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A novel phlebovirus, Punique virus (PUNV), was discovered and isolated in 2008 from sandflies from Northern Tunisia. PUNV is now classified as a unique member of the Punique phlebovirus species within the Phlebovirus genus in the Phenuiviridae family (order bunyavirales). In this study, we aimed to investigate the transmission dynamics of PUNV in Tunisia. Sandflies were collected during two consecutive years, 2009 and 2010, by CDC light traps. In 2009, a total of 873 sandflies were collected and identified to the species level. Phlebotomus perniciosus was the most abundant species. One pool of P. perniciosus females collected in autumn contained PUNV RNA, yielding an infection rate of 0.11%. The population densities of circulating sandfly species were assessed during May–November 2010 in Northern Tunisia by using sticky traps. Phlebotomus (Larroussius) perniciosus (71.74%) was the most abundant species, followed by Phlebotumus (Larroussius) longicuspis (17.47%), and Phlebotumus (Larroussius) perfiliewi (8.82%). The densities of dominant sandfly species were found to peak in early spring and again in the autumn. In 2010, species identification was not performed, and sandflies were only discriminated on the basis of sex and collection date. Out of 249 pools, three contained PUNV RNA. Each positive pool allowed virus isolation. The three pools of female sandflies containing PUNV RNA were collected in autumn with an infection rate of 0.05%. These findings provide further evidence that P. perniciosus is the main vector of PUNV in Tunisia, and this phlebovirus is endemic in Tunisia. Our findings provided strong evidence of intensive circulation of PUNV in sandflies and hosts through a viral infection buildup process between sandfly vectors and hosts starting at the beginning of the activity of sandflies in spring to reach a maximum during the second main peak in autumn. [ABSTRACT FROM AUTHOR]

In Morocco, leishmaniases are a major public health problem due to their genetic diversity and geographical distribution. Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted typically by bite of phlebotomine sand flies. This study identifies sand fly fauna in Ibaraghen village, province of Azilal, which is a focus of CL, by combination of morphological and molecular methods (sequencing of COI gene, MALDI-TOF MS protein profiling). Nested-kDNA PCR was used to detect and identify Leishmania species within potential vector species. 432 CDC light traps were placed at different heights above ground level at four capture sites during a whole year. Traps at 1.5 m above the ground yielded capture of sand flies almost double compared to above ground level (29.33%), while the collection reached 55.09% when the traps were placed 2.5 m above ground. A total of 2,830 sand flies were collected, 2,213 unfed specimens were morphologically identified, 990 males (44.73%) and 1,223 females (55.26%) of 13 species; ten Phlebotomus species and three Sergentomyia species. Six species were analysed by MALDI-TOF MS protein profiling (4 Phlebotomus and 2 Sergentomiya species), and their identification was confirmed by COI sequencing. 1,375 unfed females were screened for the presence of Leishmania by nested-kDNA PCR in pools, 11/30 pools of P. sergenti showing a single band of 750 bp corresponding to L. tropica. Our results confirm the role of P. sergenti as a proven vector in Azilal focus of cutaneous leishmaniasis; however, the relative abundance of other species known as vectors of Leishmania species emphasizes the risk of introduction of L. infantum and L. major in this province. For the first time in Morocco, a combined approach to identify sand flies by both morphology and molecular methods based on DNA barcoding and MALDI-TOF MS protein profiling was applied. [ABSTRACT FROM AUTHOR]

