Pathogenicity of Metarhizium anisopliae (Metch) Sorok and Beauveria bassiana (Bals) Vuill to adult Phlebotomus duboscqi (Neveu-Lemaire) in the laboratory

Background & objectives: Biological control of sandflies using entomopathogenic fungi is a possible alternativeto the expensive synthetic chemical control. It is potentially sustainable, less hazardous, and relatively inexpensiveand merits further investigations. The objective of this study was to identify the most pathogenic fungal isolate(s)to sandflies in the laboratory.Methods: Isolates of entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were screenedfor their pathogenicity against Phlebotomus duboscqi. Adult flies were contaminated using the technique describedby Migiro et al (2010). Briefly, flies were exposed to 0.1 g of dry conidia evenly spread on a cotton velvet clothcovering the inner side of a cylindrical plastic tube (95 mm long × 48 mm diam). In all 25 sandflies weretransferred into the cylindrical tube and allowed to walk on the velvet for one minute, after which they weretransferred from the velvet into the cages in Perplex. Insects in the control treatments were exposed to fungusfree velvet cloth before being transferred into similar cages. The treatments were maintained at 25 ± 2°C,60–70% RH and 12L: 12D photoperiod. The experiment was replicated 5 times. The most pathogenic isolateswere selected for further studies.Results: A total of 19 isolates were screened against adult sandflies in the laboratory. Mortality in the controlswas approximately 16.8 ± 1.7 %. All the isolates were found to be pathogenic to P. duboscqi. Mortality rangedbetween 76.8 and 100% on all the fungal isolates tested. The lethal time taken to 50% (LT50) and 90% (LT90(mortality ranged from 3.0–7.8 days and from 5.3–16.2 days, respectively. The virulent isolates, causing mortalitiesof 97.5–100%, were selected for further studies.Interpretation & conclusion: The high susceptibility of sandflies to entomopathogenic fungi suggests that fungiare potential alternatives to chemical control methods. We conclude that application of entomopathogenic fungicould result in acute mortalities of sandflies and reduction of parasite transmission and subsequently, reductionof leishmaniasis risk. This method of biological control has great potential as a new strategy for leishmaniasiscontro

Laboratory and semi-field evaluation of long-lasting insecticidal nets against leishmaniasis vector, Phlebotomus (Phlebotomus) duboscqi in Kenya

Background & objectives: Phlebotomine sandflies are vectors of leishmaniases and other diseases.Long-lasting insecticidal nets (LLINs) as possible tools for control have not been widely testedagainst them. The objective of this study was to determine the efficacy of Olyset® Net and PermaNet®LLINs alongside a local brand, K-O Tab® treated net (Supanet) against Phlebotomus duboscqifemale sandflies.Methods: Four replicates of unwashed and 20x washed Olyset Nets and PermaNets, K-O Tabtreatedand untreated Supanet and ‘no net’ treatments were evaluated against sandflies within thelaboratory by tunnel tests and in semi-field conditions in the greenhouse model for their efficacy.Results: All bednets allowed entry of P. duboscqi sandflies and subsequent blood-feeding. Olysetnet’s blood feeding inhibition was significantly higher than that of Supanet in the laboratory butnot in semi-field condition. Of the LLINs, only Olyset net had sandflies that could not feedsignificantly more than those of Supanet. Additionally, no significant efficacy difference wasobserved between LLINs washed 20x and unwashed ones. The only significant difference noted innumber of sandflies that were found dead or paralyzed within bednets in the semi-field conditionwas between Olyset and K-O Tab treated Supanet. In the laboratory, unwashed Olyset had asignificantly higher number of sandflies killed than all other bednet treatments.Conclusion: Olyset net use in areas where sandflies are nuisance biters and/or disease vectorscould be more beneficial in preventing sandfly bites than other tested bednets. It is recommendedthat mesh sizes of LLINs should be smaller for control of sandflies than those used for control ofmosquitoes

Association of Phlebotomus guggisbergi with Leishmania major and Leishmania tropica in a complex transmission setting for cutaneous leishmaniasis in Gilgil, Nakuru county, Kenya

Background Phlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area. Methodology/ Principal findings An entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider’s insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans. Conclusions/ Significance The high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area