Bacterial Diversity in the Lumen of Wild Caught Leishmaniasis Vector Phlebotomus argentipes (Psychodidae: Phlebotominae)

Abstract

Leishmaniasis is a vector-borne disease transmitted through female sand flies which caused by a protozoan parasite belong to genus Leishmania. Parasite development happens mostly in the midgut of the sand fly with presence of bacterial community in lumen. In Sri Lanka, Phlebotomus argentipes is known to be the vector for leishmaniasis. Several studies have reported the presence of aerobic bacteria in the gut of sand flies which evidence potential approach to control Leishmaniasis transmission through paratransgenic strategy. However, such investigations have not been conducted in Sri Lanka. Sand flies were collected by Cattle baited collections from three selected Medical Officer of Health (MOH) areas (Polpithigama, Maho and Galgamuwa) in Kurunegala district on a monthly basis from August to December 2018. Female sand flies were immobilized on ice and sterilized in 30 µL of 70% w/v ethanol followed by phosphate-buffered saline (PBS) washing. Midgut offive unfed female sand flies were pooled and homogenized with 150 µL of sterile PBS (pH 7.3). The lysate was diluted to 500 µL with PBS and 100 µL of each homogenate was plated onto Brain Heart Infusion (BHI) agar followed by incubation at 28°C up to 2 weeks under aerobic conditions. The experiment procedures were repeated 10 times. Colony separation was done byphenotypical differences. Stab cultures of isolates were sequenced for 16S ribosomal RNA partial gene. A total of 1,969 specimens of sand flies were collected. Morphological identification revealed the presence of only one species, Phlebotomusargentipes(n=1,969). The male sand flies are the most represented with 91.4% (n= 1,800), whereas females represent only 8.6% (n=169). A total of 50 randomly selected unfed female P. argentipeswas examined. The average Colony Forming Unit (CFU) ranged from 8 x 101 – 130 x 102. A total of 11 bacterial isolates namely; Serratiamarcescens, Enterobactersp, Staphylococcus saprophyticus, Enterobacter cloacae, Staphylococcus sciuri, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Staphylococcus arlettae, Aeromonascaviae, Staphylococcus warneri, Bacillus megaterium. Sequences showed 99% - 100% identities to the existing sequences, which was assigned to the same genus and species. S. marcescens, shown to have an antileishmanial activity. Therefore, further studies are required to explore the potential use of this species for paratransgenesis.