Phlebotomus Perniciosus Research Articles

Epidemiology of Sandfly-Borne Phleboviruses in North Africa: An Overview

Eight sandfly-borne phleboviruses were found to circulate in North Africa. Phleboviruses detected in sandflies were Toscana (TOSV), Sandfly Fever Sicilian (SFSV), Sandfly Fever Naples (SFNV), Cyprus (CYPV), Punique (PUNV), Utique, Saddaguia, and Medjerda Valley (MVV) viruses, yielding an overall infection rate of 0.02–0.6%. Phlebotomus perniciosus and Phlebotomus longicuspis were the most common vector species in the region. TOSV seroprevalence in dogs from Algeria (4.56%) and Tunisia (7.5%) was low and close, unlike SFSV (38.1%) and PUNV (43.5%), which were restricted to Tunisia. SFSV (1.3–21%) and TOSV (3.8–50%) were the most prevalent among humans. TOSV was frequently detected and symptomatically confirmed in both Algeria (3.8%) and Tunisia (12.86%). Other sandfly-borne phleboviruses have also been detected but less importantly, such as SFNV in Morocco (2.9%) and Tunisia (1.1%) and PUNV (8.72%), CYPV (2.9%), and MVV (1.35%) in Tunisia. Their distribution was mainly northern. Overall, 15.9% of the healthy population were seropositive for sandfly-borne phleboviruses, with evidenced cocirculation. Noticeably, studies conducted in Morocco were mostly interested in TOSV in sandflies. Available data from Libya and Egypt were scant or historical. Further elaboration is required to check the sporadic detection of less-prevalent phleboviruses and fully elucidate the epidemiological situation.

The development of L. major, L. donovani and L. martiniquensis, Leishmania currently emerging in Europe, in the sand fly species Phlebotomus perniciosus and P. tobbi.

Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum.

Stability and suitability of housekeeping genes in phlebotomine sand flies

We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

Leishmania spp. in equids and their potential vectors in endemic areas of canine leishmaniasis.

Equids may be infected by zoonotic Leishmania spp., including Leishmania infantum, in regions where canine leishmaniasis (CanL) is endemic, and Leishmania martiniquensis, which has been reported in horses from Central Europe. This study was designed to evaluate the occurrence of both Leishmania spp. among equids living in CanL endemic areas of Italy, as well as to identify dipteran vectors from the same habitats. From March to October 2023, blood, serum and tissue samples from skin lesions were collected from equids (n = 98; n = 56 donkeys and n = 42 horses) living in Italy, as well as sand flies and biting midges. Blood samples (n = 98) and skin lesions (n = 56) were tested for Leishmania spp. by conventional and real time PCRs and sera were tested by immunofluorescence antibody tests (IFAT) for both L. infantum and L. martiniquensis. Insects were morphologically identified, and female specimens (n = 268 sand flies, n = 7 biting midges) analyzed for Leishmania DNA, as well as engorged sand flies (n = 16) for blood-meal detection. Two animals with skin lesions (i.e., one donkey and one horse) scored positive for Leishmania spp. DNA, and 19 animals (i.e., 19.4%; n = 13 donkeys and n = 6 horses) were seropositive for L. infantum, with five of them also for L. martiniquensis. Most seropositive animals had no dermatological lesions (i.e., 68.4%) while both animals molecularly positive for Leishmania spp. scored seronegative. Of the 356 sand flies collected, 12 females (i.e., n = 8 Sergentomyia minuta; n = 3 Phlebotomus perniciosus, n = 1 Phlebotomus perfiliewi) were positive for Leishmania spp. DNA, and one out of seven biting midges collected was DNA-positive for L. infantum. Moreover, engorged sand flies scored positive for human and equine DNA. Data suggest that equids living in CanL endemic areas are exposed to Leishmania spp., but their role in the circulation of the parasite needs further investigations.

Are phlebotomine sand flies (Diptera: Psychodidae) really attracted to UV-light sources?

