Molecular detection of Leishmania donovani, Leishmania major, and Trypanosoma species in Sergentomyia squamipleuris sand flies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya.

BACKGROUND: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts. METHODS: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. RESULTS: We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. CONCLUSIONS: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

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.

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 alternative to the expensive synthetic chemical control. It is potentially sustainable, less hazardous, and relatively inexpensive and 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 screened for their pathogenicity against Phlebotomus duboscqi. Adult flies were contaminated using the technique described by Migiro et al (2010). Briefly, flies were exposed to 0.1 g of dry conidia evenly spread on a cotton velvet cloth covering the inner side of a cylindrical plastic tube (95 mm long × 48 mm diam). In all 25 sandflies were transferred into the cylindrical tube and allowed to walk on the velvet for one minute, after which they were transferred 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 isolates were selected for further studies. RESULTS: A total of 19 isolates were screened against adult sandflies in the laboratory. Mortality in the controls was approximately 16.8 ± 1.7 %. All the isolates were found to be pathogenic to P. duboscqi. Mortality ranged between 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 mortalities of 97.5-100%, were selected for further studies. INTERPRETATION & CONCLUSION: The high susceptibility of sandflies to entomopathogenic fungi suggests that fungi are potential alternatives to chemical control methods. We conclude that application of entomopathogenic fungi could result in acute mortalities of sandflies and reduction of parasite transmission and subsequently, reduction of leishmaniasis risk. This method of biological control has great potential as a new strategy for leishmaniasis control.

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 tested against 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 duboscqi female sandflies. METHODS: Four replicates of unwashed and 20x washed Olyset Nets and PermaNets, K-O Tab-treated and untreated Supanet and 'no net' treatments were evaluated against sandflies within the laboratory 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. Olyset net's blood feeding inhibition was significantly higher than that of Supanet in the laboratory but not in semi-field condition. Of the LLINs, only Olyset net had sandflies that could not feed significantly more than those of Supanet. Additionally, no significant efficacy difference was observed between LLINs washed 20x and unwashed ones. The only significant difference noted in number of sandflies that were found dead or paralyzed within bednets in the semi-field condition was between Olyset and K-O Tab treated Supanet. In the laboratory, unwashed Olyset had a significantly higher number of sandflies killed than all other bednet treatments. CONCLUSION: Olyset net use in areas where sandflies are nuisance biters and/or disease vectors could be more beneficial in preventing sandfly bites than other tested bednets. It is recommended that mesh sizes of LLINs should be smaller for control of sandflies than those used for control of mosquitoes.

Comparative attractiveness of CO(2)-baited CDC light traps and animal baits to Phlebotomus duboscqi sandflies.

BACKGROUND & OBJECTIVES: In order to understand sandfly bionomics, vector species identification, and to develop methods for sandfly control, there is a need to sample sandflies in any particular habitat. This survey was aimed at determining the best method of sampling Phlebotomus (Phlebotomus) duboscqi (Diptera: Psychodidae) in the field. METHODS: Different animal baits and CO2-baited CDC light traps were used to attract sandflies released in an insect-proof screen-house located in the sandfly's natural habitat in Marigat, Baringo district of Kenya. RESULTS: Attraction of hungry P. duboscqi female sandflies by the goat (Capra hircis) was significantly higher than that of hamster (Mesocricetus auretus), Nile grass rat (Arvicanthis niloticus), gerbil (Tatera robusta) and chicken (Gallus domestica). However, two rodent species, A. niloticus and T. robusta did not differ significantly. A linear regression analysis of weights of animal baits and number of sandflies attracted revealed an insignificant result. The fluorescent dyes used to distinguish sandflies of different day experiments seemed not to influence the sandfly numbers in relation to the studied sandfly behaviour. INTERPRETATION & CONCLUSION: The similar attraction pattern of P. duboscqi in semi-field environment by CO(2)-baited CDC light trap and the goat provides hope for solution to the problem of fast dissipating dry ice (CO(2) source) in the field. Goats can, therefore, also be utilized as deflectors of vectors of cutaneous leishmaniasis from humans in zooprophylaxis in Leishmania major endemic areas where the sandfly is found.

Isolation and characterization of flagellates from rodents and canids in Masinga, Machakos District, Kenya.

A total of 728 animals comprising of 633 rodents and 95 canids were examined for leishmanial parasites. Flagellates were isolated from 67 out of 111 (60.4%) Acomys subspinosus (spiny mouse), 12 out of 143 (8.4% ) Mastomys natalensis (multimammate rat), 2 out of 50 (4.0%) Lemniscomys striatus (striped mouse), 2 out of 6 (33.3%) Herpestes sanguineus (slender mongoose), 1 of 1 Helogale parvula (dwarf mongoose) and 1 out of 84 Canis familiaris (domestic dog). All isolates were characterized by Isoenzyme analysis using nine enzymes, namely, malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), malic enzyme (ME), 6-phosphogluconate dehydrogenase (GPGD) and mannose phosphate isomerase (MPI). Enzyme profiles of these isolates were compared with those of five WHO Leishmania reference strains and five well characterized rodent trypanosomes of the subgenus Herpetosoma. The profiles of the isolates were found to be different from those of the Leishmania and Trypanosoma reference strains but the parasites were morphologically similar to rodent trypanosomes. These results suggest that Leishmania parasites were not among the isolates. The enzymes profiles of the three mongoose isolates were identical but differed from profiles of isolates from rodents and dog. This is the first time in Kenya that a high prevalence of nonpathogenic trypanosomes is reported in rodents and canids. From the epidemiological point of view, these trypanosomes must be differentiated from the pathogenic species of trypanosomes and Leishmania that infect man and other animals. The results of this study suggest that rodents do not seem to play a role as reservoirs of Leishmania parasites in Masinga Location, Kenya.