Studies on the behaviour and control of phlebotomine sandflies using experimental houses.

Programmes for the control of phlebotomine sandflies (Diptera: Psychodidae), the vectors of leishmaniases, mainly target adults because larval breeding sites are generally unknown or inaccessible. To determine how blood-questing sandfly females enter homes and to develop means for their control, an experimental house (EH) was constructed in a village endemic for cutaneous leishmaniasis. Initially, carbon dioxide (CO2 )-baited suction traps were installed inside the EH to attract and capture sandflies. For other experiments, the windows of the EH were fitted with CO2 -baited window entrance traps (WETs) that allow each window to be considered as a separate unit. The majority of captures inside the EH and in WETs consisted of Phlebotomus sergenti, a species that enters inhabited houses relatively infrequently. Analyses of collections in WETs and in sticky traps on external walls showed that sandflies entered windows having landed previously on the wall below or either side of the window. Shelves constructed below windows significantly reduced the numbers of sandflies that entered both the EH and inhabited houses. The lining of internal walls with insecticide-impregnated fabric significantly increased mortality rates of sandflies captured inside the EH. To reduce the biting burden imposed by phlebotomine sandflies, several control measures must be integrated and sustained.

Insecticide-treated vertical mesh barriers reduce the number of biting mosquitoes.

Mosquitoes foraging for blood sources normally fly relatively close to the ground where wind velocities do not exceed their flight speed. An experiment designed to block foraging mosquitoes from reaching inhabited houses was conducted in a rural settlement flanked by agricultural fields. Mosquitoes were collected during 9 nights using 30 carbon dioxide-baited traps deployed along the external walls of six houses in the row closest to the settlement's perimeter fence. Thereafter, a deltamethrin-impregnated mesh was draped along 400 m of the perimeter fence to a height of 2 m opposite three of the monitored houses. Mosquitoes were trapped for a further 11 nights. A significant difference in the numbers of mosquitoes caught before and after the intervention was demonstrated near protected houses, whereas no significant difference was observed in catches near control houses. The percentage of Culex perexiguus (Diptera: Culicidae), an important vector of West Nile virus, was significantly lower near protected houses (13%) than around control houses (45%). By contrast, the percentage of Culex pipiens was not significantly affected (16% at experimental and 18% at control houses). Although the results presented here are preliminary, the data demonstrate the potential efficacy of vertical insecticidal barriers for mosquito control.

Exclusion of phlebotomine sand flies from inhabited areas by means of vertical mesh barriers.

Vector control constitutes an important component of integrated disease control campaigns. Source reduction is not an option for phlebotomine sand fly vectors of leishmaniasis, because larval breeding sites remain either unknown or inaccessible. Thus, all control efforts are directed against the adult sand flies, mostly attempting to limit their contact with humans. We describe experiments using an insecticide-treated vertical barrier to prevent sand flies from reaching inhabited areas of an agricultural settlement. A 400 meter long section of the peripheral fence of Kibbutz Sde Eliyahu, Jordan Valley, Israel was draped with a deltamethrin-impregnated net that is impenetrable to sand flies (polyester net, 450 holes/inch(2)). Sand flies were captured before and after construction of the barrier using CO(2)-baited CDC traps. Sand fly numbers, as monitored around three houses internal to the barrier, exhibited an 84.9% decrease once the barrier was erected (P=0.003). Concurrently, the neighboring control group of three houses, not protected by the barrier, exhibited a 15.9% increase in sand fly numbers (P=0.974). These results corroborate previous findings of field tests conducted on a smaller scale in an arid suburban setting. Campaigns for reducing the burden of sand fly bites and curtailing the transmission of leishmaniasis, should consider integrating vertical fine-mesh nets with other sand fly control measures.

Studies on the flight patterns of foraging sand flies.

