Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia

From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize.     

Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis

We analyzed the development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. In addition, we compared sandfly infections initiated with axenic amastigotes or promastigotes. Our data showed no important difference between Lu. longipalpis infection rates resulting from either type of infections. Furthermore, development of infection was equivalent in both cases. All promastigote forms were found inside the sandfly and, after blood digestion, most of the population consisted of procyclics and nectomonads. A low percentage of metacyclic forms was coincident with a high number of nectomonads during late stages of infection, but which form gives rise to metacyclic forms in L. infantum chagasi is unknown. These results also show that the promastigote infection model, at least for this situation, is suitable for obtaining of infected sandflies because it is easier and less laborious.     

Biochemical characterization and zymodeme classification of Leishmania isolates from patients, vectors, and reservoir hosts in Kenya.

A total of 407 Leishmania and other Leishmania-like isolates obtained from patients, other vertebrates, sand fly vectors, and other arthropods from Kenya and other countries were characterized and compared with several World Health Organization and other well-characterized reference strains of Leishmania, Trypanosoma, Crithidia, Herpetomonas, and Leptomonas by cellulose acetate electrophoresis (CAE), using 20 enzyme systems. Analysis of the isoenzyme banding patterns (IBP) of the isolates generated isoenzyme profiles that were resolved as zymodemes and tabulated. Isolates that produced similar isoenzyme profiles in all 20 enzyme systems were placed into a particular Leishmania isoenzyme taxon, with the zymodeme designated numerically as Zn. A total of 66 zymodemes were recorded for the 407 isolates studied. To obviate the need to draw all 66 representative IBP for each of the 20 enzyme systems, the 66 zymodemes (Z1-Z66) were again placed into similarity groups represented by pattern number or Pn. This resulted in 23-50 IBP (Pn) per enzyme system. The highest number of IBP scored was for malate dehydrogenase (MDH) (P1-50) and the lowest score was for glucose-6-phosphate isomerase (GPI) (P1-23). From these different isoenzyme profiles or zymodemes, IBP of 14 (MDH, GPI, nucleoside hydrolase, phosphoglucomutase, malic enzyme, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, mannose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, glutamate oxaloacetate transferase/aspartate aminotransferase, glutathione reductase, superoxide dismutase, fumarase, and glyceraldehyde-3-phosphate dehydrogenase) of the 20 enzyme systems were selected for computer-calculated numerical taxonomy. Consistent individual isoenzyme bands with similar relative mobilities of the 14 enzyme systems were scored into groups (allelomorphs, allozymes, or electromorphs) and used in cluster analysis. For each pattern in every profile, the presence of a consistent band was entered as 1 and its absence as 0. A total of 419 allozyme characters (variables) were scored for the 14 enzyme systems. Lastly, all different zymodemes sharing a particular IBP (Pn) within an enzyme system were counted and the total number was shown as a zymodeme frequency (Zf). Final analysis of the CAE isoenzyme profiles and cluster-dendrograms resulted in the identification of several potentially new species and subspecies of Leishmania and other Leishmania-like isolates from patients, sand flies, and animal reservoir hosts collected from Kenya and other locations in Africa. Zymodeme analysis of the Kenyan visceral and cutaneous leishmaniasis isolates resulted in the identification of 11 subpopulations of the L. donovani species complex and six subpopulations of the L. tropica species complex endemic to different geographic areas of Kenya.     

Characterization of Kenyan Leishmania spp. and identification of Mastomys natalensis, Taterillus emini and Aethomys kaiseri as new hosts of Leishmania major

A survey to examine rodents for leishmanias was initiated in the Perkerra Settlement Scheme, Marigat, Baringo District, Kenya, 789 rodents of ten different species were trapped and examined. Leishmanial parasites were isolated from the spleen and liver of 17 animals of five different species: seven from Tatera robusta, five from Arvicanthis niloticus, two from Mastomys natalensis, two from Taterillus emini and one from Aethomys kaiseri. These were identified as L. major by enzyme electrophoresis, using 12 enzymes, and by excreted factor (EF) serotyping. The isolation of L. major from Mastomys natalensis, Taterillus emini and Aethomys kaiseri represent newly recorded hosts of L. major.     

