Planning for wolf-livestock coexistence: landscape context predicts livestock depredation risk in agricultural landscapes.

Extensive pastoral livestock systems in Central Europe provide multiple ecosystem services and support biodiversity in agricultural landscapes but their viability is challenged by livestock depredation (LD) associated with the recovery of wolf populations. Variation in the spatial distribution of LD depends on a suite of factors, most of which are unavailable at the appropriate scales. To assess if LD patterns can be predicted sufficiently with land use data alone at the scale of one federal state in Germany, we employed a machine-learning-supported resource selection approach. The model used LD monitoring data, and publicly available land use data to describe the landscape configuration at LD and control sites (resolution 4 km * 4 km). We used SHapley Additive exPlanations to assess the importance and effects of landscape configuration and cross-validation to evaluate the model performance. Our model predicted the spatial distribution of LD events with a mean accuracy of 74%. The most influential land use features included grassland, farmland and forest. The risk of livestock depredation was high if these three landscape features co-occurred with a specific proportion. A high share of grassland, combined with a moderate proportion of forest and farmland, increased LD risk. We then used the model to predict the LD risk in five regions; the resulting risk maps showed high congruence with observed LD events. While of correlative nature and lacking specific information on wolf and livestock distribution and husbandry practices, our pragmatic modelling approach can guide spatial prioritisation of damage prevention or mitigation practices to improve livestock-wolf coexistence in agricultural landscapes.

Microsatellite based molecular epidemiology of Leishmania infantum from re-emerging foci of visceral leishmaniasis in Armenia and pilot risk assessment by ecological niche modeling.

BACKGROUND: Visceral leishmaniasis (VL) is re-emerging in Armenia since 1999 with 167 cases recorded until 2019. The objectives of this study were (i) to determine for the first time the genetic diversity and population structure of the causative agent of VL in Armenia; (ii) to compare these genotypes with those from most endemic regions worldwide; (iii) to monitor the diversity of vectors in Armenia; (iv) to predict the distribution of the vectors and VL in time and space by ecological niche modeling. METHODOLOGY/PRINCIPAL FINDINGS: Human samples from different parts of Armenia previously identified by ITS-1-RFLP as L. infantum were studied by Multilocus Microsatellite Typing (MLMT). These data were combined with previously typed L. infantum strains from the main global endemic regions for population structure analysis. Within the 23 Armenian L. infantum strains 22 different genotypes were identified. The combined analysis revealed that all strains belong to the worldwide predominating MON1-population, however most closely related to a subpopulation from Southeastern Europe, Maghreb, Middle East and Central Asia. The three observed Armenian clusters grouped within this subpopulation with strains from Greece/Turkey, and from Central Asia, respectively. Ecological niche modeling based on VL cases and collected proven vectors (P. balcanicus, P. kandelakii) identified Yerevan and districts Lori, Tavush, Syunik, Armavir, Ararat bordering Georgia, Turkey, Iran and Azerbaijan as most suitable for the vectors and with the highest risk for VL transmission. Due to climate change the suitable habitat for VL transmission will expand in future all over Armenia. CONCLUSIONS: Genetic diversity and population structure of the causative agent of VL in Armenia were addressed for the first time. Further genotyping studies should be performed with samples from infected humans, animals and sand flies from all active foci including the neighboring countries to understand transmission cycles, re-emergence, spread, and epidemiology of VL in Armenia and the entire Transcaucasus enabling epidemiological monitoring.

History of the E.I. Martsinovsky Institute of Medical Parasitology and Tropical Medicine: research on malaria and leishmaniasis.

This review presents the 100-year history of the Martsinovsky Institute of Medical Parasitology and Tropical Medicine in Moscow, Russia, starting with its foundation and early activities, and also describes the impact of its leading scientists, some of whom became internationally known. The institute headed a network of nine tropical institutes in the various Soviet republics from the 1920s to 1990. The extensive body of literature on the history and research accomplishments of this institute has mainly been published in Russian; our goal here is to introduce these achievements and this expertise to the international scientific and medical community, focusing on malaria and leishmaniasis and the development of measures to control and monitor these diseases in the USSR.