Phlebotomine sand flies transmit Leishmania parasites that cause leishmaniasis in humans. We report experimental results that improve our understanding of how foraging sand flies proceed over flat or sloping ground and how they negotiate vertical obstacles. Three rows of traps were suspended at different heights on a wire fence. Those just above ground level captured 87% of all flies, traps set at one meter captured 11% while only 2% of the flies were captured in traps set two meters above ground. When traps were deployed on a vertical support wall, the mean catch per trap was much higher than for traps suspended on the fence. Traps suspended just above ground level captured 57% of all flies, traps set at one meter above ground captured 27% of the flies and even traps set at two meters captured 16% of the flies. Although, most flies were still captured close to the ground, a higher percentage reached the second and third rows of traps. Sticky traps on a vertical wall produced similar results with significantly more flies alighting on the lower sections of the trap closest to the ground. On a vertical sand fly-proof net the overall dispersal of the flies was more like on a wall than in open space. Traps suspended just above ground level captured 49%, traps set at one meter above ground captured 36% and traps set at two meters captured 15% of the flies. Following spraying of the net with deltamethrin (1%), fewer sand flies were captured but the reduction was not statistically significant. Our conclusions are that being small and frail, sand flies tend to fly close to the ground probably in order to avoid being swept away by gusts of wind. When they encounter a vertical obstacle, they proceed upwards close to the obstacle with intermittent stops. Therefore, insecticide-sprayed walls or vertical nets should be effective for controlling sand flies approaching human habitation.

Control of phlebotomine sand flies with vertical fine-mesh nets.

Insecticide-treated vertical net barriers were used to intercept foraging sand flies. Two different nets were draped on fenced enclosures (10 by 10 m; 2 m high) in the central Jordan Valley. One enclosure was draped with a deltamethrin-impregnated net (PermaNet, 225 holes/in2). The holes of this net are sufficiently large to allow sand flies to pass through but not without coming in close contact with the mesh. The other enclosure was covered with SpiderNet+ (1,240 holes/in2) and sprayed with beta-cyfluthrin. Sand flies were captured inside and outside the enclosures before and after draping with the nets using CO2-baited CDC traps or CDC light traps. Both barrier types exhibited > 90% efficacy in blocking sand flies from entering the enclosures (P < 0.01). The Spider-Net+ exhibited high efficiency even before being sprayed with insecticide because the small mesh size physically prevented flies from passing through. In Ma'ale Adumim, a 60-m-long, 2-m-high PermaNet barrier was erected to intercept sand flies approaching houses from their natural habitats. Sand flies were monitored on all sides of the barrier using CO2-baited CDC traps or CDC light traps. Results showed a 60% reduction in the mean number of sand flies trapped behind the net compared with the untreated areas adjacent to it (P < 0.05). Integrated vector control campaigns for reducing the burden of sand fly bites should consider vertical fine-mesh nets to reduce the numbers of sand flies arriving at inhabited areas.

Control of phlebotomine sandflies in confined spaces using diffusible repellents and insecticides.

The control of phlebotomine sandflies (Diptera: Psychodidae), the vectors of leishmaniasis, is directed mostly against adults as larvae develop in unknown or inaccessible habitats. In the current study we tested geraniol, a natural plant-derived product, as a space repellent and the synthetic pyrethroid prallethrin as a diffusible insecticide. Geraniol was dispersed in the air using diffusers with an electric fan and prallethrin was evaporated using electrically heated evaporators. Both substances were tested in inhabited bedrooms and in tents. Geraniol failed to effect significant reductions in the numbers of either Phlebotomus papatasi Scopoli in rooms or Phlebotomus sergenti Parrot in tents. In laboratory experiments, geraniol proved ineffective in preventing sandflies from feeding. By contrast, prallethrin was highly effective in reducing the number of sandflies in rooms as well as in tents. Exposure of sandflies to prallethrin in laboratory experiments caused 97% mortality rates. Both prallethrin and, to a lesser extent, geraniol reduced the number of Culex mosquitoes captured in tents. Electric liquid-vaporizers with 1.5% prallethrin are highly effective in protecting people from sandfly bites in confined spaces and may be useful in combating cutaneous leishmaniasis.

Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous leishmaniasis in northern Israel.

The bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied for three years (2001-2003) in the Galilee focus of cutaneous leishmaniasis in northern Israel, where the causative Leishmania tropica (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus (Adlerius) arabicus Theodor and Phlebotomus (Paraphlebotomus) sergenti Parrot, comprising 22% and 8%, respectively, of the local sandfly fauna sampled by light traps. The predominant species overall was Phlebotomus (Larroussius) tobbi Adler & Theodor (51%) with lesser numbers of Phlebotomus (Adlerius) simici Theodor (11%), Phlebotomus (Larroussius) syriacus Adler & Theodor (5%), Phlebotomus (Larroussius) perfiliewi Perfil'ev (3%) and Phlebotomus (Phlebotomus) papatasi Scopoli (0.05%). Sandfly adult populations were prevalent from April to November and peaked between June and August, being more abundant through the summer in irrigated habitats, such as gardens and orchards, than in open grassland. Of the two cutaneous leishmaniasis vectors, P. sergenti preferred boulder mounds located at the outskirts of settlements, whereas P. arabicus was more abundant overall and near houses in particular. Females of all these sandfly species displayed a peak of activity after sunset (20.00-22.00 hours), whereas activity of males persisted longer through the night. Another slight increase in activity was noted before dawn (02.00-04.00 hours). Phlebotomus arabicus appears to be the main vector of L. tropica in the Galilee focus, due to its denser populations, more endophily and preference for peridomestic habitats than shown by P. sergenti in northern Israel.

Seasonal abundance patterns of the sandfly Phlebotomus papatasi in climatically distinct foci of cutaneous leishmaniasis in Israeli deserts.

Among foci of cutaneous leishmaniasis in Israel, population densities of the vector sandfly Phlebotomus papatasi Scopoli (Diptera: Psychodidae) were assessed during April-October 1999 in the mesic Negev desert and the hyper-xeric Arava valley, using sticky traps placed overnight near host burrows of the fat sand rat, Psammomys obesus Cretzschmar (Cricetidae: Gerbillinae). Population dynamics of Ph. papatasi differed between the Negev (study sites on sand near Mount Keren and on loess at Nizzana ruins) and the Arava valley (study sites on sand at Shezaf and in a fallow field near irrigation at wadi Arava). At the Negev sites, sandfly abundance peaked in spring (April or May), whereas at Arava sites Ph. papatasi population densities were bi-modal, with peaks in both spring and autumn (September or October). This might be conducive to sustaining enzootic Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae). In both areas, Ph. papatasi densities were much higher at the site with moister soil, raising transmission risks of zoonotic cutaneous leishmaniasis.

The ecology of cutaneous leishmaniasis in Nizzana, Israel: infection patterns in the reservoir host, and epidemiological implications.

We conducted an extensive interdisciplinary study in an emerging focus of cutaneous leishmaniasis in the Western Negev Desert of Israel between July 1998 and February 2000. The aims of the this study were to determine (1) the reservoir hosts, (2) the distribution of the pathogen within the host range, (3) the associations of host, vector, and pathogen within defined habitats, (4) the demographic distribution of the pathogen within the host populations, and (5) to apply the newly acquired epizootiological data to explain morbidity patterns in humans. Fourteen square (60 m width) sampling plots were delimited in three types of habitats each with a different kind of substrate: loess, sand, and sand-loess ecotone. Rodents and sand flies were trapped and several environmental variables were measured. Leishmania infections in rodents were detected microscopically in stained smears of ear tissue and by a Leishmania-specific polymerase chain reaction. Results indicate that, contrary to previous reports, Psammomys obesus and not Meriones crassus is the main reservoir host in the region. Additional rodents (12 Gerbillus dasyurus and two M. crassus) were also found positive for Leishmania DNA. Prevalence of Leishmania infections amongst P. obesus was highest in loess habitats (65%), intermediate in the sandy-loess ecotone (20%), and 0% in the sandy habitats. Psammomys obesus individuals in the loess habitat of the Nizzana ruins were larger, on average (probably older), than those in the sandy habitat of the Mt. Keren junction. Sand fly density was positively correlated to soil moisture being higher in the relatively humid plots of Nizzana ruins and much lower in the drier sandy soil of Mt. Keren. Elucidation of fundamental ecological factors affecting this disease has helped explain an apparent discrepancy between the distribution of the disease in the zoonotic system and among humans.

Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages.

Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins.