Development of a molecular tool for the identification of Leishmania reservoir hosts by blood meal analysis in the insect vectors

The transmission of parasites of the genus Leishmania involves a large diversity of mammalian reservoir hosts. However, many of these are yet to be identified, mainly in isolated biotopes such as the Amazonian rain forest. Furthermore, the trophic preferences of insect vectors have major epidemiologic implications. In this study, we developed a molecular tool for the identification of blood meals of phlebotomine sand flies. This assay is based on specific amplification and sequencing of the blood meal-derived single copy prepronociceptin (PNOC) gene, which is used as a target in phylogenetic studies of mammals. Sand flies were identified simultaneously with the blood-meal identification, using molecular analysis of a ribosomal locus. After a systematic assessment of the sensitivity and specificity of polymerase chain reaction amplification of the PNOC gene using human fed sand flies, the assay was tested on wild-caught sand flies. This work has important implications for the discovery of new Leishmania reservoir hosts and for a better understanding of complex parasite life cycles.     

Induction by specific T lymphocytes of intracellular destruction of Leishmania major in infected murine macrophages

The following cell populations derived from lymph nodes of mice primed in vivo with living Leishmania major promastigotes were tested for their capacity to induce parasiticidal activity in L. major-infected macrophages: a L. major-primed lymph node cells, draining lymph node cells from mice primed by a subcutaneous injection of living L. major in Freund's Complete Adjuvant; b L. major-specific T blasts, i.e. blast T cells resulting from in vitro challenge of primed lymph node cells with L. major, c propagated L. major specific T blasts, i.e. blast T cells after propagation in vitro in antigen-free medium containing interleukin-2. Results indicate that cocultivation of these L. major specific lymphocyte populations with infected peritoneal exudate macrophages induced progressive destruction of intracellular L. major. This effect was antigen specific since similar populations obtained from mice primed either with ovalbumin or bovine serum albumin did not induce significant parasite killing. The various lymphocyte populations examined did not express cytolytic activity for syngeneic macrophages infected with L. major when tested in a short-term 51Cr release assay. These negative results could not be attributed to an inability of infected macrophages to be lysed by cytolytic lymphocytes since cytolytic T lymphocytes directed to H-2 alloantigens present on macrophages were perfectly capable of lysing these infected macrophages as revealed in a 4 h 51Cr release assay. Interestingly, infected macrophages from either BALB/c (H-2d), NZB (H-2d) or CBA (H-2k) mice were lysed by cytolytic T lymphocytes specific for their respective H-2 alloantigens as well as uninfected macrophages. These results suggest that H-2 expression on the surface of infected macrophages from either L. major susceptible or resistant mouse strains is sufficient to be detected by allogeneic cytolytic T lymphocytes.     

Leishmania major: antileishmanial activity of methylbenzethonium chloride

Methylbenzethonium chloride (MBCl) decreased the growth of Leishmania major promastigotes and amastigotes in vitro. This decrease occurred during 4 days of exposure to the drug at concentrations of 0.1 to 2.5 micrograms ml-1. MBCl at 2 micrograms ml-1 killed almost 100% of the free living promastigotes and 87% of amastigotes within 4 days of treatment. Electron microscopy studies showed marked swelling of mitochondria in treated parasites. A possible additional effect on the parasite surface membrane is discussed.     

利什曼原虫的传播机制及传媒蛉种的研究进展

该文综述了人体利什曼原虫的传播机制及传媒蛉种研究的现状和进展，并通过分析媒介白蛉与利什曼原虫的相互关系，认为利什曼原虫虫种的鉴定应在分析原虫DNA的基础上，结合该利什曼原虫与其他虫种在致病特征和流行病学上的差异综合考虑，这样才具有价值。     

Lectins and toxins in the plant diet of Phlebotomus papatasi (Diptera: Psychodidae) can kill Leishmania major promastigotes in the sandfly and in culture

