Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal.

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples. The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals

Environmental risk mapping of canine leishmaniasis in France

<p>Abstract</p> <p>Background</p> <p>Canine leishmaniasis (CanL) is a zoonotic disease caused by <it>Leishmania infantum</it>, a Trypanosomatid protozoan transmitted by phlebotomine sandflies. Leishmaniasis is endemic in southern France, but the influences of environmental and climatic factors on its maintenance and emergence remain poorly understood. From a retrospective database, including all the studies reporting prevalence or incidence of CanL in France between 1965 and 2007, we performed a spatial analysis in order to i) map the reported cases in France, and ii) produce an environment-based map of the areas at risk for CanL. We performed a Principal Component Analysis (PCA) followed by a Hierarchical Ascendant Classification (HAC) to assess if the locations of CanL could be grouped according to environmental variables related to climate, forest cover, and human and dog densities. For each group, the potential distribution of CanL in France was mapped using a species niche modelling approach (Maxent model).</p> <p>Results</p> <p>Results revealed the existence of two spatial groups of CanL cases. The first group is located in the Cévennes region (southern Massif Central), at altitudes of 200-1000 m above sea level, characterized by relatively low winter temperatures (1.9°C average), 1042 mm average annual rainfall and much forest cover. The second group is located on the Mediterranean coastal plain, characterized by higher temperatures, lower rainfall and less forest cover. These two groups may correspond to the environments favoured by the two sandfly vectors in France, <it>Phlebotomus ariasi </it>and <it>Phlebotomus perniciosus </it>respectively. Our niche modelling of these two eco-epidemiological patterns was based on environmental variables and led to the first risk map for CanL in France.</p> <p>Conclusion</p> <p>Results show how an ecological approach can help to improve our understanding of the spatial distribution of CanL in France.</p

Geographical distribution and epidemiological features of Old World Leishmania infantum and Leishmania donovani foci, based on the isoenzyme analysis of 2277 strains

A series of 2277 Leishmania strains from Old World visceral leishmaniasis foci, isolated between 1973 and 2008, were studied by isoenzyme analysis. The strains were obtained from humans, domestic and wild carnivores, rodents and phlebotomine sandflies, and came from 36 countries. In all, 60 different zymodemes were identified and clustered by a phenetic analysis into 3 different groups corresponding to the typically visceralizing species L. donovani (20 zymodemes, 169 strains), L. archibaldi (3 zymodemes, 46 strains) and L. infantum (37 zymodemes, 2,062 strains). The taxonomic position of these isoenzymatic groups is discussed in view of contradictory results obtained from recent molecular studie

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

Background: Leishmania (L.) killicki is responsible for the chronic cutaneous leishmaniasis. The taxonomic status of this parasite is still not well defined. It was suggested on one hand to include this taxon within L. tropica complex but also on the other hand to consider it as a distinct phylogenetic complex. The present work represents the more detailed study on the evolutionary history of L. killicki relative to L. tropica and the taxonomic implications. Methods: Thirty five L. killicki and 25 L. tropica strains isolated from humans and from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches. Results: The genetic and phylogenetic analyses strongly support that L. killicki belongs to L. tropica complex. The study suggests the emergence of L. killicki by a funder effect followed by an independent evolution from L. tropica, but does not validate the species status of this taxon. In this context, we suggest to call this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination. Conclusions: These findings provided taxonomic and phylogenetic informations on L. killicki and helped to better know the evolutionary history of this taxon

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy.

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (20%). Both the four-country and France-only models performed reasonably well for predicting correctly the 20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping

Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1

Visceral leishmaniasis is caused by protozoan parasites of the genus Leishmania. This disease is a public health problem in countries bordering the Mediterranean, in China, and South America. Until now, isoenzyme analysis, a method with several advantages but also some limitations, is the gold standard for typing the causative agent L. infantum. We have developed a new method based on hypervariable DNA markers, the microsatellites. Its higher discriminatory power, genotype-based analysis, the possibility to use biological material instead of parasite cultures, and the fast analysis are the major improvements. We could demonstrate for the first time that there exist different geographically determined populations within the predominant zymodeme of L. infantum, which has important epidemiological implications. We also tested for relationships between genotype and clinical picture and/or host background. Leishmania is considered to reproduce mainly clonally; however, we found some indication for recombination in our study. Our work constitutes a solid basis for further population and epidemiological studies of L. infantum by completing the existing microsatellite database by analysing strains from other endemic foci