Effect of sand fly saliva on Leishmania uptake by murine macrophages.

Leishmania promastigotes are introduced into the skin by blood-sucking phlebotomine sand flies. In the vertebrate host, promastigotes invade macrophages, transform into amastigotes and multiply intracellularly. Sand fly saliva was shown to enhance the development of cutaneous leishmaniasis lesions by inhibiting some immune functions of the host macrophages. This study demonstrates that sand fly saliva promotes parasite survival and proliferation. First, macrophages gravitated towards increasing concentrations of sand fly saliva in vitro. Secondly, saliva increased the percentage of macrophages that became infected with Leishmania promastigotes and exacerbated the parasite load in these cells. Thus, during natural transmission, saliva probably reduces the exposure of promastigotes to the immune system by attracting macrophages to the parasite inoculation site and by accelerating the entry of promastigotes into macrophages. Saliva may also enhance lesion development by shortening the generation time of dividing intracellular amastigotes.

Adenosine, AMP, and protein phosphatase activity in sandfly saliva.

As they probe the skin for blood, sand flies inject saliva that prevents hemostasis. Sand fly saliva also promotes leishmaniasis by suppressing immunologic functions of macrophages. Saliva of Phlebotomus papatasi, the vector of Old World cutaneous leishmaniasis, contains adenosine and AMP. We show that Ph. papatasi saliva as well as pure adenosine down-regulate the expression of the inducible nitric oxide (NO) synthase gene in activated macrophages. In addition Ph. papatasi, but not Lutzomyia longipalpis, saliva inhibits the production of NO. Taken together, these data suggest that salivary adenosine is responsible for the down-regulation of NO synthesis. Saliva of both genera Phlebotomus and Lutzomyia contains significant levels of endogenous protein phosphatase-1/2A-like activity that is heat labile, inhibitable by okadaic acid and calyculine a, and does not require divalent cations.

Interaction of Plasmodium gallinaceum ookinetes and oocysts with extracellular matrix proteins.

Plasmodium ookinetes are elongate, motile and invasive while inside the mosquito gut but promptly metamorphose into spherical immobile oocysts upon coming in contact with the basement membrane surrounding the midgut. There they begin a prolonged growth period characterized by massive DNA synthesis for the production of sporozoites. Living Plasmodium gallinaceum ookinetes attached avidly to the murine extracellular matrix proteins, laminin and collagen type IV. In ELISA-type assays, the main ookinete surface protein, Pgs28 was implicated as a mediator of parasite attachment to these basement membrane constituents. Laminin and collagen IV adhered to ookinete and oocyst lysates spotted onto nitrocellulose membranes. Receptor-ligand blot assays demonstrated that Pgs28 and an oocyst-specific antigen recognized by the mAb 10D6 interact with murine collagen IV and laminin. 10D6 antigen was also recognized by monospecific antiserum against the human epidermal growth factor receptor. Mosquito-derived laminin was incorporated into oocyst capsules of P. gallinaceum growing in Aedes aegypti. We hypothesize that contact with the mosquito basement membrane triggers the transformation of ookinetes into oocysts. Coalescence of basement membrane proteins onto the capsules masks developing oocysts from the mosquito's immune system and facilitates their prolonged extracellular development in the mosquito body cavity.

Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex.

Maxadilan is an approximately 7kDa peptide that occurs in the saliva of the sand fly Lutzomyia longipalpis. This peptide is a potent vasodilator and may also have immunomodulatory effects related to the pathogenesis of leishmanial infections. Variation in the primary DNA and inferred amino acid sequence of maxadilan is reported. Differences were found within and among natural field populations as well as among sibling species. Extensive amino acid sequence differentiation, up to 23%, was observed among maxadilan from different populations. This is a remarkable degree of polymorphism considering the small size of this peptide. The vasodilatory activity of maxadilan was equivalent among recombinant maxadilan variants. All maxadilan variants induce interleukin-6. Predicted secondary structure and hydrophobicity plots suggest that these characteristics are conserved among variant peptides. However, profiles based on the antigenic index do differ among peptides.

Salivary glands of the sand fly Phlebotomus papatasi contain pharmacologically active amounts of adenosine and 5'-AMP.