Leishmania major promastigotes are agglutinated and die in their vector, Phlebotomus papatasi, after the sandflies feed on some plants that are found in their natural habitat. In in-vitro assays, extracts of Ricinus communis (Euphorbiaceae), Capparis spinosa (Capparaceae), Prosopis farcta (Mimosaceae) and Tamarix nilotica (Tamaricaceae) agglutinated and killed the parasites. This activity could be inhibited by specific carbohydrates, indicating that it was the result of various lectins in the extracts. An extract of Solanum luteum (Solanaceae) lysed the promastigotes under similar conditions and this cytotoxicity was not abated by the sugars tested. High mortality of promastigotes occurred in infected flies after they ingested an extract of R. communis, even when the extract fed to the flies had been pre-mixed with glucose, a carbohydrate that inhibited the agglutination caused by such an extract in vitro. The results indicate that the lectins and toxins found in the vegetation in L. major foci may decrease the transmission of the parasite.     

The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts

The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of the Leishmania infectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPG in vivo. Here we used an Leishmania major lpg1- mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1- was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion for Leishmania macrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanism in vitro and in vivo, unlike human hosts. In contrast, lpg1- parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG.     

Nucleoside transporters in Leishmania major: diversity in adenosine transporter expression or function in different strains.

Cytotoxic nucleoside derivatives may become useful in the treatment of parasitic infections. As part of our drug development studies, the effect of a number of nucleosides (100 microM) on the cellular transport of 3H-adenosine and 3H-inosine (each at 1 microM) in promastigotes from four Leishmania major strains was investigated. When 3H-inosine was used as permeant, all strains exhibited essentially the same inhibition profile, with unlabeled inosine, guanosine, formycin B, and 3'-deoxyinosine being strongly inhibitory, and adenosine-related compounds such as 2'-deoxyadenosine and tubercidin being inactive. However, when 3H-adenosine was used as permeant, considerable differences in the inhibition profiles were noted among strains. Thus, both inosine transporter-selective nucleosides such as inosine and guanosine and adenosine transporter-selective nucleosides such as 2'-deoxyadenosine and tubercidin showed variable activity as inhibitors of 3H-adenosine transport in different strains. These observations indicated that an adenosine transporter was variably expressed in different strains, and that inhibition profiles for adenosine transport indicated cellular entry via both the inosine and adenosine transporters. The existence of different types of adenosine transporters as an alternative explanation could not be ruled out. The apparent uniform expression of an inosine transporter among different species and strains of Leishmania suggests that inosine derivatives may be useful as anti-leishmanial drugs.     

Leishmania mexicana and Leishmania major: attenuation of wild-type parasites and vaccination with the attenuated lines

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type parasites.     

The amygdala regulates the antianxiety sensitization effect of flumazenil during repeated chronic ethanol or repeated stress

BACKGROUND: The benzodiazepine receptor antagonist flumazenil reduces anxiety-like behavior and sensitization of anxiety-like behavior in various models of ethanol withdrawal in rodents. The mechanism and brain region(s) that account for this action of flumazenil remain unknown. This investigation explored the potential role of several brain regions (amygdala, raphe, inferior colliculus, nucleus accumbens, and paraventricular hypothalamus) for these actions of flumazenil. METHODS: Rats were surgically implanted with guide cannulae directed over the brain region of interest and then treated with an ethanol diet for three 7-day dietary cycles (5 days on ethanol diet followed by 2 days on control diet). At approximately 4 hours, flumazenil was administered intracranially into each of the first 2 withdrawals. Examinations of anxiety-like behavior followed 1 week later during a third withdrawal. In other animals, restraint stress sessions or intra-amygdala DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) injections, preceded by intraperitoneal flumazenil injections, were substituted for the first 2 ethanol treatment cycles to assess the potential anxiety-sensitizing action of stress or a benzodiazepine receptor inverse agonist, respectively. RESULTS: Flumazenil treatment of the amygdala during the first 2 withdrawals blocked the development of sensitized anxiety seen during a third withdrawal. Similar actions of flumazenil were found when stress sessions substituted for the first 2 cycles of ethanol exposure and withdrawal. Amygdala treatment with DMCM magnified the anxiety response to the single subthreshold chronic ethanol treatment, and prophylactic flumazenil blocked this effect. CONCLUSIONS: Intra-amygdala flumazenil inhibits the development of anxiety sensitized by repeated ethanol withdrawal, stress/ethanol withdrawal, or DMCM/ethanol withdrawal. These actions suggest that site-specific and persistent effects of flumazenil on gamma-aminobutyric acid-modulatory processes in this brain region are relevant to sensitized behavioral effects seen in alcoholism.     

