Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae).

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied-despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.

Message in a vesicle - trans-kingdom intercommunication at the vector-host interface.

Vector-borne diseases cause over 700,000 deaths annually and represent 17% of all infectious illnesses worldwide. This public health menace highlights the importance of understanding how arthropod vectors, microbes and their mammalian hosts interact. Currently, an emphasis of the scientific enterprise is at the vector-host interface where human pathogens are acquired and transmitted. At this spatial junction, arthropod effector molecules are secreted, enabling microbial pathogenesis and disease. Extracellular vesicles manipulate signaling networks by carrying proteins, lipids, carbohydrates and regulatory nucleic acids. Therefore, they are well positioned to aid in cell-to-cell communication and mediate molecular interactions. This Review briefly discusses exosome and microvesicle biogenesis, their cargo, and the role that nanovesicles play during pathogen spread, host colonization and disease pathogenesis. We then focus on the role of extracellular vesicles in dictating microbial pathogenesis and host immunity during transmission of vector-borne pathogens.

An Overview on Target-Based Drug Design against Kinetoplastid Protozoan Infections: Human African Trypanosomiasis, Chagas Disease and Leishmaniases.

The protozoan diseases Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases span worldwide and therefore their impact is a universal concern. The present regimen against kinetoplastid protozoan infections is poor and insufficient. Target-based design expands the horizon of drug design and development and offers novel chemical entities and potential drug candidates to the therapeutic arsenal against the aforementioned neglected diseases. In this review, we report the most promising targets of the main kinetoplastid parasites, as well as their corresponding inhibitors. This overview is part of the Special Issue, entitled "Advances of Medicinal Chemistry against Kinetoplastid Protozoa (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) Infections: Drug Design, Synthesis and Pharmacology".

CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission.

The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.

Sandfly surveillance and investigation of Leishmania spp. DNA in sandflies in Kosovo.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. The presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance.

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts.

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.

Increasing putative vector importance of Trichophoromyia phlebotomines (Diptera: Psychodidae).

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.

The second species of Biskratrombium (Trombidiformes: Microtrombidiidae) ectoparasitic on phlebotomine sandflies (Diptera: Psychodidae) from Iran.

A new species of Biskratrombium (Trombidiformes: Microtrombidiidae), B. persicumn. sp. is described and illustrated, from Fars province, southern Iran. Biskratrombium persicum larvae are ectoparasites of the adults of Phlebotomus (Paraphlebotomus) alexandri (Sinton, 1928), P. (Phlebotomus) papatasi (Scopoli, 1786) and Sergentomyia (Sergentomyia) mervynae (Pringle, 1953) (Diptera: Psychodidae). Fars province is considered as a significant focus of leishmaniasis, a disease which is mostly associated with rural areas; these areas offer favorable habitats to the phlebotomine sandflies due to limited sanitation. This study was performed to identify the natural enemies of sandflies (as leishmania disease vectors) in this region. In this research, B. persicum larvae were removed from their sandfly hosts collected from foci of leishmaniasis (e.g., sheep and goat keeping locations) using sticky traps. Also, the abundance of sandflies infested with the parasitic mite was calculated. Some morphological abnormalities in the species are noted and world parasitengone mites parasitizing phlebotomine sandflies are reviewed.

Identification of Leishmania Species in Naturally Infected Sand Flies from Refugee Camps, Greece.

High infection rates of Leishmania donovani and L. tropica were detected in Phlebotomus spp. sand flies collected from refugee camps in Greece, indicating increased risk of infection among local populations. Detection and treatment of leishmaniasis, community education, and vector control are essential measures to prevent pathogen transmission and protect public health.

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control.

Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions.

Leishmania HASP and SHERP Genes Are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host.

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation.

Transmission of leishmaniasis from human to other vertebrates: a rapid zooanthroponotic evolution.

Research regarding zoonotic diseases often focuses on those diseases that are transferred from animals to humans. However, humans are also transmitting pathogens to animals but research on this topic is not given priority and importance. I have tried to draw the attention of researchers to this area also which is equally important. The aim of this letter was to provide a brief overview of published literature regarding reverse zoonoses or zooanthroponosis in the field of leishmaniasis and highlight the need for future work in this area. Scientific research must be conducted in the field of reverse zoonoses to provide an enriched understanding of emerging disease threats to animals and should not be neglected.

An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

Leishmaniasis: current challenges and prospects for elimination with special focus on the South Asian region.