Phlebotomine sand flies are the sole confirmed vector of leishmaniosis, a group of endemic and re-emerging diseases in southern European countries such as Spain. Light traps are the major surveillance method currently being employed. Nevertheless, color light attraction by sand flies remains mostly unknown. Following prior research, the effect of UV-LED light sources, and its synergic effect with different color lights has been evaluated employing a modified Flebocollect (FC) light trap model. Results suggest that female Phlebotomus perniciosus sand flies are more attracted to a FC trap lured with the combination of blue and UV LED light sources than commercial CDC (Center for Disease Control and prevention) traps (Bonferroni post-hoc test; p < 0.08; blue/UV mean = 0.50; CDC mean = 0.13), while the combination of red and UV modified-traps excel in capturing Sergentomyia minuta sand flies (Bonferroni post-hoc test; p < 0.04; blue/UV mean = 1.19; CDC mean = 0.66). However, based on our prior studies, incorporating UV light sources into sand fly traps does not seem to enhance their attractiveness, as it has not resulted in higher capture rates. These findings contribute to understanding how sand flies’ vision and light color detection is. Further research is recommended to standardize trap construction procedures and explore variations in different endemic regions according to different sand fly species.

Analysis of the current risk of Leishmania infantum transmission for domestic dogs in Spain and Portugal and its future projection in climate change scenarios.

Canine leishmaniosis, caused by the protozoan parasite Leishmania infantum, is a cosmopolitan vector-borne zoonosis, transmitted principally by Phlebotomus perniciosus in Spain and Portugal, where it is considered an endemic disease. Ecoinformatics tools such as ecological niche models (ENM) have been successfully tested to model the distribution of the risk of infection of different parasitosis as they take into account environmental variables vital for their survival. The risk map proposed in this study combines the potential distribution of Ph. perniciosus in the Iberian Peninsula and the calculation of the infection rate of the parasite in the vector to model the risk of contracting the disease in a more realistic way. In fact, this weighting strategy improves the predictive power of the resulting model (R2 = 0.42, p = < 0.01) compared to the Ph. perniciosus ENM model alone (R2 = 0.13, p > 0.05). The places with the highest risk of transmission are the southwest and central peninsular area, as well as the Mediterranean coast, the Balearic Islands and the Ebro basin, places where the ideal habitat of Ph. perniciosus and the infection rate is also high. In the case of future projections under climate change scenarios, an increase in the risk of infection by L. infantum can be observed in most of the territory (4.5% in 2040, 71.6% in 2060 and 63% in 2080), mainly in the northern part of the peninsula. The use of ENMs and their weighting with the infection rate in Ph. perniciosus is a useful tool in predicting the risk of infection for L. infantum in dogs for a given area. In this way, a more complete model can be obtained to facilitate prevention and control.

The correspondence between the physiological cold tolerance and the distribution border of Mediterranean sandflies in Southeast Europe.

The distribution of sandflies is influenced by several climatic factors, like winter temperature limits, summer humidity, and drought. Only a few studies were performed to search for the connection between the physiological tolerance of sandflies and the result of the Climate Envelope Models projected distribution borders. It was studied how the depth of rodent burrows could determine the occurrence of five European Phlebotomus species based on their known physiological cold tolerances. The modelled distributions based on the monthly minimum temperatures showed notable differences by soil depth and species. The observed and modelled European distribution areas of sandflies according to - 20 cm soil depth showed good coincidence with the observed occurrences of Phlebotomus neglectus , Ph. papatasi , and Ph. perfiliewi in East-Central, Southeast, and East Europe. Also, a good coincidence was found in the case of the modelled and observed occurrences of Phlebotomus perniciosus in Western Europe. It can be concluded that in their north-eastern distribution borders, sandflies can overwinter in rabbit and rodent burrows at depths of - 20 cm below the surface. The physical cold tolerance of Ph. ariasi could be underestimated. In more than - 50 cm of depth, the stable and continuously cold environment of the shelters (mean temperature less than 10°C) could inhibit the post-winter diapause activation of sandflies in the northern regions.

Co-Circulation of Leishmania Parasites and Phleboviruses in a Population of Sand Flies Collected in the South of Portugal.

