Leishmania Infection and Gut Homeostasis in the Sand Fly Lutzomyia longipalpis.

Lura, Taylor Anne (2015) Leishmania Infection and Gut Homeostasis in the Sand Fly Lutzomyia longipalpis. PhD thesis, Lancaster University.

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Abstract

Adult female sand flies are the vectors for the medically important protistan Leishmania spp. It is known that Leishmania develops within the midgut of Phlebotomine sand flies, however, very little research exists pertaining to the immune response and physical effects of Leishmania infection within the sand fly. We have studied two interlinked aspects of the sand flies response to the parasite; immune response and gut cell proliferation. This is a vital area for research as a detailed understanding of the Leishmania -sand fly relationship could potentially aid the prevention and control of leishmaniasis. To explore the immune response of sand flies to Leishmania infection two immune genes were investigated. The Protein Inhibitor of Activated Stat (PIAS), which plays a major role as a negative regulator of JAK/STAT, and Dual Oxidase (Duox),which plays an important role in infection-induced reactive oxygen species (ROS) generation in insects including sand flies. Quantitative real-time PCR (qPCR) was utilised to measure relative gene expression of the two genes. Gene expression for Leishmania-infected blood-fed flies were conducted for various time points and entomopathogenic bacteria Serratia marcescens was used for comparison. There was no significant difference in PIAS and Duox expression in Leishmania or Serratia infected flies. However, Duox and PIAS expression showed a significant increase at 48 h after blood feeding regardless of being infection or uninfected. The potentially pivotal role of PIAS and Duox in maintaining gut homeostasis during blood digestion is discussed. The physical response of the gut towards Leishmania infection, measured by midgut epithelial cell proliferation was also investigated. Anti-phosphohistone3 (PH3) antibody was used to assess midgut cell proliferation within sand flies after Leishmania or Serratia infection. Cell proliferation was significantly different between all life stages investigated, with a peak percentage in newly emerged adult females. There was an increase in cell proliferation after Leishmania infection in contrast to Serratia infection which resulted in a decrease in cell proliferation. It is likely that damaged gut cells due to parasite attachment are replaced via cell proliferation. It is suggested that Serratia infection may actively prevent cell proliferation alternatively the extent of intestinal stem cell (ISC) damage may be prohibitive to cell proliferation. Risk assessment relies on accurate information on the vector-disease relationship and surveillance. Surveillance plays an important role in vector control programs. Therefore, access to affordable and efficient surveillance traps and diagnostics is necessary. A diagnostic tool, InBios CL strips, has proven to be an efficient means to identify cutaneous leishmaniasis at the clinical level. The ability to utilise this tool to identify Leishmania infected flies within the field would be greatly beneficial to sand fly control programs. The present study found that CL strips can detect Leishmania within a single infected fly. The efficiency of the strips to detect Leishmania in pools of flies was variable, depending on number of infected flies and day post infection. Only 75% of strips gave a positive detection in pools of 10 flies. Additionally, only 75% of infected flies 2-4 days post infection were detected with the CL strips. It was concluded that the CL strips, as currently presented, are not sensitive enough for detection of a Leishmania infection in a single sand fly. The potential for further development of these strips are discussed.