Leishmania donovani, the most virulent species of Leishmania, is found in the South Asian region that harbours the majority of visceral leishmaniasis (VL) cases in the world. The traditionally accepted relationships between the causative species of Leishmania and the resultant disease phenotype have been challenged during recent years and have underscored the importance of revisiting the previously established taxonomy with revisions to its classification. The weak voice of the afflicted with decades of neglect by scientists and policy makers have led to the miserably inadequate and slow advancements in product development in the fields of diagnostics, chemotherapeutics and vector control that continue to hinder the effective management and control of this infection. Limitations notwithstanding, the regional drive for the elimination of VL initiated over a decade ago that focused on India, Nepal and Bangladesh, the three main afflicted countries in the Indian subcontinent is therefore, commendable, with the subsequent status reviews and restructuring of strategies possibly even more so. However, the renewed efforts would need to be combined with plans to combat new challenges in the South-Asian region that includes the emergence of atypical parasite variants, in order to realistically achieve the set goal of regional elimination of VL.

Leishmania infection in rodents in Greece.

OBJECTIVE: To investigate the prevalence of Leishmania infection in rodents from various areas of northern Greece. METHODS: Ninety-seven rodents (66 Mus musculus, 19 Rattus norvegicus and 12 R. rattus) were collected during pest control programmes and examined by cytology (spleen and liver smears), serology (ELISA) and PCR (real-time and gel-based) for Leishmania. Date, environment, sex, existence of dogs in the close environment were recorded for each rodent. RESULTS: All cytological preparations were negative, whereas specific IgG was detected in 54.5% in total; 70% of R. norvegicus; 50% of R. rattus; and 50% of M. musculus. In at least one molecular method, 19.6% of the samples in total were positive: 25% of R. rattus, 24% of M. musculus, but no R. norvegicus was found positive. Environment (semi-urban areas, P = 0.037) and species (M. musculus, P = 0.032) were associated with positive PCR. All infected animals showed evidence of low parasite burden, demonstrated by the negative cytological examinations and the high Ct values observed in real-time PCR. CONCLUSION: Due to the proximity of rodents to humans and dogs, these animals may be important in the epidemiology of leishmaniosis, especially if proven that they can infect sand flies.

A systematic review and meta-analysis of the prevalence of Leishmania infection in blood donors.

BACKGROUND: The risk of transfusion transmitted leishmaniasis (TTL) from apparently healthy persons or asymptomatic individuals, should not be ignored. Lack of a comprehensive review, encouraged us to design a systematic review with meta-analysis approach to assess the prevalence of Leishmania infection in healthy blood donors. METHODS: For this purpose, 6 English databases (PubMed, Scopus, Web of Sciences, Science Direct, EMBASE and CINAHL) were browsed from January 1990 to July 2016. RESULTS: Due to significant heterogeneity, the random-effects model was used (I2=98.04% and 94.68%, for serological and molecular methods, respectively). A total of 496 papers were found through searching in which 17,816 apparently healthy blood donors were examined for Leishmania infection. The weighted overall prevalence of Leishmania infection in this group was estimated 4% (95% CI=2-7) and 8.7% (95% CI=4.2-14.3) using serological and molecular methods, respectively. CONCLUSIONS: High serological prevalence does not justify widespread donor screening. Leukodepletion filters would substantially decrease the risk of TTL, hence they are potentially proposed in endemic areas specifically for high-risk recipients. To better enlighten the epidemiological aspects of Leishmania infection in blood donors, it is suggested to perform high-level stewardship and more precise studies with regard to involved risk factors.

Shifts in the ecological niche of Lutzomyia peruensis under climate change scenarios in Peru.

The Peruvian Andes presents a climate suitable for many species of sandfly that are known vectors of leishmaniasis or bartonellosis, including Lutzomyia peruensis (Diptera: Psychodidae), among others. In the present study, occurrences data for Lu. peruensis were compiled from several items in the scientific literature from Peru published between 1927 and 2015. Based on these data, ecological niche models were constructed to predict spatial distributions using three algorithms [Support vector machine (SVM), the Genetic Algorithm for Rule-set Prediction (GARP) and Maximum Entropy (MaxEnt)]. In addition, the environmental requirements of Lu. peruensis and three niche characteristics were modelled in the context of future climate change scenarios: (a) potential changes in niche breadth; (b) shifts in the direction and magnitude of niche centroids, and (c) shifts in elevation range. The model identified areas that included environments suitable for Lu. peruensis in most regions of Peru (45.77%) and an average altitude of 3289 m a.s.l. Under climate change scenarios, a decrease in the distribution areas of Lu. peruensis was observed for all representative concentration pathways. However, the centroid of the species' ecological niche showed a northwest direction in all climate change scenarios. The information generated in this study may help health authorities responsible for the supervision of strategies to control leishmaniasis to coordinate, plan and implement appropriate strategies for each area of risk, taking into account the geographic distribution and potential dispersal of Lu. peruensis.

Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population.

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.