In the Old World, phlebotomine sand flies from the genus Phlebotomus are implicated in the transmission of Leishmania spp. parasites (Kinetoplastida: Trypanosomatidae) and viruses belonging to the genus Phlebovirus (Bunyavirales: Phenuiviridae). Two of the five sand fly species known to occur in Portugal, Phlebotomus perniciosus and Ph. ariasi, the former being the most ubiquitous, are recognized vectors of Leishmania infantum, which causes visceral leishmaniasis, the most prevalent form of leishmaniasis in the country. Phlebotomus perniciosus is also the vector of the neurotropic Toscana virus, which can cause aseptic meningitis. Entomological surveillance is essential to provide fundamental data about the presence of vectors and the pathogens they can carry. As such, and given the lack of data in Portugal, an entomological survey took place in the Algarve, the southernmost region of the country, from May to October 2018. Polymerase chain reaction assays were performed in order to detect the presence of the above-mentioned pathogens in sand fly pools. Not only were both Leishmania parasites and phleboviruses detected during this study, but more importantly, it was the first time their co-circulation was verified in the same sand fly population collected in Portugal.

Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1100, n = 11), MON-80 (NP1130, n = 6), MON-24 (NP1140, n = 1) and MON-331 (NP1150, n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.

The Phlebotomine sand fly fauna of Switzerland revisited.

Sand flies (Diptera: Psychodidae, Phlebotominae; Newstead, 1911) are widespread in Europe, being particularly common in the Mediterranean region but rare north of the Alps. Thus, Switzerland is an opportune place to investigate the sand fly fauna on both sides of the Alpine crest, in southern sub-Mediterranean climate and northern oceanic temperate climate. We reinvestigated the Swiss sand fly fauna with the aim to assess changes in composition, altitudinal distribution, abundance and seasonality. Thirty-eight sites were investigated with light traps and/or interception sticky traps in 4 years. Ninety and 380 specimens were caught by light traps and sticky traps, respectively, at 15 collecting sites. Four species were identified. Phlebotomus mascittii (Grassi, 1908), Phlebotomus perniciosus (Newstead, 1911) and Sergentomyia minuta (Rondani, 1843) were confirmed in Ticino, and P. mascittii for the first time in neighbouring Grisons. Also, Phlebotomus neglectus(Tonnoir, 1921) is for the first time reported, though at a very low density compared to P. perniciosus at the same site. Its presence in Ticino supports the northward spread observed in Italy. Sand flies were detected north of the Alps at one site only, endorsing a historical report. Overall, the low density of P. perniciosus and very low density of P. neglectus suggest that canine leishmaniosis may not be an important disease risk in Switzerland.

On abnormal Phlebotomus perniciosus (Diptera: Psychodidae: Phlebotominae) from the center of Tunisia.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

Effect of high-volume insecticide spraying on sand fly vectors in household gardens in Spain.

This study investigated the efficacy of high-volume spraying with the adulticide α-cypermethrin alone and in combination with the larvicide diflubenzuron on the density of sand flies in gardens of three detached households in periurban areas in southeast Spain. Treatments were applied four times between June and August 2016, and four nearby sites, two households and two non-urbanized sites, were untreated controls. The number of sand flies collected between May and October 2016 using sticky interception and light attraction traps, was 4446 specimens. Species identified morphologically included Sergentomyia minuta (n = 2101; 48%), Phlebotomus perniciosus (n = 1922; 44%), Phlebotomus papatasi (n = 173; 4%), Phlebotomus sergenti (n = 161; 4%) and Phlebotomus ariasi (n = 36; 1%). Sand flies were detected in both treated and untreated sites. The proportion of positive sticky traps and the median (range) density of sand flies in positive traps were 61% traps and 7 (2-172) sand flies/m2 /day in untreated sites, and 43% traps and 4 (1-56) sand flies/m2 /day in treated sites (p < 0.05). Similarly, for light traps, it was 96% traps and 30 (3-168) flies/trap/day, and 83% traps and 3 (1-12) sand flies/trap/day, respectively (p < 0.05). However, sand fly density followed a comparable seasonal pattern in untreated and treated sites and did not consistently decrease following insecticide applications. These results were confirmed with mixed negative binomial modelling of sand fly density adjusted for time since application, month, environmental setting and site. The limited efficacy of the treatments, added to their cost, the impact of insecticides on non-target organisms and human health, and the risk of development of insecticide resistance, should dissuade similar outdoor applications to control sand fly vector populations in residential areas.

