Feeding behavior and activity of Phlebotomus pedifer and potential reservoir hosts of Leishmania aethiopica in southwestern Ethiopia

Background Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. Methods Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. Principal findings A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. Conclusion In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.

222 attractiveness towards the sand flies. The experimental set-up was covered by a plastic canvas to 223 avoid interference of wind and other potential feeding sources in the surroundings. 224 Fig 2: Host choice experiment set-up. Female non-fed sand flies captured from caves were transferred to the 225 middle cage, where they were left for 30 minutes to adapt. Then, the connecting tubes to the lateral cages, 226 where a hyrax and human hand were exposed, were opened to allow sand flies to obtain their preferred blood 227 meal during four hours. The hosts themselves and their places were changed for each iteration of the 228 experiment.
229 After 30 minutes adaptation in the middle cage, the connecting tubes to the two lateral cages were 230 opened for four hours to allow sand flies to bite their preferred host. Blood fed sand flies were 231 collected using a mouth aspirator and stored in 97% ethanol at -20°C until further analysis. The 232 experiment was conducted eight times and for each iteration, the position of the hosts was changed 233 and new subjects were used. Hyraxes were released at their trapping site after the experiment. The 234 blood meal sources and sand fly species were determined by sequencing a fragment of the Cyt B and 235 COI gene respectively, according to the methods described above (blood meal analysis and sand fly 236 identification).
237 Data analysis 238 All statistical analyses were carried out in R version 3.5.0, using packages "lme4" and "lmerTest" 239 [27,28]. P-values < 0.05 were assumed statistically significant. 242 The number of sand flies that fed on a particular host, within a specific habitat, during a certain 243 month was included as the response variable. The habitat where sand flies were captured (indoors, 244 outdoors, cave), the host group they acquired their blood meal from and the interaction between 245 habitat and host type were included as fixed effects. In order to correct for monthly variation in sand 246 fly presence, we incorporated the collection as a random effect in the model. A post hoc test, 247 specified as Tukey test, was applied to compare the hosts groups with each other [29].
248 After the previous general model, GLMMs were made similarly for each habitat separately to 249 determine the important blood meal sources in each habitat. The model was constructed as 250 described above, but only the host group was included as a fixed variable.  Table 1. Overall, humans 290 were the most important blood meal source (p < 0.001), accounting for 59.4% of the identified 291 origins, followed by bovines (13.9%), bush hyraxes (10.6%), goats (7.2%) and rodents (5.0%).
292 Residual blood meals were acquired from a wide variety of vertebrates, together covering 4.0% of 293 the determined sources. From the sand flies that fed on humans, five out of 137 (two collected from 294 caves and three from indoors) were positive for Leishmania kDNA. 295  3A). Significantly more sand flies (65.9%, p < 300 0.001) had fed on humans (S1 Table)