Integrative Approach to Phlebotomus mascittii Grassi, 1908: First Record in Vienna with New Morphological and Molecular Insights.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arthropod-borne (arbo) viruses. While in Mediterranean parts of Europe the sand fly fauna is diverse, in Central European countries including Austria mainly Phlebotomus mascittii is found, an assumed but unproven vector of Leishmania infantum. To update the currently understudied sand fly distribution in Austria, a sand fly survey was performed and other entomological catches were screened for sand flies. Seven new trapping locations of Ph. mascittii are reported including the first record in Vienna, representing also one of the first findings of this species in a city. Morphological identification, supported by fluorescence microscopy, was confirmed by two molecular approaches, including sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) protein profiling. Sand fly occurrence and activity were evaluated based on surveyed locations, habitat requirements and climatic parameters. Moreover, a first comparison of European Ph. mascittii populations was made by two marker genes, cytochrome c oxidase subunit 1 (COI), and cytochrome b (cytb), as well as MALDI-TOF mass spectra. Our study provides new important records of Ph. mascittii in Austria and valuable data for prospective entomological surveys. MALDI-TOF MS protein profiling was shown to be a reliable tool for differentiation between sand fly species. Rising temperatures and globalization demand for regular entomological surveys to monitor changes in species distribution and composition. This is also important with respect to the possible vector competence of Ph. mascittii.

History of the E.I. Martsinovsky Institute of Medical Parasitology and Tropical Medicine: research on malaria and leishmaniasis / História do Instituto E.I. Martsinovsky de Parasitologia Médica e Medicina Tropical: pesquisa sobre malária e leishmaniose

Abstract This review presents the 100-year history of the Martsinovsky Institute of Medical Parasitology and Tropical Medicine in Moscow, Russia, starting with its foundation and early activities, and also describes the impact of its leading scientists, some of whom became internationally known. The institute headed a network of nine tropical institutes in the various Soviet republics from the 1920s to 1990. The extensive body of literature on the history and research accomplishments of this institute has mainly been published in Russian; our goal here is to introduce these achievements and this expertise to the international scientific and medical community, focusing on malaria and leishmaniasis and the development of measures to control and monitor these diseases in the USSR.

Resumo O artigo analisa a história centenária do Instituto Martsinovsky de Parasitologia Médica e Medicina Tropical em Moscou, Rússia, desde sua fundação e primeiras atividades, e descreve a influência de seus principais cientistas, alguns dos quais viriam a conquistar renome internacional. O instituto liderou uma rede de nove institutos tropicais em diversas repúblicas soviéticas entre as décadas de 1920 e 1990. A vasta literatura sobre o trabalho de história e pesquisa desse instituto foi publicada sobretudo em russo; nosso objetivo aqui é apresentar esse trabalho e conhecimento à comunidade médica e científica internacional, concentrando-se na malária e na leishmaniose e no avanço de medidas de controle e monitoramento dessas doenças na URSS.

History of the E.I. Martsinovsky Institute of Medical Parasitology and Tropical Medicine: research on malaria and leishmaniasis

This review presents the 100-year history of the Martsinovsky Institute of Medical Parasitology and Tropical Medicine in Moscow, Russia, starting with its foundation and early activities, and also describes the impact of its leading scientists, some of whom became internationally known. The institute headed a network of nine tropical institutes in the various Soviet republics from the 1920s to 1990. The extensive body of literature on the history and research accomplishments of this institute has mainly been published in Russian; our goal here is to introduce these achievements and this expertise to the international scientific and medical community, focusing on malaria and leishmaniasis and the development of measures to control and monitor these diseases in the USSR

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent.

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.

Phylogenetic Studies.

Phylogenetics is an important component of the systems biology approach. Knowledge about evolution of the genus Leishmania is essential to understand various aspects of basic biology of these parasites, such as parasite-host or parasite-vector relationships, biogeography, or epidemiology. Here, we present a comprehensive guideline for performing phylogenetic studies based on DNA sequence data, but with principles that can be adapted to protein sequences or other molecular markers. It is presented as a compilation of the most commonly used genetic targets for phylogenetic studies of Leishmania, including their respective primers for amplification and references, as well as details of PCR assays. Guidelines are, then, presented to choose the best targets in relation to the types of samples under study. Finally, and importantly, instructions are given to obtain optimal sequences, alignments, and datasets for the subsequent data analysis and phylogenetic inference. Different bioinformatics methods and software for phylogenetic inference are presented and explained. This chapter aims to provide a compilation of methods and generic guidelines to conduct phylogenetics of Leishmania for nonspecialists.