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.     

Leishmania infections damage the feeding mechanism of the sandfly vector and implement parasite transmission by bite.

Leishmania parasites are transmitted by the bites of infected female sandflies by a mechanism that has not been clarified. Leishmania infections in the vector develop only in the gut, and the parasites' exit is through the food channel in the proboscis. The problem is how during the bite, when blood flows in, parasites are emitted through the same channel in the opposite direction. It is well documented that infected sandflies maintained on sugar diets are potent vectors, whereas transmission fails after constant feeding on blood. Hence to study the mechanism of transmission, we fed these diets to Phlebotomus papatasi infected with L. major. Histological examination demonstrated that only in the sugar-fed flies did the cuticle lining of the cardiac valve detach and other valve tissues degenerate gradually. The injury of the main valve of the food pumps hindered gorging of most flies when force-fed from capillaries, and they regurgitated the gut contents with fluids from the capillaries. We suggest that infections are caused by parasites regurgitated from the stomach that are deposited in the host tissue. We found that secretion of chitinolytic enzymes by cultured L. major parasites is inhibited by blood or hemoglobin, and hence these enzymes are apparently absent from the blood-fed infected flies, where the cardiac valve appears undamaged. We therefore presume that lysis of the chitin in the cuticle lining of the valve leads to exposure and degeneration of the underlying tissues.     

Experimental Leishmania major infection in mice: role of IL-10

L. major infection of mice induces polarized Th1 and Th2 responses that are correlated with healing of the infection (Th1) or a fatal disease (Th2). The Th subset specific cytokines, IFNgamma and IL-4, themselves were shown to be important factors for the differentiation into the Th1 and Th2 pathways during infection. We studied the role of the Th2 cytokine IL-10 during leishmania infection: removal of endogenous IL-10 by anti-IL-10 treatment did not alter the Th2 cytokine pattern in non-healer mice nor did it modulate DTH reactivity, IgE production or fatal disease progression, but partially blocked the IFNgamma inhibiting effect of rIL-4 in healer mice. During chronic infection similar amounts of IL-10 were produced in both healer and non-healer mice. However, at early time-points during infection IL-10 production was significantly higher in the non-healer Th2 responder animals. IL-10 production in vitro caused significant inhibition of in vitro IFNgamma production. In conclusion IL-10, unlike IL-4 and IFNgamma, does not seem to play a readily detectable role in the Th subset differentiation during L. major infection. However, the high production of IL-10 early during infection in non-healer mice and inhibition of leishmania-specific IFNgamma production may contribute to drive the immune response towards a Th2 response.     

The parasiticidal effect of electricity on Leishmania major, both in vitro and in vivo

The effects of low electrical potentials on Leishmania major (MRHO/IR/75/ER) were investigated both in culture (in terms of promastigote viability) and in experimentally infected BALB/c and NMRI mice (in terms of the cure of pre-existing skin lesions). Exposure to direct-current potentials of 3, 6, 9 and 12 V (at 0.2-10.7 mA) killed all promastigotes in <15, <10, <10 and <10 min, respectively. When electrodes were used to pass similar direct currents across skin lesions on the tails of infected mice, all but the lowest voltage (3 V) caused unwanted ulceration. At 3 V, however, 3 weeks of electrotherapy, for 10 min twice weekly, initially appeared to cure all the lesions and the therapy was then halted. If given no electrotherapy, the BALB/c mice showed much greater Leishmania-attributable morbidity and mortality than the NMRI mice, and it was only in the treated BALB/c mice that relapses were observed, about 3 weeks after electrotherapy had ceased. The possible clinical use of electrotherapy in the treatment of human cutaneous leishmaniasis is discussed.