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.

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host.

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.

Sporogonic development of a malaria parasite in vitro.

The sporogonic cycle of the avian malaria parasite Plasmodium gallinaceum was completed in vitro. Ookinetes (motile zygotes) were seeded onto a murine basement membrane-like gel (Matrigel) in coculture with Drosophila melanogaster cells (Schneider's L2). Transformation into oocysts as well as subsequent growth and differentiation were observed in parasites attached to Matrigel and depended on the presence of L2 cells. Sporozoites were first observed on day 10 in culture. Specific circumsporozoite protein antigenicity was identified in mature oocysts and in sporozoites. It is now possible to follow the entire life cycle of Plasmodium in vitro.

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.

Transmission and scanning EM-immunogold labeling of Leishmania major lipophosphoglycan in the sandfly Phlebotomus papatasi.

Previous studies using immunostaining and light microscopy demonstrated expression of Leishmania major lipophosphoglycan (LPG) on parasites developing in the sandfly gut from 2 days post infection. By days 4 to 7 post infection, there appeared to be large amounts of parasite-free LPG deposited on/in the microvilli and epithelial cells lining the thoracic midgut, while forward migration of parasites and the morphological changes which accompany metacyclogenesis were associated with developmental modification of the LPG molecules. Studies presented here examine this process with much greater precision using electron microscopy and immunogold labeling techniques to study the different developmental forms (nectomonads, haptomonads, paramastigotes, and metacyclics) of promastigotes in the sandfly gut. Results obtained using LPG-specific monoclonal antibodies (WIC79.3, 45D3 and the metacyclic-specific 3F12) show (1) gold labeling over the cell surface, within the flagellar pocket, and extending along the entire length of the flagellum of electron-dense nectomonads observed in the abdominal and thoracic midgut regions on days 4 and 7 post infection, and of electron-lucid haptomonads in the foregut, (2) dense labeling around the flagellar tips, by which nectomonad forms bind to the midgut microvilli, but not on the microvilli themselves or within the epithelial cells lining the midgut, (3) significant metacyclic-specific (3F12) labeling on nectomonad forms in the lumen of the midgut and attached to the microvilli, and (4) dense labeling on the cell surface of electron-lucid paramastigotes in the esophagus and in the filamentous matrix surrounding paramastigote and metacyclic forms in the esophagus and pharynx. These results are discussed in the light of the proposed roles for LPG in parasite attachment to, and survival in, the sandfly gut.

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.

Host-parasite relationships of Ascogregarina chagasi (Eugregarinorida, Aseptatorina, Lecudinidae) in Lutzomyia longipalpis (Diptera: Psychodidae).

The life cycle of Ascogregarina chagasi in larvae and in adult female Lutzomyia longipalpis was studied by light and electron microscopy. Sporozoites and young gamonts were attached to the epithelial lining of the larval midgut via an osmiophilic contact zone. The mucron of young gamonts was bordered by an invaginated pellicular fold, and an electron-opaque vesicular structure was observed adjacent to it. Sporozoites possessed an apical complex and were bound by a double membrane with underlying subpellicular microtubules. The gamont pellicle was uniformly corrugated and consisted of two cortical membranes and a plasma membrane. Mature gamonts and gametocysts were found in the posterior ectoperitrophic space of third and fourth instar larval midguts and in the haemocoel of adult flies. Gametocysts in adult females adhered to the genital accessory glands, where they were encased in electron-dense capsules secreted by the fly through haemocyte-mediated humoral immune reactions. Oocytes were spindle-shaped and bound by a double-layered wall with a discernible polar plug at each end. Sporulation was endogenous, occurring within gametocysts in the midguts of larvae or the accessory glands of adult females. FITC-phalloidine staining of all stages of A. chagasi except mature gametocysts produced bright fluorescence, indicating the presence of a diffuse, actin-like protein in the cytoplasm.

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.

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.

The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major.

