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.     

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.     

Trypsin and chymotrypsin-Iike enzymes of the sandfly Phlebotomus papatasi infected with Leishmania and their possible role in vector competence

Phlebotomus papatasi (Scopoli) is susceptible to infection with Leishmania major Yakimov & Schokov and resistant to L. donovani Laveran & Mesnil. The possibility that susceptibility depends on midgut levels of trypsin and chymotrypsin-like (esterolytic) enzymes was investigated. Infection with L. major reduced the trypsin-like activity to 93.5% and 86% of the control value at 20 and 30 h post feeding and increased it to 106% at 52 h. Infection with L. donovani reduced trypsin-like activity to 64% and 73% of the control value at 30 and 52 h post feeding. The overall amount of trypsin and chymotrypsin-like enzymes in L. major infections was reduced to 50% and 34% of the control value at 20 and 30 h post feeding and increased to 184% at 52 h. Only one of the enzymes separated by gel electrophoresis was lower throughout, i.e. peak D. Overall, the midgut enzyme level with L. donovani infection was 86% of the control value at 30 h post feeding and 105% at 52 h; their relative amounts changed throughout. Soybean trypsin inhibitor enabled L. donovani to survive and multiply in P. papatasi. It is suggested that a specific component of the trypsin-like activity prevents the survival of L. donovani in P. papatasi and that modulation of this factor enables L. major to survive.     

The efficacy of indoor CDC light traps for collecting the sandfly Phlebotomus argentipes, vector of Leishmania donovani

The efficacy of light traps for collecting sandflies (Diptera: Psychodidae) varies both inter-specifically and intra-specifically (by gender and physiological status) as a result of significant differences in phototropic and other behavioural characteristics. The efficacy of miniature CDC light traps for collecting Phlebotomus argentipes Annandale & Brunetti, a vector of Leishmania donovani Laveran & Mesnil (Kinetoplastida: Trypanosomatidae), was assessed in the Indian state of Bihar. Sandflies were collected during the night from 16 houses in each of three villages over 3 months (four times at fortnightly intervals) using CDC light traps indoors, and by aspirator collection (carried out by one person for 30 min/house) from the walls of the same houses the following morning. Incidence rate ratios (IRRs) between CDC light trap collections and aspirator collections were obtained through a negative binomial regression with household as random effect. CDC light traps were especially effective in catching males (IRR 3.08, 95% confidence interval [CI] 2.12-4.46) and unfed females (IRR 3.50, 95% CI 2.37-5.16) of P. argentipes, and to a lesser extent gravids (IRR 1.77, 95% CI 1.07-2.93). However, only a relatively small proportion of all blood-fed P. argentipes were collected by light trap (IRR 0.45, 95% CI 0.28-0.73). Despite its limitations in collecting blood-fed female sandflies, the CDC light trap appears to trap a sufficient proportion of the indoor population of sandflies for sampling purposes, and as this light trap is also more convenient and more easily standardized than the aspirator method, we conclude that it is the most efficient method for monitoring P. argentipes populations in the Indian subcontinent.     

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.     

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.     

Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector

Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between patients in our study (245 m).     

Leishmania in phlebotomid sandflies. VII. On the taxonomic status of Leishmania peruviana, causative agent of Peruvian 'uta', as indicated by its development in the sandfly, Lutzomyia longipalpis.

The name Leishmania peruviana was given by Velez (1913) to the parasite responsible for a form of cutaneous leishmaniasis known as 'uta'; this disease occurs in the Peruvian Andes. Clinical similarities between uta and 'oriental sore', which is caused by Leishmania tropica of the Eastern Hemisphere, have, however, led to the suggestion that uta is simply due to L. tropica, which was introduced into Latin America by African slaves or European immigrants. Leishmania species are divisible into three distinct sections, according to their pattern of development in their natural (phlebotomine) vectors. One of these sections, the PERIPYLARIA, contains the subspecies of Leishmania braziliensis, and is characterized by parasites that undergo a phase of development attached to the wall of the hindgut (pylorus and ileum), in addition to multiplication in the midgut and subsequent invasion of the foregut. Such development is unknown in any other group of leishmaniae, including those groups of the section SUPRAPYLARIA, which includes parasites of the L. tropica complex. Three isolates of L. peruviana were studied in laboratory-bred sandflies, Lutzomyia longipalpis (Lutz & Neiva), and all showed consistent and prolific development of rounded or stumpy flagellates attached to the wall of the hindgut and, in some instances, growth of free, elongate promastigotes throughout the midgut. Development of both L. tropica and L. major, in the same insect, was restricted to massive development of free flagellates in the midgut, up to the cardial valve. From the behaviour of L. peruviana in the sandfly, its slow growth in hamster skin and the small size of its amastigotes, it is concluded that this parasite is (a) distinctly different from both L. tropica and L. major, and (b) closely related to subspecies of L. braziliensis within the section PERIPYLARIA. On this evidence it is also concluded that L. peruviana is indigenous to the American continent. The specific name is best retained for the time being (rather than L. braziliensis peruviana).     

