Typing of four genetic loci discriminates among closely related species of New World Leishmania

All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.     

A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.     

Old World and New World Cupressus pollen: morphological and cytological remarks

The aim of this work was to collect new information about pollen morphology and pollen wall structure comparing Cupressus species from the Old World and New World. Cupressus is a nonmonophyletic genus that includes species that appear to be divided into Old World and New World clades. Observations in this study on cypress pollen indicate that grain size and composition of intine β-glucans are different between the considered Old World and New World species. Different from all the other American cypress species, pollen of C. macrocarpa reacted to dying in a similar manner to Old World species. Rehydrated pollen grains collected from 20 Asian, Afro-Mediterranean and American cypress species were measured under a light microscope. The size of the pollen grains and the percentage of intine in relation to the pollen grain diameter were significantly different between Old World and New World species. Pollen wall composition was tested after addition of different dyes to the hydration solution, and subsequent observations were carried out by light and fluorescence microscopy. Lugol and calcofluor staining showed differences in composition of the middle and inner intine layers between New World and Old World species.     

Sesquiterpene lactones and systematics of the genus Artemisia

The sesquiterpene lactones isolated from species in the genus Artemisia have been reviewed in an attempt to better understand the phylogeny and systematics of the four sections (subgenera), Abrotanum, Absinthium, Dracunculus and Seriphidium, proposed by Besser in 1829. The absence of hair on the receptacle is the only morphological characteristic separating species of Abrotanum from the species of Absinthium. There are no chemical characteristics segregating the species in these two subgenera since both produce eudesmanolides and guaianolides that are identical or biosynthetically similar. This suggests that the two subgenera could be combined into one (Artemisia) as proposed by Poljakov. The subgenus Seriphidium is composed of two geographical groups, one in the Old World and the other in the New World. The Old World species almost exclusively produce sesquiterpene lactones in the eudesmanolide class whereas the New World species (section Tridentatae) produce eudesmanolides and guaianolides, many of the latter being identical or structurally related to the sesquiterpene lactones in New World Abrotanum species. The chemical data in conjunction with geographic distributions suggest that the subgenus Seriphidium is polyphyletic and that the section Tridentatae originated from Abrotanum. Consequently, the Tridentate should be recognized as a subgenus separate and distinct from the Old World Seriphidium. There was insufficient information from the subgenus Dracunculus for interpretation.     

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Background

Three major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures.

Methods/Principal Findings

High resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 µl DNA sample, i.e., less than a single parasite per reaction.

Conclusions/Significance

This new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys.     

Comparative genomics of Leishmania isolates from Brazil confirms the presence of Leishmania major in the Americas

Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named “L. major-like”, and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major-like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major-like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World.     

Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition

Background

A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes.

Methods/Principal Findings

We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.

Conclusions/Significance

These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.     

Chromosomal rearrangements differentiating the ryegrass genome from the Triticeae, oat, and rice genomes using common heterologous RFLP probes

An restriction fragment length polymorphism (RFLP)-based genetic map of ryegrass (Lolium) was constructed for comparative mapping with other Poaceae species using heterologous anchor probes. The genetic map contained 120 RFLP markers from cDNA clones of barley (Hordeum vulgare L.), oat (Avena sativa L.), and rice (Oryza sativa L.), covering 664 cM on seven linkage groups (LGs). The genome comparisons of ryegrass relative to the Triticeae, oat, and rice extended the syntenic relationships among the species. Seven ryegrass linkage groups were represented by 10 syntenic segments of Triticeae chromosomes, 12 syntenic segments of oat chromosomes, or 16 syntenic segments of rice chromosomes, suggesting that the ryegrass genome has a high degree of genome conservation relative to the Triticeae, oat, and rice. Furthermore, we found ten large-scale chromosomal rearrangements that characterize the ryegrass genome. In detail, a chromosomal rearrangement was observed on ryegrass LG4 relative to the Triticeae, four rearrangements on ryegrass LGs2, 4, 5, and 6 relative to oat, and five rearrangements on ryegrass LGs1, 2, 4, 5, and 7 relative to rice. Of these, seven chromosomal rearrangements are reported for the first time in this study. The extended comparative relationships reported in this study facilitate the transfer of genetic knowledge from well-studied major cereal crops to ryegrass.     

A direct method for the chromosomal assignment of DNA markers in Leishmania

A simple method for the chromosomal assignment of any DNA marker would be an important tool for the ongoing project to map the genome of the protozoan parasite Leishmania. The Leishmania chromosomes enter pulsed field gel electrophoresis (PFGE) gels under current electrophoretic conditions, but their direct identification in a given strain is hampered by their stacking in a few chromosomal bands, and by the very frequent size variations of the same chromosome among parasite strains. To overcome these problems, we determined the complete karyotypes of 12 Old World Leishmania cloned strains. This enabled us to select three of these strains that display great chromosome size polymorphisms, such that every chromosome can be individualized by a specific pattern after hybridization onto these three karyotypes. The complete resolution of the genomes of these three strains can be carried out with only three electrophoretic conditions. This makes a series of three blots sufficient for the assignment of any new marker on a particular Leishmania chromosome.     

