Fig 1.
Map of Kenya (inset) and Gilgil Sub-county showing the location of the trapping sites.
Gilgil covers an area of approximately 1348.4 Km2 and has a population of 138,448 people. Lowest temperatures are experienced in June and July while the highest temperatures occur from December to February. The map was designed using QGIS (v 3.0.3).
Table 1.
Sandfly density, relative abundance (%) and sex ratios in Gilgil.
Fig 2.
Sandfly species diversity across the sampling sites in Gilgil.
M: male; F: female.
Fig 3.
Phylogenetic analysis of the sandfly mitochondrial COI sequences by Maximum Likelihood method.
The analysis was performed in MEGA v.7 based on the Kimura 2-parameter model. Bootstrap support values based on 1000 replicates are shown at the nodes. Other reference phlebotomine species belonging to different subgenera were included for comparison. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sample identities are shown in red while sequences retrieved from the GenBank are indicated in black. Representative CO1 sequences for each species are available in the GenBank under accession numbers: MK169221-MK169223.
Fig 4.
Leishmania parasite identification by ITS1-PCR-RFLP.
M: 100 bp ladder;1 and 2: Leishmania spp. isolated from sandflies; 3 and 4: L. major (Friedlin strain) and L. tropica (Lv357 strain) positive controls; 5: negative control. Molecular sizes of RFLP product for each species are shown; L. major (220 and 140 bp); L. tropica (200 and 60 bp).
Fig 5.
Real-time PCR-HRM analysis of Leishmania infection in female sandflies.
A derivative dissociation curve of 179 bp L. tropica (Tm = 84.2°C) and 195 bp L. major (Tm = 85.8°C) ITS1-PCR amplicons. Infection was inferred by comparing the melting profiles of the samples to the controls.
Fig 6.
Map of Gilgil showing the proportions of bloodmeal sources per trapping site.
The map was designed using ArcGIS Online version.
Table 2.
Bloodmeal sources of female sandflies identified by real-time PCR-HRM analysis of CYT-B.
Fig 7.
HRM profiles of vertebrate hosts present in sandfly bloodmeals using CYT-B.
Positive controls are shown in the legend; human, rock hyrax, goat and rabbit. The melting profiles of mixed bloodmeals varied slightly compared to those of pure bloodmeals. Bloodmeal source detection failed in two partially blood-fed sandflies (2.70%). This could be due to the degradation of DNA in the bloodmeal. The e-values of BLAST search matches to GenBank sequences, percentage identities and GenBank accessions for some of the exclusive blood-fed sandflies are provided in Table 3.
Table 3.
Vertebrate species represented in sand fly bloodmeals.