Re-Emerging foci of visceral leishmaniasis in Armenia - first molecular diagnosis of clinical samples.

Visceral leishmaniasis (VL) was firstly reported in Armenia in 1913. Following a considerable increase of the number of cases until the mid 1950s, the disease disappeared after 1969 and re-emerged in 1999. Scientific literature about VL in Armenia is available only in Russian or Armenian. This paper presents a historical overview about leishmaniasis in Armenia based on this literature as well as an epidemiological update since the re-emergence of the disease. In 1999-2016, 116 indigenous VL cases were recorded mainly in children in 8 of the 11 districts, however, VL is underreported because of lack of trained medical personal and diagnostic facilities. The aim of this work was to apply for the first time molecular diagnosis of VL in Armenia. Out of 25 VL suspected patients, 22 were positive by microscopy and polymerase chain reaction (PCR). Genotyping using internal transcribed spacer 1-PCR-restriction fragment length polymorphism and sequencing identified the causative agent of VL in Armenia as Leishmania infantum. The present work is an important step towards the inclusion of molecular techniques in the current diagnosis of VL in Armenia and the establishment of local molecular diagnostic facilities.

A pilot study on fingerprinting Leishmania species from the Old World using Fourier transform infrared spectroscopy.

Leishmania species are protozoan parasites and the causative agents of leishmaniasis, a vector borne disease that imposes a large health burden on individuals living mainly in tropical and subtropical regions. Different Leishmania species are responsible for the distinct clinical patterns, such as cutaneous, mucocutaneous, and visceral leishmaniasis, with the latter being potentially fatal if left untreated. For this reason, it is important to perform correct species identification and differentiation. Fourier transform infrared spectroscopy (FTIR) is an analytical spectroscopic technique increasingly being used as a potential tool for identification of microorganisms for diagnostic purposes. By employing mid-infrared (MIR) spectral data, it is not only possible to assess the chemical structures but also to achieve differentiation supported by multivariate statistic analysis. This work comprises a pilot study on differentiation of Leishmania species of the Old World (L. major, L. tropica, L. infantum, and L. donovani) as well as hybrids of distinct species by using vibrational spectroscopic fingerprints. Films of intact Leishmania parasites and their deoxyribonucleic acid (DNA) were characterized comparatively with respect to their biochemical nature and MIR spectral patterns. The strains' hyperspectral datasets were multivariately examined by means of variance-based principal components analysis (PCA) and distance-based hierarchical cluster analysis (HCA). With the implementation of MIR spectral datasets we show that a phenotypic differentiation of Leishmania at species and intra-species level is feasible. Thus, FTIR spectroscopy can be further exploited for building up spectral databases of Leishmania parasites in view of high-throughput analysis of clinical specimens. Graphical abstract For Leishmania species discrimination, sample films of intact parasites and their extracted DNA were analyzed by FTIR micro-spectroscopy. Hyperspectral datasets that comprise mid-infrared fingerprints were submitted to multivariate analysis tools such as principal components analysis (PCA) and hierarchical cluster analysis (HCA).

Spatiotemporal and molecular epidemiology of cutaneous leishmaniasis in Libya.

BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health problem in Libya. In this paper, we describe the eco-epidemiological parameters of CL during the armed conflict period from January 2011 till December 2012. Current spatiotemporal distributions of CL cases were explored and projected to the future using a correlative modelling approach. In addition the present results were compared with our previous data obtained for the time period 1995-2008. METHODOLOGY/PRINCIPAL FINDINGS: We investigated 312 CL patients who presented to the Dermatology Department at the Tripoli Central Hospital and came from 81 endemic areas distributed in 10 districts. The patients presented with typical localized lesions which appeared commonly on the face, arms and legs. Molecular identification of parasites by a PCR-RFLP approach targeting the ITS1 region of the rDNA was successful for 81 patients with two causative species identified: L. major and L. tropica comprised 59 (72.8%) and 22 (27.2%) cases, respectively. Around 77.3% of L. tropica CL and 57.7% of L. major CL caused single lesions. Five CL patients among our data set were seropositive for HIV. L. tropica was found mainly in three districts, Murqub (27.3%), Jabal al Gharbi (27.3%) and Misrata (13.7%) while L. major was found in two districts, in Jabal al Gharbi (61%) and Jafara (20.3%). Seasonal occurrence of CL cases showed that most cases (74.2%) admitted to the hospital between November and March, L. major cases from November till January (69.4%), and L. tropica cases mainly in January and February (41%). Two risk factors were identified for the two species; the presence of previously infected household members, and the presence of rodents and sandflies in patient's neighborhoods. Spatiotemporal projections using correlative distribution models based on current case data and climatic conditions showed that coastal regions have a higher level of risk due to more favourable conditions for the transmitting vectors. CONCLUSION: Future projection of CL until 2060 showed a trend of increasing incidence of CL in the north-western part of Libya, a spread along the coastal region and a possible emergence of new endemics in the north-eastern districts of Libya. These results should be considered for control programs to prevent the emergence of new endemic areas taking also into consideration changes in socio-economical factors such as migration, conflicts, urbanization, land use and access to health care.