Tropical Asian species show that the Old World clade of ‘spiny solanums’ (Solanum subgenus Leptostemonum pro parte: Solanaceae) is not monophyletic

The tropical Asian taxa of the species‐rich genus Solanum (Solanaceae) have been less well studied than their highly diverse New World relatives. Most of these tropical Asian species, including the cultivated brinjal eggplant/aubergine and its wild progenitor, are part of the largest monophyletic Solanum lineage, the ‘spiny solanums’ (subgenus Leptostemonum or the Leptostemonum clade). Here we present the first phylogenetic analysis of spiny solanums that includes broad sampling of the tropical Asian species, with 42 of the 56 currently recognized species represented. Two nuclear and three plastid regions [internal transcribed spacer (ITS), waxy, ndhF‐rpL32, trnS‐trnG and trnT‐trnF] were amplified and used to reconstruct phylogenetic relationships using maximum likelihood and Bayesian methods. Our analyses show that Old World spiny solanums do not resolve in a single clade, but are part of three unrelated lineages, suggesting at least three independent introductions from the New World. We identify and describe several monophyletic groups in Old World solanums that have not been previously recognized. Some of these lineages are coherent in terms of morphology and geography, whereas others show considerable morphological variation and enigmatic distribution patterns. Tropical Asia occupies a key position in the biogeography of Old World spiny solanums, with tropical Asian taxa resolved as the closest relatives of diverse groups of species from Australia and Africa.     

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.     

Historical evidence for a pre-Columbian presence of <Emphasis Type="Italic">Datura</Emphasis> in the Old World and implications for a first millennium transfer from the New World

Datura (Solanaceae) is a small genus of plants that, for long, was thought to occur naturally in both the New and Old Worlds. However, recent studies indicate that all species in the genus originated in the Americas. This finding has prompted the conclusion that no species of Datura could have been present in the Old World prior to its introduction there by Europeans in the early 16th century CE. Further, the textual evidence traditionally cited in support of a pre-Columbian Old World presence of Datura species is suggested to be due to the misreading of classical Greek and Arabic sources. As a result, botanists generally accept the opinion that Datura species were transferred into the Old World in the post-Columbian period. While the taxonomic and geographic evidence for a New World origin for all the Datura species appears to be well supported, the assertion that Datura species were not known in the Old World prior to the 16th century is based on a limited examination of the pre-Columbian non-Anglo sources. We draw on old Arabic and Indic1 texts and southern Indian iconographic representations to show that there is conclusive evidence for the pre-Columbian presence of at least one species of Datura in the Old World. Given the systematic evidence for a New World origin of the genus, the most plausible explanation for this presence is a relatively recent but pre-Columbian (probably first millennium CE) transfer of at least one Datura species, D. metel, into the Old World. Because D. metel is a domesticated species with a disjunct distribution, this might represent an instance of human-mediated transport from the New World to the Old World, as in the case of the sweet potato (Ipomoea batatas).     

Phosphoenolpyruvate Carboxykinase 1 Gene (Pck1) Displays Parallel Evolution between Old World and New World Fruit Bats

Bats are an ideal mammalian group for exploring adaptations to fasting due to their large variety of diets and because fasting is a regular part of their life cycle. Mammals fed on a carbohydrate-rich diet experience a rapid decrease in blood glucose levels during a fast, thus, the development of mechanisms to resist the consequences of regular fasts, experienced on a daily basis, must have been crucial in the evolution of frugivorous bats. Phosphoenolpyruvate carboxykinase 1 (PEPCK1, encoded by the Pck1 gene) is the rate-limiting enzyme in gluconeogenesis and is largely responsible for the maintenance of glucose homeostasis during fasting in fruit-eating bats. To test whether Pck1 has experienced adaptive evolution in frugivorous bats, we obtained Pck1 coding sequence from 20 species of bats, including five Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our molecular evolutionary analyses of these sequences revealed that Pck1 was under purifying selection in both Old World and New World fruit bats with no evidence of positive selection detected in either ancestral branch leading to fruit bats. Interestingly, however, six specific amino acid substitutions were detected on the ancestral lineage of OWFBs. In addition, we found considerable evidence for parallel evolution, at the amino acid level, between the PEPCK1 sequences of Old World fruit bats and New World fruit bats. Test for parallel evolution showed that four parallel substitutions (Q276R, R503H, I558V and Q593R) were driven by natural selection. Our study provides evidence that Pck1 underwent parallel evolution between Old World and New World fruit bats, two lineages of mammals that feed on a carbohydrate-rich diet and experience regular periods of fasting as part of their life cycle.     

Detection of parasite-derived tRNA and rRNA fragments in the peripheral blood of mice experimentally infected with Leishmania donovani and Leishmania amazonensis using next-generation sequencing analysis

All prokaryotes and eukaryotes, including parasites, release extracellular vesicles or exosomes that contain selected proteins, lipids, nucleic acids, glycoconjugates, and metabolites. Leishmania exosomes are highly enriched in small RNAs derived from the rRNAs and tRNAs of the protozoan parasite species. Here, using plasma exosomes isolated by a kit and next-generation sequencing, we report the detection of fragments of parasite-derived rRNAs and tRNAs in the peripheral plasma samples of mice experimentally infected with Leishmania donovani and Leishmania amazonensis, the causative agents of Old World visceral leishmaniasis and New World disseminated cutaneous leishmaniasis, respectively. Detected RNA molecules of 28S rRNA, 5.8S rRNA, tRNA-Glu, and tRNA-Thr were common to both plasma samples of mice inoculated with L. donovani and L. amazonensis, whereas tRNA-Ile and tRNA-Trp were only detected in L. amazonensis-infected mice. The detected rRNAs and tRNA isotypes were matched with the exosomal components reported in a previous key study. Our preliminary results suggested that parasite-derived small RNAs were circulating in the blood of mice infected with Leishmania species, providing a better understanding of the roles of exosomal components in leishmaniasis and also new insights into exosome-based biomarkers for Leishmania infection.     

Easy Identification of Leishmania Species by Mass Spectrometry

Background

Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management.

Methodology

We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania.

Principal Findings

Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level.

Conclusions/Significance

Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.     

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.