Vit Dvorak

Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel.

TOC summary for table of contents: Infection with Leishmania tropica is emerging because of encroachment of rock hyraxes and transmission by multiple vector species.

The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region

An updated view of the establishment and spread of the leishmaniases in Europe is presented, mostly with respect to newly emerging and re-emerging foci and the incrimination of neglected as well as new reservoir hosts. At the same time, a concept of specific versus permissive vectors reassesses the potential role of various sandfly species in Leishmania transmission and considers the risk of introduction of exotic Leishmania species in Europe. The leishmaniases are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural hosts and vectors, immunosuppressive conditions (like infection with human immunodeficiency virus (HIV) or organ transplantation-associated therapies in humans) and the consequences of war, all contribute to the transformation of the epidemiology of leishmaniasis. Such changes should be considered when studying the spread of the disease throughout Europe for targeted control measures to safeguard public health.

Sergentomyia schwetzi is not a competent vector for Leishmania donovani and other Leishmania species pathogenic to humans.

BackgroundSand fly species of the genus Sergentomyia are proven vectors of reptilian Leishmania that are non-pathogenic to humans. However, a consideration of the role of Sergentomyia spp. in the circulation of mammalian leishmaniasis appears repeatedly in the literature and the possibility of Leishmania transmission to humans remains unclear. Here we studied the susceptibility of colonized Sergentomyia schwetzi to Leishmania donovani and two other Leishmania species pathogenic to humans: L. infantum and L. major.MethodsFemales of laboratory-reared S. schwetzi were infected by cultured Leishmania spp. by feeding through a chicken membrane, dissected at different time intervals post bloodmeal and examined by light microscopy for the abundance and location of infections.ResultsAll three Leishmania species produced heavy late stage infections in Lutzomyia longipalpis or Phlebotomus duboscqi sand flies used as positive controls. In contrast, none of them completed their developmental cycle in Sergentomyia females; Leishmania promastigotes developed within the bloodmeal enclosed by the peritrophic matrix (PM) but were defecated together with the blood remnants, failing to establish a midgut infection. In S. schwetzi, the PM persisted significantly longer than in L. longipalpis and it was degraded almost simultaneously with defecation. Therefore, Leishmania transformation from procyclic to long nectomonad forms was delayed and parasites did not attach to the midgut epithelium.ConclusionsSergentomyia schwetzi is refractory to human Leishmania species and the data indicate that the crucial aspect of the refractoriness is the relative timing of defecation versus PM degradation.

Re-Emergence of Visceral and Cutaneous Leishmaniasis in the Greek Island of Crete

Leishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50 m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica).

Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani.

Background Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ). Methodology/Principal Findings Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×103 per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding. Conclusions Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.

Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry

BackgroundPhlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches.MethodsSix laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes.ResultsSpecimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens.ConclusionsSpecies identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.

A comparison of the intraspecific variability of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae)

ABSTRACT Phlebotomus sergenti populations from different areas of the Mediterranean basin are known to exhibit high intraspecific variability. Previous studies of ITS2 revealed the presence of two branches that may represent sibling species. To corroborate this finding by other tools, two colonies of P. sergenti originating from Turkey and Israel, each belonging to a different ITS2 branch, were compared by three different methods: geometric morphometric analysis of wing shape, RAPD (random amplified polymorphic DNA), and cross-mating study. For geometric morphometric analysis, two-dimensional Cartesian coordinates of 16 landmarks from the wings were digitized and analyzed. Significant shape differences were found between colonies but not between sexes within each colony. RAPD results formed two distinctive clades corresponding to the origin of the colony but also showed heterogenity among members of both colonies. In cross-mating studies, viable hybrid F1 and F2 progeny were obtained when both Turkish males/Israeli females and Israeli males/Turkish females were crossed. F1 progeny was included in RAPD analysis and these hybrids formed a distinctive clade with an intermediate position between the two parental clades. No significant differences were found in egg production of crossed sand flies. The cross-mating study showed that there is no reproductive barrier between P. sergenti from different geographical areas. On the other hand, RAPD and geometric morphometric analysis revealed a significant difference between colonies and confirmed the suitability of previous ITS2 analysis for discrimination among sand fly populations. Further development of molecular markers should resolve a possible existence of sibling species within Phlebotomus sergenti.

Intraspecific variability of natural populations of Phlebotomus sergenti, the main vector of Leishmania tropica

ABSTRACT: An intraspecific study of Phlebotomus sergenti was performed on populations from Turkey, Syria, Israel, and Uzbekistan by four different approaches: geometric morphometries, RAPD analysis, internal transcribed spacer 2 (ITS2) sequencing (nuclear marker), and cytochrome B sequencing (mitochondrial marker). In RAPD analysis, distinct clades were formed in accordance with the geographical origin of the specimens. There was no distinct grouping according to place of origin within the Turkish samples from various localities in south-eastern Anatolia, which suggests a gene flow between populations separated spatially by the Amanos mountains, a mountain range of a considerable altitude. The results of ITS2 rDNA sequencing complied with the previously published intraspecific division of P. sergenti into two branches, northeastern and southwestern. However, mtDNA haplotypes formed three lineages with specimens from Turkey and Israel, sharing a common clade. A previously postulated hypothesis about a complex of sibling species within P. sergenti is therefore questionable. Cytochrome B seems to be a more discriminative marker for intraspecific variability assessment.

A taxonomic study of Phlebotomus (Larroussius) perfiliewi s. l.

Phlebotomus (Larroussius) perfiliewi is one of the main vectors of Leishmania infantum in the Mediterranean basin and in Central Asia. Its taxonomic status remains doubtful. It usually includes three taxa of specific or subspecific value, depending on the authors: P. perfiliewiParrot, 1930 s. st. in North Africa, Western Europe and the Balkans, Crimea; Phlebotomus galilaeusTheodor, 1958 in Israel/Palestine and Cyprus and Phlebotomus transcaucasicusPerfiliev, 1937 in Caucasus and Central Asia. While the identification of the females of P. perfiliewi s. l. is relatively easy, based on the particular morphology of their spermathecal ducts, the specific identification of the females of Phlebotomus perfiliewi perfiliewi, Phlebotomus perfiliewi galilaeus and Phlebotomus perfiliewi transcaucasicus is impossible. The differentiation of males, based mainly on characters on aedeagus, is also difficult. The goal of the present study is to carry out a taxonomic update based on a morphological study of specimens coming from various populations from Asia, Europe and North Africa coupled with their molecular analysis.

Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents

Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia, possibly as reservoir hosts.