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

An approach to telmophagous Nematocera (Ceratopogonidae, Psychodidae, and Simuliidae) of Spain, with emphasis on its medical and veterinary importance

Nematocera are a suborder of Diptera which historically influenced human history more than any other arthropod group. Four families show a hematophagous behaviour, one being solenophagous (Culicidae), feeding directly on blood vessels, and three being telmophagous (Ceratopogonidae, Psychodidae, and Simuliidae), feeding on blood which pools at the site where their mouthparts have formed a laceration. Although mosquitoes rank first in importance, the telmophagous are also of great interest. Objectives: to update the status of these nematocerans through a transdisciplinary approach, reviewing the main characteristics of each family, the situation of the main vector-borne diseases transmitted by them in the country, especially during the 21st century, and the most relevant species or species groups from a medical and veterinary perspective. Methods: a literature search of databases was conducted and supplemented by browsing specialized journals and citation searching. Results were reviewed and filtered. Results: to date, 84 species of biting midges (Ceratopogonidae), 13 species of sand flies (Psychodidae), and 53 species of black flies (Simuliidae) have been reported in Spain. Culicoides imicola and the Obsoletus complex stand out as the most important biting midges, as they are incriminated in the transmission of bluetongue and Schmallemberg virus; Phlebotomus perniciosus and Phlebotomus ariasi are the vectors of Leishmania infantum; and Simulium erythrocephalum and the Ornatum complex cause the greater part of nuisance and bites to humans in Spain. Conclusions: there is a need to increase research capacity in Spain in order to address several health challenges arising from the presence of telmophagous Nematocera in particular, and of blood-sucking atropods in general.

MALDI-TOF MS Limits for the Identification of Mediterranean Sandflies of the Subgenus Larroussius, with a Special Focus on the Phlebotomus perniciosus Complex.

Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph.&amp;nbsp;papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

Distribution and Ecology of Phlebotomine Sand Flies (Diptera: Psychodidae) in Endemic and Nonendemic Area of Leishmaniasis in Northern Morocco.

Northern Morocco is endemic for cutaneous and visceral leishmaniasis. Our entomological investigations aim to evaluate the risk of Leishmania transmission by determining the species composition, the density, and seasonal fluctuation of sand fly populations in endemic and nonendemic areas of leishmaniasis in Tetouan province (North-Western Morocco). Using Sticky-paper traps, 8,370 specimens were collected between May and November 2015 in two localities: peri-urban area of Tetouan city, where leishmaniasis is endemic and that of the Oued Laou village where no cases of leishmaniasis have been recorded. Six sand fly species were identified. The genus Phebotomus was represented by five species: Phlebotomus ariasi, Phlebotomus. longicuspis, Phlebotomus perniciosus, Phlebotomus kazeruni, and Phlebotomus sergenti, while the genus Sergentomyia was represented by only one species Sergentomyia minuta. Phlebotomus perniciosus was dominant in the nonendemic area (47%) while Phlebotomus sergenti was dominant in the endemic area (51%). The spatio-temporal distribution of sand fly populations is discussed according to biotic and abiotic variables. Seasonal fluctuation in sand fly density showed a bimodal pattern for the subgenus Larroussius and a unimodal pattern for the subgenus Paraphlebotomus in Tetouan city. But, in Oued Laou village, a unimodal density distribution for species of the Larroussius subgenus and a bimodal seasonal distribution for species of the subgenus Paraphlebotomus were identified. We affirm the coexistence, in the study area, of vectors of both cutaneous and visceral leishmaniasis, namely P. ariasi, P. longicuspis and P. perniciosus vectors of Leishmania infantum and P. sergenti vector of L. tropica. However, the geographic distribution, the specific abundance, and the activity reveal significant differences between endemic and nonendemic areas in the region.

Retrospective Analysis of Leishmaniasis in Sicily (Italy) from 2013 to 2021: One-Health Impact and Future Control Strategies.