Salivary gland homogenates of the sand fly Phlebotomus papatasi contain large amounts of adenosine and 5'-AMP, of the order of 1 nmol per pair of glands, as demonstrated by liquid chromatography, ultraviolet spectrometry, mass spectrometry and bioassays. These purines, 75-80 % of which are secreted from the glands following a blood meal, have vasodilatory and anti-platelet activities and probably help the fly to obtain a blood meal. Salivary 5'-AMP is also responsible for the previously reported protein phosphatase inhibitor in the salivary glands of P. papatasi, which is shown to be artifactual in nature as a result of allosteric modification by AMP of the phosphatase substrate used (phosphorylase a).

Genetic variation among natural and laboratory colony populations of Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera: Psychodidae) from Colombia.

Genetic diversity among three field populations of Lutzomyia longipalpis in Colombia was studied using isozyme analysis. Study sites were as much as 598 km apart and included populations separated by the eastern Cordillera of the Andes. Genetic variability among populations, estimated by heterozygosity, was within values typical for insects in general (8.1%). Heterozygosity for field populations were compared with a laboratory colony from Colombia (Melgar colony) and were only slightly lower. These results suggest that establishment and long term maintenance of the Melgar colony has had little effect on the level of isozyme variability it carries. Genetic divergences between populations was evaluated using estimates of genetic distance. Genetic divergence among the three field populations was low (D = 0.021), suggesting they represent local populations within a single species. Genetic distance between field populations and the Melgar colony was also low (D = 0.016), suggesting that this colony population does not depart significantly from natural populations. Finally, comparisons were made between Colombian populations and colonies from Brazil and Costa Rica. Genetic distance values were high between Colombian and both Brazil and Costa Rica colony populations (D = 0.199 and 0.098 respectively) providing additional support for our earlier report that populations from the three countries represent distinct species.

Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages.

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.

Saliva of Lutzomyia longipalpis sibling species differs in its composition and capacity to enhance leishmaniasis.

Leishmania donovani chagasi parasites, transmitted by sandflies of the Lutzomyia longipalpis species complex, normally cause visceral leishmaniasis. However, in Central America infections frequently result in cutaneous disease. We undertook experiments to investigate the possible influence of sandfly saliva on the course of infection. Erythemas caused by feeding sandflies correlated well with the levels of the erythema-inducing peptide, maxadilan, in their saliva. Saliva of Brazilian flies was the most potent, that of Colombian flies less so, and Costa Rican saliva had very little maxadilan and lacked activity. Nucleotide sequence differences in the maxadilan gene of the three species were detected by 'single strand conformational polymorphism' electrophoresis. Leishmania infections proliferated fastest when coinjected with the saliva of Costa Rican flies. Brazilian flies had less influence, and Colombian flies only a slight effect. Thus Costa Rican Lutzomyia longipalpis, vectors of non-ulcerative cutaneous disease, have very low vasodilatory activity and very little maxadilan, but their saliva strongly enhances cutaneous proliferation of Leishmania infections. Conversely, flies from Colombia and Brazil, vectors of visceral disease, have more maxadilan, but exacerbate cutaneous infections to a lesser degree. These coincidental observations suggest that species of Lutzomyia longipalpis differ in their propensity to modulate the pathology of the disease they transmit.

Lutzomyia longipalpis is a species complex: genetic divergence and interspecific hybrid sterility among three populations.

The sand fly Lutzomyia longipalpis is the vector of Leishmania donovani chagasi in Latin America. An analysis of genetic variability at 27 enzyme coding loci among three laboratory populations of Lu. longipalpis revealed substantial genetic polymorphism. Levels of genetic distance between all pairwise comparisons of colonies were very high, and consistent with those previously reported among separate species in the genus Lutzomyia. Between 7% and 22% of the loci studied were diagnostic for any two of the colony populations. Experimental hybridization between colonies resulted in the production of sexually sterile male progeny. Our results provide strong evidence that Lu. longipalpis exists in nature as a complex of at least three distinct species. The possible effects of colonization on the genetic makeup of laboratory populations is considered in extending our results to natural populations.