We report the study of sandfly Leishmania infection in an area of low incidence of visceral leishmaniasis in Tunisia. Sandflies were collected monthly using CDC light-traps set in houses and animal shelters during May–November 2016 and 2017. All males were identified at the species level. A sample of 878 females including all gravid specimens was subjected to kDNA qPCR for Leishmania detection and parasite load estimation. Leishmania species were determined by ITS1 PCR sequencing, and species identification of infected sandflies was performed by DNA barcoding. Phlebotomus perfiliewi and P. perniciosus were the dominant species during the two-year period. However, comparison of their relative abundances showed that P. perniciosus was more abundant during peaks of 2017 with longer activity duration. Real-time kDNA PCR did not detect Leishmania infection in 2016, although it identified four positive specimens (0.7%) in 2017. All four infected specimens were identified as P. perniciosus. ITS1 PCR sequencing allowed L. infantum identification in one kDNA qPCR-positive specimen. This was a P. perniciosus gravid female with a high parasite load caught during the long-lasting peak of 2017. This work highlights the usefulness of multi-seasonal studies of sandfly dynamics and kDNA qPCR in screening Leishmania infection and determining L. infantum vectors in hypo-endemic foci of human leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is a lethal parasitic disease, transmitted by sand fly vectors. Immunomodulatory properties of sand fly saliva proteins and their protective effects against Leishmania infection in pre-exposed animals suggest that a combination of an antigenic salivary protein along with a Leishmania antigen can be considered for designing a vaccine against leishmaniasis Methods: Three different fusion forms of L. infantum hypothetical protein (LiHyV) in combination with Phlebotomus kandelakii salivary apyrase (PkanAp) were subjected to insilico analyses. Major Histocompatibility Complex (MHC) class I and II epitopes in both humans and BALB/c mice were predicted. Antigenicity, immunogenicity, epitope conservancy, toxicity, and population coverage were also evaluated. Results: Highly antigenic promiscuous epitopes consisting of truncated LiHyV (10285) and full-length PkanAp (21-329) were identified in human and was named Model 1. This model contained 25 MHC-I and 141 MHC-II antigenic peptides which among them, MPANSDIRI and AQSLFDFSGLALDSN were fully conserved. LALDSNATV, RCSSALVSI, ALVSINVPL, SAVESGALF of MHC-I epitopes, and 28 MHC-II binding epitopes showed 60% conservancy among various clades. A population coverage with a rate of >75% in the Iranian population and >70% in the whole world was also identified. Conc lusion: Based on this in-silico approach, the predicted Model 1 could potentially be used as a vaccine candidate against VL. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the ecological aspects of sand flies in southwestern North Khorasan, in which cutaneous leishmaniasis caused by Leishmania major has been reported with the highest annual incidence in Iran. Sampling was carried out in four localities including: Khorasha (natural), Ghamiteh (natural), Jorbat (semi urban) and Brick kilns (urban), twice a month using 105 sticky paper traps from indoors and outdoors dwellings during May-December 2017. Specimens were removed from sticky papers, washed in acetone, preserved in 80% ethanol, mounted on microscopic slides by Puri's medium, and identified using valid morphological keys. Simpson (D), richness (S), Menhinick (DMg), Margalef (DMn), Shannon-Weiner (H′), evenness (J') were calculated for species diversity. The synanthropic index was determined for the first time in the area. Totally 517 specimens were collected, 47% in outdoors and 30.4% in human indoor dwellings and 22.6% in animal. Eight species of sand flies including 5 species of the genus Phlebotomus and 3 species of the genus Sergentomyia were identified. Phlebotomus papatasi and Sergentomyia sintoni were the most common and Eudominant species, active in all months, collected in the maximum number and percentage in September and August, respectively, and showed the highest abundance in outdoors. The synanthropic index ranged from 6.25 to 38.9 in the study area. The Shannon-Wiener index was estimated to be up to 1.4 and 1.37 in Khorasha and November, respectively, which showed the highest diversity due to maximal richness and evenness compared to other areas. High abundance of Ph. papatasi, as the main vector of cutaneous leishmaniasis, can enhance the potential risk of emerging CL in new areas, the data can be equally important when vector control measures are considered. [ABSTRACT FROM AUTHOR]

Dogs are reservoir hosts of leishmaniasis caused by Leishmania infantum and transmitted by phlebotomine vectors. The effect of dinotefuran, pyriproxyfen and permethrin spot‐on solution (Vectra®3D, Ceva Santé Animale, Libourne, France) on Leishmania transmissibility by naturally infected dogs via reared Phlebotomus perniciosus, was assessed. Dogs affected by leishmaniasis were submitted to xenodiagnosis and 6 infecting >10% of insects were treated topically on day 0. Antifeeding, insecticidal and anti‐transmissibility effects were evaluated through xenodiagnoses performed on days 1, 7 and 28, using individual pre‐treatment parameters as control. Feeding and mortality rates were assessed at 24 h, whereas promastigote infection, maturation and burden were assessed up to 96 h post blood meal (potentially infectious rate). On day 1, the anti‐feeding efficacy was >95% in 4 dogs, insecticidal efficacy 100% in 4 dogs, and anti‐transmissibility effect 100% in 6 dogs. Efficacy rates recorded on day 7 were very similar to day 1. On day 28, anti‐feeding and insecticidal efficacy values were much broader, ranging 32.6–100% and 7.7–94.4%, respectively. Potentially infectious insects were recorded from two dogs, with sharp decrease in transmissibility rate as compared with pre‐treatment condition. Altogether, Vectra®3D abrogated by >98% the potential Leishmania transmissibility by the examined pool of infected dogs over 1 month. [ABSTRACT FROM AUTHOR]

