Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE(2)/IL-10 sequential pathway

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen-induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge-induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP-1 alpha, TNF-alpha, and leukotriene B-4 (LTB4). Furthermore, SGE treatment enhanced the production of anti-inflammatory mediators, IL-10 and PGE(2). SGE treatments failed to inhibit neutrophil migration and MIP-1 alpha and LTB4 production in IL-10(-/-) mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE-induced IL-10 production, and PGE(2) release triggered by SGE remained increased in IL-10(-/-) mice, suggesting that prostanoids are acting through an IL-10-dependent mechanism. SGE treatments in vivo reduced the OVA-induced lymphoproliferation of spleen-derived cells. Further, the in vitro incubation of bone marrow-derived dendritic cells (DC) with SGE inhibited the proliferation of CD4(+) T cells from OVA-immunized mice, which was reversed by indomethacin and anti-IL-10 antibody treatments. Supporting these results, SGE induced the production of PGE(2) and IL-10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC-membrane expression of MHC-II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation-induced neutrophil migration by an autocrine DC sequential production of PGE(2)/IL-10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Leishmaniasis transmission: distribution and coarse-resolution ecology of two vectors and two parasites in Egypt

Introduction: In past decades, leishmaniasis burden has been low across Egypt; however, changing environment and land use has placed several parts of the country at risk. As a consequence, leishmaniasis has become a particularly difficult health problem, both for local inhabitants and for multinational military personnel. Methods: To evaluate coarse-resolution aspects of the ecology of leishmaniasis transmission, collection records for sandflies and Leishmania species were obtained from diverse sources. To characterize environmental variation across the country, we used multitemporal Land Surface Temperature (LST) and Normalized Difference Vegetation Index (NDVI) data from the Moderate Resolution Imaging Spectroradiometer (MODIS) for 2005-2011. Ecological niche models were generated using MaxEnt, and results were analyzed using background similarity tests to assess whether associations among vectors and parasites (i.e., niche similarity) can be detected across broad geographic regions. Results: We found niche similarity only between one vector species and its corresponding parasite species (i.e., Phlebotomus papatasi with Leishmania major), suggesting that geographic ranges of zoonotic cutaneous leishmaniasis and its potential vector may overlap, but under distinct environmental associations. Other associations (e.g., P. sergenti with L. major) were not supported. Mapping suitable areas for each species suggested that northeastern Egypt is particularly at risk because both parasites have potential to circulate. Conclusions: Ecological niche modeling approaches can be used as a first-pass assessment of vector-parasite interactions, offering useful insights into constraints on the geography of transmission patterns of leishmaniasis.

Bionomics of phlebotomine sand flies (Diptera: Psychodidae) in the province of Al-Baha, Saudi Arabia

The bionomics of phlebotomine sand flies (Diptera: Psychodidae) were studied for two successive years (January 1996-December 1997) at 12 collecting stations representing six sectors of the province of Al-Baha, Saudi Arabia. The predominant species was Phlebotomus bergeroti (41.7%), followed by lesser numbers of Phlebotomus sergenti (11%), Phlebotomus arabicus (10.6%), Sergentomyia tiberiadis (10.5%), Phlebotomus papatasi (10.2%), Sergentomyia antennata (9.6%), Phlebotomus alexandri (3%), Phlebotomus orientalis (2.3%) and Sergentomyia clydei (1.1%). The distribution of the collected species including species that are elsewhere known to act as vectors of human cutaneous leishmaniasis were distributed across different altitudes in Al-Baha. P. bergeroti, P. papatasi and P. arabicus were more abundant indoors; however, P. sergenti was more abundant outdoors. Sand fly populations exhibited three patterns of seasonal abundance in terms of their monthly activity. P. bergeroti, P. sergenti and P. arabicus were found to be naturally infected with Leishmania-like flagellates at an infection rate of 0.2%.

Sandflies (Diptera: Psychodidae) in a focus of visceral leishmaniasis in White Nile, Sudan

