DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major

Epidemiological field studies on leishmaniasis have been hampered by the laborious, and often inefficient, methods used to assess the rates of infection of sandfly vectors (Diptera; Phlebotominae) by species of the causative disease organisms, protozoal parasites of the genus Leishmania (Kinetoplastida; Trypanosomatidae). We report the rapid and accurate identification of both sandfly vector (Phlebotomus (Phlebotomus) papatasi (Scopoli] and infecting Leishmania major Yakimov & Schokov by DNA hybridizations to squash-blotted sandflies. Large numbers of whole (infected) sandflies can be quickly squashed on to nylon hybridization filters and (following standard procedures) the filter-bound DNA can be hybridized sequentially to cloned, multicopy genomic sequences that are specific for species of Leishmania (kinetoplast DNA) or for the sandfly (ribosomal (r) DNA). Our sandfly probe consists of a 3.2 kb fragment of the intergenic 'non-transcribed' spacer of rDNA of P. papatasi that we have detected only in this species: it is present in all six geographically isolated populations tested (from Tunisia through to India) but cannot be detected in the morphologically similar P. (Phlebotomus) duboscqi Neveu-Lemaire, the vector of Leishmania major south of the Sahara; it also cannot be detected in Phlebotomus species of the subgenera Larroussius and Paraphlebotomus that together with P. papatasi are the dominant man-biting sandflies in north African foci of zoonotic cutaneous leishmaniasis, where (as in many arid regions of western Asia) P. papatasi is believed to be the sole vector of L. major.     

Bionomics of Phlebotomus papatasi (Diptera: Psychodidae) in an endemic focus of zoonotic cutaneous leishmaniasis in central Iran.

Following an epidemiological survey of zoonotic cutaneous leishmaniasis (ZCL) in several villages of Badrood, a rural district north of the city of Natanz, central Iran, Phlebotomus (Phlebotomus) papatasi Scopoli were found to be naturally infected with Leishmania (Leishmania) major zymodeme MON-26. Sand flies were collected and dissected biweekly from rodent burrows from May to October 2001. Leptomonad infection rates varied between 6.7% and 22.0%, being greatest in September, coinciding with peak activity of P. papatasi, two-three months before the highest incidence of ZCL human cases in November-December. The leptomonad infection rate was 1.1% of the 94 P. papatasi captured indoors. In ELISA testing of 520 P. papatasi blood meals during Sept. 2001 and Aug. 2002, the proportion giving positive reactions for human, sheep, cow, goat, rodent, and bird were 31.2%, 69.6%, 63%, 38.8%, 24.7%, and 21.8%, respectively. This report thus incriminates P. papatasi as the vector of zoonotic cutaneous leishmaniasis in this part of Iran.     

Incrimination of Phiebotomus papatasi as vector of Leishmania major in the southern Jordan Valley

he status of sandflies as vectors of cutaneous leishmaniasis in the southern Jordan Valley was investigated during 1992. Sandflies were collected from domestic habitats and from burrows of Psammomys obesus . Of 686 Phlebotomus papatasi females collected from burrows, fourteen harboured promastigotes in their guts. On the other hand, none of 1446 P.papatasi females collected from domestic habitats were found infected. The highest infection rate (5.5%) was recorded in November at the end of the sandfly season. Six leishmanial stocks isolated from P.papatasi females were typed by cellulose acetate electrophoresis using the six enzymes G6PDH, 6PGDH, PGI, PGM, FK and ME. Five of the leishmanial stocks were identical to a Leishmania major reference strain (MHOMISU/73/5-ASKH). The sixth isolate was a 6PGDH variant of L.major . These findings present the first direct evidence of the role of P.papatasi as a vector of L.major in Jordan.     

Chitinase secreted by Leishmania functions in the sandfly vector.

Leishmania major parasites ingested with host blood by the sandfly Phlebotomus papatasi multiply confined within the peritrophic membrane. This membrane consists of a chitin framework and a protein carbohydrate matrix and it is secreted around the food by the insect midgut. Histological sections of infected flies show lysis of the chitin layer in the anterior region of the peritrophic membrane that permits the essential forward migration of a concentrated mass of parasites. Both the location and the nature of this disintegration are specific to infected flies. At a later stage the parasites concentrate in the cardiac valve region and subsequently this segment of the fore gut loses its cuticular lining. We have found that chitinase and N-acetylglucosaminidase are secreted by cultured L. major promastigotes, but not by sandfly guts. Hence lysis of the chitin layer of the peritrophic membrane could be catalysed by these enzymes of the parasites. Activity of both enzymes was also observed in other trypanosomatids, including L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculata and Trypanosoma lewisi.     

Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.     

