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BACKGROUND: Antibody responses to sand fly saliva have been suggested to be a useful marker of exposure to sand fly bites and Leishmania infection and a potential tool to monitor the effectiveness of entomological interventions. Exposure to sand fly bites before infection has also been suggested to modulate the severity of the infection. Here, we test these hypotheses by quantifying the anti-saliva IgG response in a cohort study of dogs exposed to natural infection with Leishmania infantum in Brazil. METHODS: IgG responses to crude salivary antigens of the sand fly Lutzomyia longipalpis were measured by ELISA in longitudinal serum samples from 47 previously unexposed sentinel dogs and 11 initially uninfected resident dogs for up to 2 years. Antibody responses were compared to the intensity of transmission, assessed by variation in the incidence of infection between seasons and between dogs. Antibody responses before patent infection were then compared with the severity of infection, assessed using tissue parasite loads and clinical symptoms. RESULTS: Previously unexposed dogs acquired anti-saliva antibody responses within 2 months, and the rate of acquisition increased with the intensity of seasonal transmission. Over the following 2 years, antibody responses varied with seasonal transmission and sand fly numbers, declining rapidly in periods of low transmission. Antibody responses varied greatly between dogs and correlated with the intensity of transmission experienced by individual dogs, measured by the number of days in the field before patent infection. After infection, anti-saliva antibody responses were positively correlated with anti-parasite antibody responses. However, there was no evidence that the degree of exposure to sand fly bites before infection affected the severity of the infection. CONCLUSIONS: Anti-saliva antibody responses are a marker of current transmission intensity in dogs exposed to natural infection with Leishmania infantum, but are not as

Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists' attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA ( approximately 120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.

In nature the prevalence of Leishmania infection in whole sand fly populations can be very low (<0.1%), even in areas of endemicity and high transmission. It has long since been assumed that the protozoan parasite Leishmania can manipulate the feeding behavior of its sand fly vector, thus enhancing transmission efficiency, but neither the way in which it does so nor the mechanisms behind such manipulation have been described. A key feature of parasite development in the sand fly gut is the secretion of a gel-like plug composed of filamentous proteophosphoglycan. Using both experimental and natural parasite–sand fly combinations we show that secretion of this gel is accompanied by differentiation of mammal-infective transmission stages. Further, Leishmania infection specifically causes an increase in vector biting persistence on mice (re-feeding after interruption) and also promotes feeding on multiple hosts. Both of these aspects of vector behavior were found to be finely tuned to the differentiation of parasite transmission stages in the sand fly gut. By experimentally accelerating the development rate of the parasites, we showed that Leishmania can optimize its transmission by inducing increased biting persistence only when infective stages are present. This crucial adaptive manipulation resulted in enhanced infection of experimental hosts. Thus, we demonstrate that behavioral manipulation of the infected vector provides a selective advantage to the parasite by significantly increasing transmission.

Leishmaniasis is a vectorborne disease transmitted to human and other mammalian hosts by sand fly bite. In the present study, we show that immunization with Leishmania mexicana promastigote secretory gel (PSG) or with a chemically defined synthetic glycovaccine containing the glycans found in L. mexicana PSG can provide significant protection against challenge by the bite of infected sand flies. Only the glycan from L. mexicana was protective; those from other species did not protect against L. mexicana infection. Furthermore, neither PSG nor the glycovaccine protected against artificial needle challenge, which is traditionally used in antileishmanial vaccine development. Conversely, an antigen preparation that was effective against needle challenge offered no protection against sand fly bite. These findings provide a new target for Leishmania vaccine development and demonstrate the critical role that the vector plays in the evaluation of candidate vaccines for leishmaniasis and other vectorborne diseases.

An expressed sequence tag library has been generated from a sand fly vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed from whole adults and 16,608 clones were sequenced from both ends and assembled into 10,203 contigs and singlets. Of these 58% showed significant similarity to known genes from other organisms, < 4% were identical to described sand fly genes, and 42% had no match to any database sequence. Our analyses revealed putative proteins involved in the barrier function of the gut (pelitrophins, microvillar proteins, glutamine synthase), digestive physiology (secreted and membrane-anchored hydrolytic enzymes), and the immune response (gram-negative binding proteins, thioester proteins, scavenger receptors, galectins, signating pathway factors, caspases, serpins, and peroxidases). Sequence analysis of this transcriptome dataset has provided new insights into genes that might be associated with the response of the vector to the development of Leishmania.

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in

In this study, disease progression after intravenous or subdermal infection of dogs with Leishmania infantum JPC strain was monitored. A challenge performed on 14 dogs via the intravenous route with 5 x 10(7) stationary phase promastigotes of the L. infantum JPC strain was 100% successful. During a follow up period of 1.5 years, several parameters were evaluated in order to find the most reliable disease markers. Parasite detection by culture and histology were found to be very sensitive (100%). Additionally, regular physical examination, serology and serum gamma-globulin levels were found to be useful parameters in the evaluation of disease severity and are recommended for inclusion in vaccination-challenge experiments. Although this intravenous challenge model has practical limitations, the data set confirms it is the best experimental model currently available for vaccine development. Two intravenously infected dogs were treated with corticosteroids for 5 months. This treatment was shown to enhance all aspects of a Leishmania infection. Five more dogs were infected by sub-dermal injection of promastigotes mixed with a proteophosphoglycan-matrix (PSG) secreted by Leishmania that assists in transmission and infection by sand fly bite. The resulting parasite burdens were low and the animals remained asymptomatic during a 2-year follow up period. However, this procedure did result in infection in 80% of the dogs and is appealing for future development as a natural challenge model in vaccine development.

Sand flies are the exclusive vectors of the protozoan parasite Leishmania, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel. Here we analyse the inocula from Leishmania mexicana-infected Lutzomyia longipalpis sand flies. Analysis revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of promastigote secretory gel found to accompany the parasites during transmission. Collectively these results have important implications for understanding the relationship between the parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasize that to fully understand transmission of vector-borne diseases the interaction between the parasite, its vector and the mammalian host must be considered together.

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.