Dogs are reservoir hosts for Leishmania infantum, a protozoan parasite transmitted by phlebotomine sand flies. The anti‐feeding and fast‐killing efficacy of Scalibor® deltamethrin collars against experimental Phlebotomus perniciosus challenges on dogs was determined over 1 year. Two groups of 8 dogs each were fitted with placebo (control) or deltamethrin collars (treated) on Day 0 and exposed to sand flies approximately every 28 days up to Day 364. After each exposure, anti‐feeding and fast‐killing efficacy rates were determined by comparing blood‐fed or live insects, respectively, in the treated vs. the control group. Blood‐fed and live sand flies were significantly less in treated dogs as compared to control dogs at each assessment. The anti‐feeding efficacy rate exceeded 90% except on Day 337 (89%) but increased again (96%) on Day 364. Fast killing efficacy was <74% over the study when considering all flies. However, this value increased cumulatively to 98% when only blood‐fed flies were compared between groups. Scalibor® collars are highly effective at preventing P. perniciosus blood‐feeding and in fast‐killing flies taking a blood meal for up to 1 year after application. These strong and long‐lasting effects are an important strategic component for L. infantum transmission control. [ABSTRACT FROM AUTHOR]

Former studies proposed that the speciation of the subgenus Paraphlebotomus happened in the Neogene Epoch in the circum-Mediterranean region due to the geographical segregation effect of the former Paratethys Sea. It was aimed to study whether the modelled Neogene ranges of Phlebotomus sergenti and Phlebotomus similis support or contradict this barrier role of the Paratethys in the speciation of Paraphlebotomus sandfly. For this purpose, the potential Neogene geographical ranges of Phlebotomus sergenti and Phlebotomus similis were modelled based on the present climatic requirements of the taxa. The Miocene models do not support the circum-Paratethyan migration of the ancestor of Phlebotomus similis. In general, Phlebotomus similis shows a low affinity to the North Paratethyan shorelines during the entire Miocene epoch. The only exceptions are the Tortonian and early Messinian periods when the climatic conditions could be suitable for Phlebotomus similis in the North Paratethyan shorelines. It was found that neither the modelled late Miocene, Pliocene nor the mid-Pleistocene period distributions of Phlebotomus sergenti and Phlebotomus similis shows notable differences in the suitability values in the Balkans and the Middle East. It is most plausible that the divergence of the Phlebotomus similis and its relatives was related to the tectonic subsidence of the Hellene Orogenic Belt and Phlebotomus similis specialised in the Balkan Peninsula and the present-day North Pontic area during the middle-late Miocene epoch. The Messinian desiccations of the Mediterranean Basin and the Zanclean re-flood caused the migration, but not the speciation of Phlebotomus similis and its sister taxa. [ABSTRACT FROM AUTHOR]