Visceral leishmaniasis (VL) has been known to occur since the 1980s on the western bank of the White Nile River (Central Sudan), 150 km south of Khartoum, and has resulted in high mortality. The most recent outbreak of the disease in this area began in 2006. Entomological surveys were carried out during May 2008, June 2010 and May and July 2011 in the White Nile area. Sandflies were collected using Centers for Disease Control light traps and sticky oil traps in the village of Kadaba and the nearby woodland. Phlebotomus females were dissected for the presence of Leishmania promastigotes. A total of 17,387 sandflies, including six species of Phlebotomus and 10 species of Sergentomyia, were identified. The Phlebotomus species recorded were Phlebotomus orientalis, Phlebotomus papatasi, Phlebotomus bergeroti, Phlebotomus duboscqi, Phlebotomus rodhaini and Phlebotomus saevus. P. orientalis was collected in both habitats. The relative abundance of P. orientalis in the woodland habitat was higher than that recorded in the village habitat. In the woodland habitat, there was a notable increase in the relative abundance of P. orientalis during the surveys conducted in 2008 and 2010 compared to 2011. None of the 311 P. orientalis females dissected were infected with Leishmania promastigotes, although relatively high parous rates were recorded in both habitats. Based on the distribution of P. orientalis recorded in this study, this species is the most likely vector of VL in the endemic focus in the White Nile area. Further investigation is required to elucidate the seasonal abundance and distribution of the vector, as well as the transmission season of VL in both habitats so that appropriate control strategies for the vector can be designed.

Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis

Individual, naturally occurring Phlebotomus mongolensis and Phlebotomus caucasicus from Iran were screened for infections with the maternally inherited intracellular Rickettsia-like bacterium Wolbachia pipientis via targeting a major surface protein gene (wsp). The main objective of this study was to determine if W. pipientis could be detected in these species. The sandflies were screened using polymerase chain reaction to amplify a fragment of the Wolbachia surface protein gene. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes. Two strains of Wolbachia were found. Strain Turk 54 (accession EU780683) is widespread and has previously been reported in Phlebotomus papatasi and other insects. Strain Turk 07 (accession KC576916) is a novel strain, found for first time in the two sister species. A-group strains of W. pipientis occur throughout much of the habitat of these sandflies. It is possible that Wolbachia is transferred via horizontal transmission. Horizontal transfer could shed light on sandfly control because Wolbachia is believed to drive a deleterious gene into sandflies that reduces their natural population density. With regard to our findings in this study, we can conclude that one species of sandfly can be infected with different Wolbachia strains and that different species of sandflies can be infected with a common strain.

Ecology of cutaneous leishmaniasis in Sinai: linking parasites, vectors and hosts

Cutaneous leishmaniasis (CL) is a neglected clinical form of public health importance that is quite prevalent in the northern and eastern parts of Egypt. A comprehensive study over seven years (January 2005-December 2011) was conducted to track CL transmission with respect to both sandfly vectors and animal reservoirs. The study identified six sandfly species collected from different districts in North Sinai: Phlebotomus papatasi, Phlebotomus kazeruni, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia antennata and Sergentomyia clydei. Leishmania (-)-like flagellates were identified in 15 P. papatasi individuals (0.5% of 3,008 dissected females). Rodent populations were sampled in the same districts where sandflies were collected and eight species were identified: Rattus norvegicus (n = 39), Rattus rattus frugivorous (n = 13), Rattus rattus alexandrinus (n = 4), Gerbillus pyramidum floweri (n = 38), Gerbillus andersoni (n = 28), Mus musculus (n = 5), Meriones sacramenti (n = 22) and Meriones crassus (n = 10). Thirty-two rodents were found to be positive for Leishmania infection (20.12% of 159 examined rodents). Only Leishmania major was isolated and identified in 100% of the parasite samples. The diversity of both the vector and rodent populations was examined using diversity indices and clustering approaches.

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K-m=0.59 +/- 0.04 mM, V-max=0.123 +/- 0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38 +/- 4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.

The effect of climate change on the potential distribution of the European Phlebotomus species

Our study intended to explore the potential distributionshif of Phlebotomusariasi, P. neglectus, P. perfiliewi, P. perniciosus, and P. tobbi, and some other sandfly species: P. papatasi, P. sergenti, and P. similis. We used climate envelope modeling (CEM) method to determine the ecological requirements of the species and to model the potential distribution for three periods (1961-1990, 2011-2040, and 2041- 2070). We found that by the end of the 2060’s the Southern UK, Germany, entire France and also the western part of Poland can be colonized by sandfly species, mostly by P. ariasi and P. pernicosus. P. ariasishowe the greatest potential northward expansion, from 49°N to 59°N. For all of the studied sand fly species the entire Mediterranean Basin, the Balkan Peninsula, the Carpathian Basin, and northern coastline of the Black Sea are potentially suitable. The length of the predicted active period of the vectors will increase with one or two months.

