the world. Nevertheless, other researchers have hypothesized that these arthropods could be involved in the transmission of other pathogens such as Schmallenberg virus, Plasmodium and Leishmania parasites. Identification of the Culicoides’ potential vector competence is crucial in understanding the worldwide Culicoides/Leishmania life cycle.FindingsBlood fed and parous females of biting midges Culicoides spp. were collected between 2009 and 2010 in Central Tunisia. DNA was extracted from individual blood fed Culicoides and used as a template in a genus-specific PCR. Leishmania DNA was detected in 14 Culicoides imicola specimens and one Culicoides circumscriptus. In a second step, parasite identification was performed based on a single copy Topo-isomerase II gene specific amplification and sequencing. Leishmania infantum was identified in two infected Culicoides spp.ConclusionThis is the first report of Leishmania DNA detection from naturally infected wild caught Culicoides spp. Our finding supports the assumption that Culicoides spp. are a potential vector for L. infantum.

Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications

BackgroundThe taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica.MethodsThirty-five L. killicki and 25 L. tropica strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches.ResultsThe results of the genetic and phylogenetic analyses strongly support the hypothesis that L. killicki belongs to the L. tropica complex. Our data suggest that L. killicki emerged from a single founder event and that it evolved independently from L. tropica. However, they do not validate the hypothesis that L. killicki is a distinct complex. Therefore, we suggest naming this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination.ConclusionsThis study provides taxonomic and phylogenetic information on L. killicki and improves our knowledge on the evolutionary history of this taxon.

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.