Potential distributions of phlebotomine sandfly vectors of human visceral leishmaniasis caused by Leishmania infantum in Morocco

Zoonotic visceral leishmaniasis is a common vector-borne systemic disease caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). In Morocco the situation is complex: many sandfly species have been collected in areas in which the disease is endemic, but only Phlebotomus ariasi, Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) have been confirmed to have vectorial roles. The objective of the present study was to ascertain the potential distribution of L. infantum and its vectors in Morocco, using ecological niche modelling. Vector records were obtained from field collections of the Laboratory team and from previously published entomological observations. Epidemiological data for L. infantum modelling were obtained from Moroccan Ministry of Health reports. The jackknife test indicated that the bioclimatic variables with the greatest influence on model development for all species were annual precipitation and precipitation in the driest quarter of the year. MaxEnt model representations for sandfly species that act as vectors of L. infantum showed the widespread geographic distribution of these species in Morocco, specifically in northern and central Morocco, where foci of visceral leishmaniasis are found. The ecological niche modelling points out areas in which the probability of occurrence of these species is higher. This information should be considered as a starting point for further research to fully elucidate the ecology and epidemiology of these species, as well as of the pathogens they transmit.     

Phosphoglycosylation of a secreted acid phosphatase from Leishmania donovani.

The secreted acid phosphatase (SAcP) of L.donovani is a heterogeneous glycoprotein that displays a wide array of N- and O-linked glycosylations. The O-linked sugars are of particular interest due to their similarity to the phosphoglycan structures of the major lipophosphoglycan surface antigen and released phosphoglycan (Turco et al., 1987; Greis et al., 1992). This study describes a structural analysis of the SAcP O-linked glycosylations using mass spectroscopy, amino acid sequencing, and enzymatic carbohydrate sequencing. Analysis of glycan chain lengths and peptide glycosylation site distribution was performed, revealing that the average O-linked structure was approximately 32 repeat units in length. Amino acid sequence analysis of glycosylated peptides showed that phosphoglycosylations did not occur randomly but were localized to specific serine residues within an array of degenerate serine/threonine-rich repeat sequences localized in the C-terminus. No evidence was obtained for modification of threonine residues. The observed pattern suggested that a consensus sequence may exist for localization of phosphoglycan structures.     

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.     

Courtship and mating by the sandfly Phlebotomus duboscqi, a vector of zoonotic cutaneous leishmaniasis in the Afrotropical region

Courtship behaviour of males of the Afrotropical sandfly Phlebotomus duboscqi Neveu-Lemaire (Diptera: Psychodidae) involved mounting the female and clasping her 'waist' with the male coxites placed between the female's thorax and abdomen. This behaviour, which we call 'piggy-backing', was preceded by male wing beating, perhaps involving mate recognition and contact pheromones. It did not seem to be pre- or postcopulatory mate guarding. Piggy-backing was attempted by P. duboscqi males on females of other species (P. papatasi and P. perniciosus) and even on other male P. duboscqi. The majority of female P. duboscqi piggy-backed by males were already inseminated, and most of the courting did not lead to copulation. This, coupled with the presence of a mating plug (semen) in each spermatheca of inseminated females, suggests that female P. duboscqi are monogamous for at least the first gonotrophic cycle. Male courtship with piggy-backing was more intense when females could feed on a hamster than when a hamster was present but the females were denied access to the host. It is suggested that, when a hamster was available to the females, the conditions in the laboratory are similar to those in rodent holes, the natural habitat of P. duboscqi.     

Incrimination of four sandfly species previously unrecognized as vectors of Leishmania parasites in Mexico

Cutaneous leishmaniasis (CL) is endemic to the Yucatan Peninsula of Mexico. The main causative agent is the parasite Leishmania mexicana (Biagi) (Kinetoplastida: Trypanosomatidae) and, based on the classic work of Dr Biagi's research team, it has been generally accepted and frequently reported that the only vector of L. mexicana in the region is the sandfly Lutzomyia olmeca olmeca (Vargas & Diáz‐Nájera) (Diptera: Psychodidae). Evidence gathered from recent entomological studies conducted mainly in Calakmul, Campeche, however, suggests that other species may also be vectors of L. mexicana. We conducted a field study in two villages in Calakmul, Campeche in the Yucatan Peninsula, where recent cases of CL have been reported, to document the species composition and relative abundances of the sandfly fauna and to identify which species are likely to be the main vectors by assessing the biting rates and parasite infection rates of the suspected vector species. Sandfly catches were conducted from November 2005 to February 2006 in Unión 20 de Junio and Dos Lagunas Sur. Sandflies were captured using Shannon (18.00–22.00 hours), Disney and CDC light traps (18.00–06.00 hours). Biting and infection rates were calculated for the four most abundant species: Lutzomyia cruciata (Coquillett), Lu. o. olmeca, Lutzomyia panamensis (Shannon) and Lutzomyia shannoni (Dyar). In Dos Lagunas Sur, Lu. panamensis and Lu. o. olmeca exhibited the highest biting rates throughout the sampling period. In Unión 20 de Junio, Lu. cruciata and Lu. o. olmeca had the highest biting rates over the same period. Regarding infection rates, we report herein the establishment of a polymerase chain reaction protocol and validation of IR1 and LM17 oligonucleotides to analyse the infection rates of sandflies. Out of 769 females analysed, the overall infection rates were 1.4% in Dos Lagunas Sur and 5.3% in Unión 20 de Junio. In Dos Lagunas Sur we found L. mexicana infections in two sandfly species, Lu. shannoni and Lutzomyia ylephiletor (Fairchild & Hertig), whereas in Unión 20 de Junio we found infections in Lu. shannoni, Lu. cruciata, Lu. o. olmeca and Lu. panamensis. The possible role of these four sandfly species in relation to L. mexicana transmission in Calakmul is discussed.