Leishmaniasis is an important vector-borne disease that represents a serious public health problem, including in Sicily (Italy), which is considered an endemic area. We collected canine, feline and human data from 2013 to 2021 in Sicily, while entomological surveys were conducted only in 2013 and 2021. Overall, 23,794/74,349 (34.4%) of dogs and 274/4774 (11.8%) of cats were positive in one or more diagnostic tests. A total of 467 cases of human Leishmaniasis were reported, with 71% showing cutaneous and 29% visceral involvement. The provinces with the largest number of patients were Agrigento (45.4%) and Palermo (37%). In 2013, Phlebotomus perfiliewi was the dominant sandfly species in Sicily (68.7%), followed by Phlebotomus perniciosus (17.2%) and Sergentomya minuta (14%). In 2021, Phlebotomus perfiliewi was confirmed as the most common species (61.6%), followed by Phlebotomus perniciosus (33.1%) and Sergentomya minuta (4.7%). Of particular interest was the identification of Phlebotomus papatasi (0.41%) in Agrigento. Our retrospective study can inform health authorities for the development of appropriate screening, treatment and control strategies to reduce Leishmania incidence rate. This study examined the present state of Leishmaniasis control, surveillance, and prevention in Sicily, but also highlighted deficiencies that could be addressed through the application of One-Health principles.

Leishmaniasis vectors in the environment of treated leishmaniasis cases in Spain.

Transmission of leishmaniasis in endemic areas is characterized by microfocality related to the presence of the vector. Most entomological studies in southwestern Europe have focused on sylvatic areas and town outskirts, very few have sampled town or urban centres, and no survey has investigated inside households. The aim of this study was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with environments in which there was no association. Sand flies were captured in 26 households associated with recently treated leishmaniasis patients, 15 neighbouring houses without associated cases, and in others environments. Overall 7495 sand flies belonging to six species were captured. The highest sand fly density was found in farmhouses where there is a great availability of blood sources and breeding sites. In the environment of human leishmaniasis cases, Sergentomyia minuta was the most prevalent species followed by Phlebotomus perniciosus. Nevertheless, lower Leishmania infantum infection rates and lower intensity of infection were detected in S. minuta sand flies than in P. perniciosus. The density of P. perniciosus in households with recently treated leishmaniasis patients varies between 0 and 108 sand flies per light trap/night, with the maximum values corresponding to farmhouses. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households.

New Microsatellite Markers for Genetic Studies on Sergentomyia schwetzi (Diptera: Psychodidae): A Suspected Vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in the Canine Leishmaniasis Focus of Mont-Rolland, Senegal.

Visceral leishmaniasis is not endemic in West Africa, but prevalence of canine leishmaniasis and seroprevalence of Leishmania infantum infection in humans are high in the Mont Rolland community (Thiès region, Senegal). Previous studies in this area showed that Sergentomyia schwetzi could be the potential vector of Le. infantum. To precisely describe the biology and population structure of this potential vector, we identified eight novel microsatellite loci to characterize Se. schwetzi populations. We tested these loci in Se. schwetzi populations from five locations at Mont Rolland (Thiès, Senegal). All the loci were polymorphic, with a mean of 17.25 alleles (observed heterozygosity: 0.455). We did not detect any evidence of scoring errors due to stuttering and large allele dropout. Moreover, several of these loci were also amplified in six other sand fly species (Sergentomyia magna, Sergentomyia dubia, Sergentomyia minuta, Phlebotomus duboscqi, Phlebotomus perniciosus, and Phlebotomus ariasi). These preliminary results demonstrate the utility of these microsatellite markers for Se. schwetzi (and for the other sand fly species) population genetic studies.

First Isolation of Punique Virus from Sand Flies Collected in Northern Algeria.

In the last decade, several phleboviruses transmitted by sand flies were detected in the Mediterranean countries, with the health impact of some of them being unknown. From September to October 2020, a total of 3351 sand flies were captured in Kherrata (Bejaia, northern Algeria) and identified by sex, grouped in 62 pools, which were tested for the presence of phlebovirus RNA using endpoint RT-PCR. Two pools (male and female, respectively) were positive. The genome sequencing and phylogenetic analysis showed that the two phleboviruses detected were closely related to the Punique virus (PUNV) isolated in Tunisia and detected in Algeria. Both PUNV strains were isolated on VERO cells from positive pools. Morphological identification of 300 sand flies randomly selected, showed a clear dominance of Phlebotomus perniciosus (98.67%). The dominance of this species in the study area was confirmed by PCR targeting the mitochondrial DNA. Our result represents the first isolation of PUNV and the second report in Algeria from two distinct regions which confirm its large circulation in the country and more broadly in North Africa. Further studies are needed to measure the impact on public health through seroprevalence studies in humans as well as animals and to investigate its potential involvement in neurological viral diseases.