Abstract 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.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania , affecting millions of people worldwide. The disease is transmitted by the bite of infected female sand flies, which act as vectors and hosts for the parasites. The interaction between Leishmania parasites and sand flies is complex and dynamic, involving various factors that influence parasite development, survival and transmission. This review examines how the immune response of sand flies affects vector competence and transmission of Leishmania parasites, and what the potential strategies are to prevent or reduce infection. The review also summarizes the main findings and conclusions of the existing literature and discusses implications and recommendations for future research and practice. The study reveals that the immune response of sand flies is a key determinant of vector competence and transmission of Leishmania parasites, and that several molecular and cellular mechanisms are involved in the interaction between parasite and vector. The study also suggests that there are potential strategies for controlling leishmaniasis, such as interfering with parasite development, modulating the vector's immune response or reducing the vector population. However, the study also identifies several gaps and limitations in current knowledge and calls for more comprehensive and systematic studies on vector-parasite interaction and its impact on leishmaniasis transmission and control., (© Indian Society for Parasitology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross-sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate-related factors into account. In addition, PCR-based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo., (© 2024 The Author(s). Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Present study was carried to determine the sand fly species composition, breeding sites ecology, seasonal abundance, and spatial distribution in district Malakand, Khyber Pakhtunkhwa, Pakistan. In addition, risk factors associated with cutaneous leishmaniasis (CL) were also evaluated. Survey of indoor and outdoor habitats was carried out using sticky traps in 31 villages of Dargai and Batkhela tehsils of Malakand. Soil from habitats of adult and immature sand flies was analysed. Questionnaire‐based household survey was also performed in these villages to assess risk factors associated with CL. Soil samples from selected CL positive households were analysed for its contents. Additionally, clinicoepidemiological data from local health centres was examined for the year 2019. Total of 3,140 sand flies belonging to 18 species were collected. Phlebotomus sergenti was the most abundant species (38.16%). Its abundance had a strong positive correlation with mean monthly relative humidity and negative correlation with average temperature. Phlebotomus sergenti and Phlebotomus papatasi were abundant at an elevation ranging from 320 to 1,120 m above sea level and in agricultural lands near human settlements. Flight height preference apparatus collected maximum sand flies at 30 cm (1ft) above the ground and all species associated negatively with height. Soil analysis from habitats of adult and immature flies showed that highest mean number of adults and immatures were recorded from silt loam which carried highest concentrations of K2O, Mg, Ca, and Zn. Number of immature sand flies correlated moderately (r =.7, p <.05) with K2O soil concentrations. There was significant similarity between organic matter contents in soil samples from positive breeding sites and CL households (Wilcoxon rank‐sum test, p =.1976). In multivariate analysis model for CL risk factors, age (26–35 and >35 years), knowledge of leishmaniasis, living in a middle and upper class, preachers visit to villages, and assumption that Afghan refugees are more prone to CL were significant. CL patient's archived data from health centres showed that majority of patients had lesions on face and hands. Patient's influx was highest in February and March. [ABSTRACT FROM AUTHOR]

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Background: In Iran, both cutaneous leishmaniases (CL) and visceral leishmaniases (VL) are endemic, recording one of the 10 highest CL prevalence in the world. Parasites are transmitted by the bite of infected Phlebotomus sand fly females. Several sand fly species have been identified as vectors in the studied region of Kerman province. Residual spraying to control adult sand flies, is the only way to decrease the spreading of the diseases but, following control treatment against malaria vectors in endemic areas in Iran, resistance or tolerance to insecticides emerged in some sand fly species. The objective of this study was to survey insecticides susceptibility levels of 3 vector species in wild sand fly populations in different foci of the diseases in Kerman province. Ph. sergenti, and Ph. papatasi respectively vectors of anthroponotic and zoonotic cutaneous leishmaniases and for the first time Ph. alexandri one of the anthroponotic visceral leishmaniases vector were tested against: deltamethrin 0.05%, malathion 5%, dichloro-diphenyl-trichloroethane (DDT) 4%. Materials and methods: In leishmaniases endemic areas species specific sand fly sites were selected in Kerman province, and specimens were collected by manual aspirators at different time intervals during the spring and summer 2019. All the susceptibility tests were performed according to the WHO tube test recommended procedure. Results: Twenty five blood-fed female sand flies from the region's prevalent species were used in each pooled test replicates. All wild specimens died within 60 min of exposure to DDT 4%, malathion 5%, and deltamethrin 0.05%, but the mortality rate for Ph. papatasi exposed to malathion and DDT was 91.6% and 66.6%, respectively. Conclusion: According to current study results, Ph. sergenti and Ph. alexandri are highly susceptible to all the evaluated insecticides in the study areas. However, Ph. papatasi was susceptible to deltamethrin (100% mortality), possibly resistant or tolerant to malathion (91.6% mortality), and confirmed to be resistant to DDT (66.6% mortality). [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) due to Leishmania tropica is a neglected tropical disease characterized by a wide geographical distribution in the Mediterranean basin and is endemic in several of its countries. In addition, the vector Phlebotomus sergenti is abundantly present all around the basin. Its transmission cycle is still subject to debate. In some countries, the presence of an animal reservoir has been confirmed. In Morocco, CL due to L. tropica has risen since the 1980s and has spread widely to become the most abundant form of leishmaniasis in the territory. However, the anthroponotic transmission is so far the only recognized mode, despite recordings of L. tropica infection in animal hosts. In this review article, we assess the situation of CL due to L. tropica in the Mediterranean basin with a focus on Morocco and gather knowledge about any potential zoonotic transmission in the country. A concomitant zoonotic transmission could explain the persistence of the disease in areas where human protective measures combined with vector management did not help reduce the disease burden. Author summary: Cutaneous leishmaniasis (CL) due to Leishmania tropica is a neglected tropical disease still considered to be anthroponotic in Morocco despite evidence of zoonotic transmission in other endemic countries of the Mediterranean basin. Its high incidence in the country makes it necessary to investigate whether or not a potential concomitant zoonotic transmission exists alongside the anthroponotic one. Moreover, several reports of natural animal infection have been noted in the country. In this paper, we review how CL due to L. tropica is present around the Mediterranean basin, with a particular focus on the Moroccan foci, and try to unveil the existence of a zoonotic transmission in the area. A deep understanding of the transmission patterns of L. tropica in the region is necessary for the implementation of effective control measures and a better assessment of the disease burden. In addition, the complexity of this neglected disease requires more fundamental and applied research to unveil potential factors involved in its spread. [ABSTRACT FROM AUTHOR]