Epidemiological analysis of Leishmania tropica strains and giemsa-stained smears from Syrian and Turkish leishmaniasis patients using multilocus microsatellite typing (MLMT).

Turkey is located in an important geographical location, in terms of the epidemiology of vector-borne diseases, linking Asia and Europe. Cutaneous leishmaniasis (CL) is one of the endemic diseases in a Turkey and according to the Ministry Health of Turkey, 45% of CL patients originate from Sanliurfa province located in southeastern Turkey. Herein, the epidemiological status of CL, caused by L. tropica, in Turkey was examined using multilocus microsatellite typing (MLMT) of strains obtained from Turkish and Syrian patients. A total of 38 cryopreserved strains and 20 Giemsa-stained smears were included in the present study. MLMT was performed using 12 highly specific microsatellite markers. Delta K (ΔK) calculation and Bayesian statistics were used to determine the population structure. Three main populations (POP A, B and C) were identified and further examination revealed the presence of three subpopulations for POP B and C. Combined analysis was performed using the data of previously typed L. tropica strains and Mediterranean and Sanliurfa populations were identified. This finding suggests that the epidemiological status of L. tropica is more complicated than expected when compared to previous studies. A new population, comprised of Syrian L. tropica samples, was reported for the first time in Turkey, and the data presented here will provide new epidemiological information for further studies.

Leishmania infections: Molecular targets and diagnosis.

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

BACKGROUND: The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. METHODOLOGY AND PRINCIPAL FINDINGS: Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? CONCLUSIONS AND SIGNIFICANCE: We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

Phylogenetic structure of Leishmania tropica in the new endemic focus Birjand in East Iran in comparison to other Iranian endemic regions.

Iran has been identified being among the countries with the highest number of cutaneous leishmaniasis (CL) cases. South Khorasan province in East Iran is an emerging focus of CL. Species identification of sixty clinical samples by ITS1 PCR-RFLP presented evidence for the dominance of Leishmania tropica (90%) in this region. Analysis of the ITS1 sequence of 19 L. tropica isolates revealed seven closely related sequence types. In addition, ITS1 sequences available in GenBank from other Iranian regions were compiled for comparison with the studied isolates. Iranian L. tropica was distributed in two main clusters. All East Iranian sequence types were grouped with strains from foci from Southeast and Central regions in cluster A, showing highly similar sequences. The highest similarity was observed between most L. tropica from East and all isolates from Southeast regions and from Savojbolagh county in Central Iran. Southwest L. tropica was shown to be paraphyletic as the isolates were distributed in both clusters A and B. All Northeastern L. tropica were part of cluster B, however they showed significant heterogeneity and were distributed in different subclusters. Distribution of L. tropica populations was to some extent congruent with genetic lineages of Phlebotomus sergenti in Iran and may be an evidence for parasite-vector co-evolution. Southeast-East L. tropica was also similar to strains from Herat province in Afghanistan at the East border of Iran. This is the first comprehensive study on population structure of L. tropica in Iran that provides a guideline for appropriate sampling for further molecular based epidemiological studies.

Leishmania donovani populations in Eastern Sudan: temporal structuring and a link between human and canine transmission.

