Ecology and seasonality of sandflies and potential reservoirs of cutaneous leishmaniasis in Ochollo, a hotspot in southern Ethiopia

Abstract

Background Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedif er is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. Methods and findings Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L . aethiopica infections only found in P . pedifer . The overall sandfly and P . pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P . pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. Conclusions This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.

Evaluation of a pan-LeishmaniaSL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

“…A 30% sugar source was permanently provided to adult sand flies. Phlebotomus pedifer sand flies were captured in a previous study in Ochollo (6° 11’ N, 37° 41’ E), a village in southwestern Ethiopia where CL is endemic [24,25]. Sand flies were captured between March 2017 and February 2018 using CDC light traps and sticky traps.…”

“…Specimens were stored in 97% ethanol at −20°C until nucleic acid isolation was carried out in March 2018. Leishmania DNA positive sand flies were all P. pedifer infected with L. aethiopica [24]. Nucleic acid extracts were stored at −20°C until analysis for the current study.…”

“…Molecular analyses revealed that all hyraxes were H. brucei infected with L. aethiopica . The original tissue samples in 97% ethanol were stored at −20°C until the current analysis [24].…”

“…Nucleic acids of experimentally infected ( L. major ) L. longipalpis were isolated with a crude extraction buffer and purified using an ethanol precipitation approach as described previously [24]. In short, individual sand fly specimens were incubated overnight in 50 µL extraction buffer (10 mM Tris-HCl pH 8, 10 mM EDTA, 0.1 % SDS, 150 mM NaCl) and 0.5 µL proteinase K (200 µg/mL) without maceration.…”

“…Seven L. aethiopica DNA positive (kDNA and ITS-1) and 15 negative hyrax tissue samples were selected from our previous study [24]. DNA and RNA were simultaneously extracted from the original tissue samples with the NucleoSpin RNA kit and additional reagents from the NucleoSpin RNA/DNA buffer set (Macherey Nagel, Germany) according to the manufacturer’s instructions.…”