Follow your gut: improved techniques for monitoring and understanding endemic leishmaniasis transmission through individual blood-fed sand flies

Authors

• PA Huffcutt
• C Meneses
• JG Valenzuela
• T Donatelli Serafim
• E Iniguez
• S Kamhawi

Abstract

Leishmaniasis is a neglected parasitic disease transmitted by female phlebotomine sand fly bites that affects up to a million people annually. With many features of transmission dynamics poorly understood, in-depth analysis of individual field-collected blood-fed sand flies can offer valuable insights. In addition to understanding host preference, we hypothesize that linking the source of the sand fly bloodmeal to infection status will shed light on directionality of transmission and point to potential animal reservoirs in natural settings. To achieve this, we are developing a field-applicable toolbox based on analysis of single blood-fed sand fly midguts preserved on FTA cards. DNA and RNA recovery per individual midgut was optimized to enable several concurrent assays. A sensitive probe-based qPCR targeting kinetoplast DNA was developed to screen samples for Leishmania infection. Using an organic bulk-RNA seq approach, we have identified several potential stage-specific gene targets to then use RT-qPCR to differentiate immature (recent pick-up) vs. mature (recent transmission) infections in the sand fly. We have also optimized host detection from single sand flies experimentally fed on blood from humans and pertinent domestic and sylvatic animals using a multiplex PCR based on mitochondrial genes. All protocols are currently being validated in blood-fed field collected sand flies. Once validated, this combination of molecular tools can enable reliable identification of leishmaniasis reservoirs, accurate assessment of the focality and intensity of ongoing transmission, and verification of vector competence of sand fly species in their natural environment, informing targeted control efforts in active or emerging foci of leishmaniasis.

Scientific Focus Area: Microbiology and Infectious Diseases

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