Bloodmeal digestion and Leishmania major infections in Phlebotomus duboscqi: effect of carbohydrates inhibiting midgut lectin activity

The carbohydrates galactosamine and heparin, previously shown to inhibit phlebotomine lectin activity in vitro, were fed to the sandfly Phlebotomus duboscqi Neveu-Lemaire (Diptera: Psychodidae) with blood, and the effects on mortality, fecundity, protease activity and susceptibility to Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae) were studied. Previous study revealed that galactosamine considerably enhanced the establishment of L. major infection in P. duboscqi and significantly increased parasite loads in late infections. This work demonstrates a similar but less pronounced effect of heparin. Heparin increased infection rates and parasite loads 3 and 9 days post-feeding but did not affect the location of Leishmania promastigotes and their anterior migration. Galactosamine supplement caused pronounced changes in bloodmeal digestion. It abolished the activity of alkaline proteases and trypsin, caused premature defecation of bloodmeal, increased mortality of female sandflies in days 1-4 post-feeding and decreased their fecundity. Heparin had a less pronounced effect on sandfly physiology. It lowered trypsin activity 12 and 72 h post-bloodmeal but did not alter defecation, mortality and oviposition. The data suggest that the enhancing effect of these carbohydrates on Leishmania infections in sandfly midgut could be explained by their interference with midgut proteases. The study supports the hypothesis that proteolytic activities of midgut proteases strongly influence the vector competence of sandflies.     

Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission

Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of "metacyclic" promastigotes in the mouthparts of the vector is discussed.     

Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Development of sandfly forms of Leishmania major in sucrose solutions

Stages of Leishmania developing in the vector include different morphs that are exposed first to ingested blood and then to sugar meals. This study sought to determine whether stages occurring in the latter medium could be induced by culturing in sugar-based media. In sucrose solutions, L. major continued to divide and multiplied by 38-46%. Paramastigotes and aflagellates are forms present in late stages of Leishmania infection in Phlebotomus papatasi. They constituted 79% of the forms in sucrose medium, but a maximum of 15% in NNN. Rate and degree of transformation varied as a function of the stage of growth of the NNN starter culture. Motility was lost in sucrose media but was retained in a mixture of sucrose and Ringer's solution. In the latter mixture, the parasites exhibited transformation as well as attachment to the substrate and morphological changes of the flagellum similar to those occurring in the sandfly vector. Parasites from sucrose medium and from P. papatasi reacted similarly, whereas those from NNN reacted differently to a monoclonal antibody. It is suggested that transformation of L. major in sucrose media resembles this process in the vector.     

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.     

Sandfly midgut lectin: effect of galactosamine on Leishmania major infections

Galactosamine, which has been shown in vitro to specifically inhibit sandfly midgut lectin activity, was fed to Phlebotomus duboscqi females with blood containing promastigotes of Leishmania major . Non-inhibitory sugar, galactose, was added in controls. For two strains of L. major (LV 561 and Neal-P), galactosamine substantially enhanced the establishment of infection in the sandfly posterior midgut and significantly increased parasite loads after defaecation, but did not affect anterior migration of Leishmania . On day 3 post-infection, most infections in galactosamine-fed sandfly groups (92% of LV 561 and 100% of Neal-P) were found in the ectoperitrophic space of the posterior midgut, whereas most infections in the galactose-fed groups of sandflies (85% in LV 561 and 96% in Neal-P) were restricted to the peritrophic sac. On day 9, however, the proportion of infections colonizing the stomodeal valve was similar in both dietary groups of sandflies for both strains of L. major . The addition of galactosamine prevented the decrease of parasite loads which occurred in controls between days 3 and 6 post-infection. On days 6 and 9, heavy infections were observed almost exclusively in galactosamine-fed females. Differences between groups were more pronounced for the Neal-P strain, which normally developed poorly in sandflies. Morphology of L. major LV 561 was not affected by galactosamine supplement: the lengths of parasite body and flagellum were similar in both sandfly groups. Two hypotheses are considered for the role of sandfly midgut lectin in Leishmania development in the vector midgut. One proposes that sandfly lectin kills Leishmania promastigotes, the other assumes that lectin blocks LPG-mediated binding of promastigotes to sandfly midgut microvilli.     

Sandfly (Diptera, Psychodidae, Phlebotominae) fauna of South-Western Pakistan. 1. Diagnostic morphology of Phlebotomus papatasi (Scopoli), Ph. bergeroti (Parrot) and Ph. salehi (Mesghali)

A survey was conducted to study the morphology of the sandfly fauna in South-Western Pakistan (Balochistan). During the revision of different genera of sandflies the specimens of Phlebotomus papatasi (Scopoli) (N = 720), Ph. bergeroti Parrot (N = 30) and Ph. salehi Mesghali (N = 70) were encountered in various localities. These localities appear to be new records of the subgenus in the literature to date. Ph. bergeroti is reported for the first time from Pakistan and Ph. salehi from Balochistan as well. Characters of these three Pakistanese Phlebotomus are compared with the published data of these species from other countries. Keys for the identification of Pakistanese Phlebotomus are also constructed. Two female Ph. papatasi collected from indoors out of 132 female flies (1.5%) were found positive with flagellate infection in pharynx and midgut. The possible vectorial role of these flies is also discussed. Further surveys are necessary in parts of the country that have not been systematically surveyed.     