The effect of climate change on the potential distribution of the European Phlebotomus species and the parasite Leishmania infantum in 2011-2070. A climate envelope modeling approach

Aims: In the Mediterranean areas of Europe, leishmanisasis is one of the most emerging vector-borne diseases. Members of genus Phlebotomus are the primary vectors of the genus Leishmania. To track the human health effect of climate change it is a very important interdisciplinary question to study whether the climatic requirements and geographical distribution of the vectors of human pathogen organisms correlate with each other. Our study intended to explore the potential effects of ongoing climate change, in particular through a potential upward altitudinal and latitudinal shift of the distribution of the parasite Leishmania infantum, its vectors Phlebotomus ariasi, P. neglectus, P. perfiliewi, P. perniciosus, and P. tobbi, and some other sandfly species: P. papatasi, P. sergenti, and P. similis. Methods: By using a climate envelope modelling (CEM) method we modelled the current and future (2011-2070) potential distribution of 8 European sandfly species and L. infantum based on the current distribution using the REMO regional climate model. Results: We found that by the end of the 2060’s most parts of Western Europe can be colonized by sandfly species, mostly by P. ariasi and P. pernicosus. P. ariasi showed the greatest potential northward expansion. For all the studied vectors of L. infantum the entire Mediterranean Basin and South-Eastern Europe seemed to be suitable. L. infantum can affect the Eastern Mediterranean, without notable northward expansion. Our model resulted 1 to 2 months prolongation of the potentially active period of P. neglectus P. papatasi and P. perniciosus for the 2060’s in Southern Hungary. Conclusion: Our findings confirm the concerns that leishmanisais can become a real hazard for the major part of the European population to the end of the 21th century and the Carpathian Basin is a particularly vulnerable area.

Risco de introdução de novas espécies de Leishmania na Região do Algarve

As leishmanioses são doenças parasitárias causadas por protozoários do género Leishmania, transmitidas pela picada de flebótomos fêmeas e que causam uma elevada morbilidade e mortalidade em África, América Latina, Ásia, e na Região Mediterrânica. A vigilância da prevalência das leishmanias e dos respectivos vectores, nas regiões endémicas e envolventes, são cruciais para prever o risco de introdução e disseminação de novas espécies de Leishmania que poderão dar origem a novos focos de doença(s). A monitorização da infecção por Leishmania nos flebótomos é importante para a compreensão da eco-epidemiologia e avaliação de programas de controlo. Por outro lado, a transmissão de Leishmania pode envolver um elevado número de hospedeiros vertebrados que podem ser identificados através da análise das refeições hemáticas dos vectores. A Região do Algarve é considerada um foco endémico de leishmaniose(s), humana e canina, desde a década de 1980. O principal objectivo deste estudo foi actualizar a distribuição, a abundância relativa, a densidade e outros aspectos vectoriais das espécies flebotomínicas na Região Algarvia para uma possível aplicação na vigilância epidemiológica. De Maio a Novembro de 2007, foram capturados 1595 flebótomos, de ambos os sexos, através de armadilhas luminosas tipo CDC, em 175 biótopos. Foram identificadas quatro espécies flebotomínicas: Phlebotomus perniciosus, P. ariasi, P. sergenti e Sergentomyia minuta. Após identificação realizaram-se PCRs com alvo no (i) kDNA e na região ITS-1 para detecção da presença de DNA de Leishmania nos flebótomos e (ii) gene do citocromo B mitocondrial para análise das refeições hemáticas. A presença de uma fêmea de P. perniciosus infectada com L. infantum (taxa de infecção total de 0,12%) demonstrou que o Algarve continua a ser um foco de leishmaniose. A presença de P. sergenti e P. papatasi (esta última, encontrada em estudos anteriores) poderão constituir indicadores de risco de introdução/disseminação de L. tropica e L. major devido à mobilidade de turistas e imigrantes (e outros) de áreas endémicas para esta região. A realização, pela primeira vez em Portugal, de uma técnica de biologia molecular para estudo das preferências hemáticas, demonstrou ser uma ferramenta útil, que no futuro poderá eventualmente contribuir para verificar se os vectores apresentam capacidade de adaptação a novos hospedeiros vertebrados.

The taxonomic status of Phlebotomus oliverioi (Diptera, Psychodidae) and a lectotype designation for Psathyromyia brasiliensis

Phlebotomus oliverioi Barretto & Coutinho, 1941 was originally described based only on the male holotype and has since been considered junior-synonym of Psathyromyia brasiliensis (Costa Lima, 1932). The study of the holotype of Ph. oliverioi allowed us to conclude that the head of this specimen belongs to the genus Psychodopygus and the wings, thorax and abdomen belong to a different species of Psathyromyia. Thus, Ph. oliverioi is a not valid species, and must be removed from the synonym and excluded from the provisions of the International Code of Zoological Nomenclature (Articles 17.2 and 23.8). The specimen was measured, drawn, photographed and the morphological characters are discussed. Lectotype and paralectotypes to Pa. brasiliensis are designated.