We have been able to provide conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. The priority of our studies in 2011 has been to determine the frequency, timing, and anatomical location of hybrid formation within the midgut, and to extend the findings of sexual competence to other sand fly vector species and other species and strains of Leishmania. We have been able to recover L. major hybrids from Lutzomyia longipalpis, demonstrating that the main genus of sand flies found in the New World are capable of supporting the sexual reproductive cycle of the Leishmania parasties that they transmit. Hybrids were recovered as early as three days post infection, indicating that early developmental stages, e.g. procyclic and/or nectomonad promastigotes, are sexually competent forms. We have also been able to successfully cross L. major sub-strains from West Africa and the Middle East that produce substantially different cutaneous pathologies in C57Bl/6 mice, with the former producing complete erosion of the ear dermis and the later producing dermal lesions that fully resolve. Backcross hybrids generated between F1 progeny and each parental line will be generated to positionally map the parasite genes controlling these clinical outcomes. To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, in 2011 we sampled many different groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic cutoff value that predicted transmission competence. Alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have important implications for transmission of Leishmania by the sand fly vector in the laboratory, as they permit predictions of transmission success in a population of infected flies to be used for critical experiments involving, for example, infected sand fly challenge to evaluate experimental vaccines. The findings also have important implications for transmission of Leishmania by the sand fly in nature, indicating that the number of organisms acquired by the sand fly from an infection reservoir may influence the dose of transmitted parasites and the clinical outcome of infection following transmission by bite.
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