Leishmaniasis in the Jordan Valley. V. Dispersal characteristics of the sandfly Phlebotomus papatasi

Several characteristics of dispersing and non-dispersing Phlebotomus papatasi (Scopoli) were quantified and compared. The majority of dispersing sandflies, trapped crossing fallow fields, were females (68.5% v. 51.1%); of the dispersing females, 55.4% were parous, 48.1% were inseminated and 11.6% were gravid. In the population of sandflies sampled exiting from burrows of the sand rat Psammomys obesus Cretschmar, these categories, respectively, represented 39%, 90% and 26% of the females examined. Leishmania promastigotes were found in 9% of females exiting from P. obesus burrows, and in 2.7% of the dispersing females. The anthrone test established that the reason for activity of gravid females is sugar feeding. These females do not disperse and are suitable targets for future control measures.     

Further evidence that deltamethrin-impregnated collars protect domestic dogs from sandfly bites

In many foci of zoonotic visceral leishmaniasis (ZVL), domestic dogs are important reservoir hosts of the causative Leishmania parasites transmitted by phlebotomine sandflies (Diptera: Psychodidae). We tested the protective value of impregnated dog collars (20 g plastic containing deltamethrin 800 mg ai) against Phlebotomus papatasi (Scopoli) sandflies in Iran. For each assay, the dog was sedated and caged in a net with 70-100 wild-caught sandflies overnight (23.30-06.30 hours). Dogs wearing the collars were bitten by approximately 80% fewer sandflies than before collars were fitted, i.e. 51% vs. 11% of hungry female flies exposed. Sandfly mortality rates following 20 h exposure to dogs with collars (18%) or without collars (17%) were not significantly different. Effects of collars were tested when dogs had been wearing them for 8 days. A previous trial against the sandfly P. perniciosus Newstead in France, using smaller dogs, showed that effects of such collars were not fully realized until they had been worn for 2 weeks or more; they remained effective for at least 8 months and killed significant proportions of the sandflies exposed. Present results with P. papatasi, confirming that this simple device provides effective protection against sandflies, are considered sufficiently encouraging to justify a community-wide field trial of deltamethrin-impregnated dog collars against ZVL vector sandflies in Iran.     

Seasonal variation of Leishmania major infection rates in sandflies from rodent burrows in Isfahan province, Iran

Abstract. In preparation for field trials of killed Leishmania major vaccine, natural infections with Leishmania promastigotes were monitored in Phlebotomine sandfly vectors from villages of Borkhar rural district, northeast of Isfahan in central Iran, where Lmajor zymodeme MON-26 (=LON-l) has been identified as causing zoonotic cutaneous leishmaniasis (ZCL). Sandflies were collected and dissected weekly, from burrows of rodent colonies, during the 'sandfly season', June-October 1991. Leptomonad infection rates were 12% of 26 Phlebotomus ansarii , 8% of 280 P.caucasicus , 11 % of 1042 P.papatasi and none of 126 Sergentomyia sintoni , being greatest during late August through September, coinciding with peak activity of the sandflies, 2–3 months before the highest incidence of ZCL human cases in November-December.     

A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.     

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranh?o, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.     

Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.     

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.     

Entomopathogens of phlebotomine sand flies: laboratory experiments and natural infections.

The susceptibility of different geographical strains of Phlebotomus papatasi to a cytoplasmic polyhedrosis virus (CPV) was determined experimentally by feeding polyhedra to larvae. Of the Indian P. papatasi, 15.6% became infected, whereas Egyptian P. papatasi were mostly refractory. Infection rates were not augmented in colony flies from the Jordan Valley, 23.8% of which were naturally infected with CPV. The infectivity of Serratia marcescens and Beauvaria bassiana to P. papatasi were determined experimentally. A suspension of B. bassiana spores or S. marcescens bacteria, ingested by P. papatasi in sucrose solution, did not significantly augment mortality rates or reduce the number of eggs oviposited. However, B. bassiana spores smeared on a filter paper constituting 1 or 5% of the surface area available to flies induced 100% mortality of P. papatasi on days 5 and 4, respectively. Mortality in Lutzomyia longipalpis reached 100% on day 4. There were markedly lower mortality rates in the control groups and more eggs were produced by these females (P. papatasi: control = 48.5; experimental = 0.9-1.6 eggs/female; L. longipalpis; control = 17.1; experimental = 0 eggs/female). From wild-caught Colombian Lutzomyia spp., a nonfluorescent pseudomonas, an Entomophthorales fungus, and a Trypanosomatid protozoon (probably Leptomonas) were isolated in culture media. Gregarines (Ascogregarina saraviae) and nematodes (Tylenchida and Spirurida) were also recorded. In laboratory-reared flies, an ectoparasitic fungus was associated with high mortality rates of first instar Lutzomyia spp. larvae. Opportunistic ectoparasitic aggregates of bacteria, yeast, and fungi on the tarsi of colonized L. longipalpis and P. papatasi hindered their mobility and were associated with reduced colony vigor. Aspergillus flavus, B. bassiana, and S. marcescens were isolated from laboratory-bred P. papatasi adults.