To test the effects of post-bloodmeal nutrition of sand flies on the transmission of Leishmania major, groups of infected P. papatasi females maintained on diets of sucrose, trehalose, albumin or a mixture of sucrose and albumin, were subjected to forced feeding with capillaries. Transmission was evaluated by counting the parasites egested; numbers ranged from 0 to over 1,000 promastigotes. Infections of the anterior midgut were seen in the majority of flies from all the experimental groups but the percentage of transmitting females was significantly higher in the group maintained on a mixture of sucrose and albumin. There were no attached parasites in the pharynx and cibarium of the flies and the presence of free promastigotes in these parts was not itself indicative of infectivity. However, transmission was positively correlated with apparent inability to engorge. The parasites egested were typical infective form promastigotes and identical to those observed in the esophagus and the anterior thoracic midgut. A mechanism by which infective stage promastigotes from the esophagus and the stomodeal valve may be transmitted by bite is proposed.

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.

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.

Leishmaniasis in the Jordan Valley II. Sandflies and transmission in the central endemic area.

Cutaneous leishmaniasis in the Jordan Valley is maintained within the close association of the rodent Psammomys obesus and sandfly Phlebotomus papatasi, which appear to be the exclusive host and vector species. The incidence of the disease was similar to the distribution of Psammomys colonies in the region, i.e., the plains of light stoneless soil. An infection rate of 93% was recorded in the very common P. obesus. Other potential host species, except for Mus musculus, were scarce and no infection with Leishmania was found in them. The only Phlebotomus species caught in significant numbers was Ph. papatasi and this was also the only species harbouring leishmanial parasites, up to 56% in one sample. All Leishmania isolates from Psammomys and from Ph. papatasi were identical to those from local human cases. The density of Ph. papatasi populations in uncultivated areas was correlated with soil conditions favouring high humidity in Psammomys burrows. A very low rate of engorged females among the Sergentomyia species collected suggests that the common species, S. antennata and S. africana asiatica, are highly autogenous.

Leishmaniasis in Israel: reservoir hosts, sandfly vectors and leishmanial strains in the Negev, Central Arava and along the Dead Sea.

The reservoir animals, sandfly vectors and strains of Leishmania from foci in the southern region of Israel were studied. The rodent host species are: Psammomys obesus, Meriones crassus and probably Nesokia indica. The vector species are Phlebotomus papatasi, which were caught at all collecting sites and Ph. sergenti, which were collected in the area of the Dead Sea and in the Central Arava. Strains of Leishmania major isolated from rodents, vectors and man were serologically and enzymologically identical with regard to their excreted factor (EF) serotypes, their malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI) and glucose-6-phosphate dehydrogenase (G6PDH) enzyme variant types, but exhibited three variant subtypes of 6-phosphogluconate dehydrogenase (6PGDH). The distribution of the 6PGDH subtypes correlates with three different geographical locations. Scarcity of water is the main factor limiting the biotopes of the sandflies and the spread of leishmaniasis. The subjects discussed are the dependence of sandfly distribution on rodent-burrow depth in arid areas and the inter-relationship between the leishmanial subtypes, vectors and hosts.

Vector compatibility of Phlebotomus papatasi dependent on differentially induced digestion.

Infection with Leishmania tropica, a strain specific to the sandfly Phlebotomus papatasi, was inhibited in sandflies fed on turkey blood. Reduction of the parasite number was correlated with the digestive process. A relatively high DNAase level was induced in the gut of the sandfly by the nucleated turkey erythrocytes. This is the first record of vector-pathogen incompatibility, thus induced, and of differentially triggered digestive processes.

Cytoplasmic polyhedrosis viruses in Phlebotomus papatasi inhibit development of Leishmania major.

Cytoplasmic polyhedrosis viruses (CPV's) were observed in wild-caught and laboratory-reared Phlebotomus papatasi. Chronic CPV pathology of the midgut, characterized by structural aberrations in the epithelium and the peritrophic membrane, interfered with blood digestion and rendered the sand flies refractory to Leishmania major infections. Rates of natural and artificial L. major infections were inversely correlated to the incidence of CPV infections. The interaction between viruses and protozoan parasites in an insect host is of basic biological interest and in this case may be of significance in the epidemiology of cutaneous leishmaniasis.

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.