BACKGROUND: Visceral leishmaniasis (VL), caused by the members of the Leishmania donovani complex, has been responsible for devastating VL epidemics in the Sudan. Multilocus microsatellite and sequence typing studies can provide valuable insights into the molecular epidemiology of leishmaniasis, when applied at local scales. Here we present population genetic data for a large panel of strains and clones collected in endemic Sudan between 1993 and 2001. METHODS: Genetic diversity was evaluated at fourteen microsatellite markers and eleven nuclear sequence loci across 124 strains and clones. RESULTS: Microsatellite data defined six genetic subpopulations with which the nuclear sequence data were broadly congruent. Pairwise estimates of FST (microsatellite) and KST (sequence) indicated small but significant shifts among the allelic repertoires of circulating strains year on year. Furthermore, we noted the co-occurrence of human and canine L. donovani strains in three of the six clusters defined. Finally, we identified widespread deficit in heterozygosity in all four years tested but strong deviation from inter-locus linkage equilibrium in two years. CONCLUSIONS: Significant genetic diversity is present among L. donovani in Sudan, and minor population structuring between years is characteristic of entrenched, endemic disease transmission. Seasonality in vector abundance and transmission may, to an extent, explain the shallow temporal clines in allelic frequency that we observed. Genetically similar canine and human strains highlight the role of dogs as important local reservoirs of visceral leishmaniasis.

Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

Genetic diversity evaluation on Portuguese Leishmania infantum strains by multilocus microsatellite typing.

Leishmania infantum is the main etiological agent of zoonotic visceral leishmaniasis in the Mediterranean region, including Portugal, but, given its low isoenzyme diversity in this country, the population structure is poorly known. A set of 14 polymorphic microsatellite markers was studied on 136 Portuguese Leishmania strains isolated from different hosts, geographic regions and different clinical forms. A total of 108 different genotypes were found, which is a degree of genetic diversity comparable to other regions, even within zymodeme MON-1. A single most common genotype was detected in 1:5 of all strains, which, with a greater number of multi-strain genotypes found in the Lisbon Metropolitan Region, particularly for human strains, was suggestive of the occurrence of clonal transmission. In addition, a high re-infection rate was found among HIV+ patients. Model based analysis by STRUCTURE uncovered two main populations (populations A and B, composed of MON-1 and non-MON-1 strains, respectively), with great genetic diversity between them, and two MON-1 sub-populations (A1 and A2). High inbreeding coefficients were found in these populations, although strains with mixed ancestry were identified, suggesting that recombination also plays a role in the epidemiology of this species in Portugal. Some but limited geographical differentiation was observed, with groups of strains from the same regions clustering together, particularly those from canine origin. Our results show that L. infantum isolates from Portugal present microsatellite diversity comparable to other regions and that different transmission models play a role in its epidemiology, from clonal transmission to recombination. In addition, although Portugal is a small country, mobility of people and animals is high and Leishmania can be probably easily disseminated between infected hosts throughout the country, two instances of seemingly local restricted transmission were identified.

Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia).

BACKGROUND/OBJECTIVES: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. METHODOLOGY/PRINCIPAL FINDINGS: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. CONCLUSIONS/SIGNIFICANCE: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.

Genetic typing reveals monomorphism between antimony sensitive and resistant Leishmania donovani isolates from visceral leishmaniasis or post kala-azar dermal leishmaniasis cases in India.

Resistance to pentavalent antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also known as kala-azar (KA), caused by Leishmania donovani. In India, over 60% of KA patients are unresponsive to the first-line drug sodium antimony gluconate (SAG). Resistance determinants in laboratory strains are partly known; however, the mechanism operating in field isolates is not well understood. In this study, we attempted to analyze the genetic polymorphism between SAG sensitive and resistant parasites using a total of 52 L. donovani isolates obtained either from bone marrow of VL patients or from skin lesions of post kala-azar dermal leishmaniasis (PKDL) patients that constitute an important reservoir of parasite. The clinical isolates were analyzed in comparison with L. donovani parasites from reference strains belonging to distinct geographical locations, at internal transcribed spacer 1 region; coding region of gp63 and nine microsatellite repeat regions. Our results demonstrated that both SAG resistant (n = 26) and sensitive (n = 19) Indian isolates, whether causing VL or PKDL, were monomorphic at all the genetic loci tested, unlike the L. donovani in East African or Leishmania infantum in Mediterranean countries where intraspecies variations exist at these loci. Further, the Indian isolates were found closest to the Kenyan isolates of L. donovani on the basis of fragment analysis of microsatellite markers.