American leishmaniasis is a group of endemic, zoonotic diseases extending from Argentina to Texas. Ulcers of the skin and mucosa are the most common manifestations of this disorder in humans, caused by protozoa of the genus Leishmania. The parasite is acquired when injected into the blood stream of the human host by Lutzomyia sand fly vectors. Efforts to control leishmaniasis are complicated by the diversity, genetic polymorphism, and sympatry of the parasites. Thus, development of vaccines is currently seen as a formidable task. Control and surveillance of the vector and its natural hosts offer an alternative for containment of the disease. However, in nature, interruption of the transmission cycle is itself complicated as humans are only incidental hosts in the parasite's life history which otherwise includes a variety of wild animals, mostly mammals, that serve as natural reservoirs. In addition, more than 350 sand fly species have been described for the Americas with 32 so far implicated as proven or suspected vectors of human leishmaniases. The long-term objective of this proposal is to develop new methods for rapid and routine identification of insect vector populations and their interactions with animal reservoirs. To that effect, this proposal recruits the tools of molecular genetics and scanning electron microscopy (SEM).
Three specific aims are pursued: 1. description of Lutzomyia genotypes useful for rapid identification of known and possibly, cryptic species 2. assessment of parasite infection rates in Lutzomyia spp. 3. identification of their natural hosts The first objective will be achieved by allocation of fine-structure character states observed by SEM to Lutzomyia spp. as well as by the development of rapid assays based on oligonucleotide DNA probes directed to genetically variable regions of the insect's genome. These regions will be initially surveyed by the Polymerase Chain Reaction (PCR) followed by direct sequencing. The second objective will be obtained by PCR-based detection of specific Leishmania DNA sequences in individual insects in a format convenient for screening thousands of flies. The third objective will be achieved by analysis of insects displaying evidence of blood meal (engorged, colored abdomen). DNA extracts from the excised abdomen of individual insects will be submitted to amplification with primers directed to the cytochrome b gene belonging to the mitochondrial genome. These primers are designed to discriminate against insect, plant, fungi, protists and bacterial homologues of the gene. Cloning of the amplified fragment(s) and sequencing of such clones will allow an assessment of the diversity of animal hosts involved. Phylogenetic analysis against a panel of cyt b sequences from mammals from the Amazonian basin will pinpoint the exact preferred host(s). Students trained under this grant will establish new connections between molecular genetics and the ecology of a tropical disease and create a precedent useful for the understanding of other vector-borne diseases. This proposal should ultimately show that DNA markers offer a window into routine measurements of otherwise intractable aspects of the natural biology of an insect vector.
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