Background: Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various human and animal pathogens such as Bartonella bacilliformis, Phlebovirus, and parasitic protozoa of the genus Leishmania, causative agent of leishmaniases that account among most significant vector-borne diseases. The Maghreb countries Mauritania, Morocco, Algeria, Tunisia, and Libya occupy a vast area of North Africa and belong to most affected regions by these diseases. Locally varying climatic and ecological conditions support diverse sand fly fauna that includes many proven or suspected vectors. The aim of this review is to summarize often fragmented information and to provide an updated list of sand fly species of the Maghreb region with illustration of species-specific morphological features and maps of their reported distribution. Materials and methods: The literature search focused on scholar databases to review information on the sand fly species distribution and their role in the disease transmissions in Mauritania, Morocco, Algeria, Tunisia, and Libya, surveying sources from the period between 1900 and 2020. Reported distribution of each species was collated using Google Earth, and distribution maps were drawn using ArcGIS software. Morphological illustrations were compiled from various published sources. Results and conclusions: In total, 32 species of the genera Phlebotomus (Ph.) and Sergentomyia (Se.) were reported in the Maghreb region (15 from Libya, 18 from Tunisia, 23 from Morocco, 24 from Algeria, and 9 from Mauritania). Phlebotomus mariae and Se. africana subsp. asiatica were recorded only in Morocco, Ph. mascitti, Se. hirtus, and Se. tiberiadis only in Algeria, whereas Ph. duboscqi, Se. dubia, Se. africana africana, Se. lesleyae, Se. magna, and Se. freetownensis were reported only from Mauritania. Our review has updated and summarized the geographic distribution of 26 species reported so far in Morocco, Algeria, Tunisia, and Libya, excluding Mauritania from a detailed analysis due to the unavailability of accurate distribution data. In addition, morphological differences important for species identification are summarized with particular attention to closely related species such as Ph. papatasi and Ph. bergeroti, Ph. chabaudi, and Ph. riouxi, and Se. christophersi and Se. clydei. [ABSTRACT FROM AUTHOR]

Background: Dogs are the reservoir host of Leishmania infantum, the agent of zoonotic visceral leishmaniasis (VL), which is transmitted by the bite of phlebotomine sand flies. The sand fly Phlebotomus perniciosus is the main vector of zoonotic VL in the western Mediterranean region. Fluralaner has been shown to effectively kill this vector. The aim of this study was to evaluate the insecticidal efficacy of oral fluralaner in dogs bitten by P. perniciosus. Methods: Two parallel-group, negative-controlled, randomized, masked laboratory trials with equivalent designs were performed in two different locations using two different pathogen-free laboratory-bred P. perniciosus strains for the challenge. In each trial, 12 purpose-bred beagles, initially ranked on natural attractiveness to sand flies, were randomly allocated to two groups (6 animals/group). Dogs in one group received fluralaner orally at the approved dose on day 0, and dogs in the control group were not treated. Each dog was subsequently exposed to an average of 70 unfed live sand fly females on days 1, 28, 56 and 84. Viability of blood-fed females was then evaluated for up to 96 h after exposure, and insecticidal efficacy was measured as the survival rate of flies fed on the fluralaner-treated dogs versus that of dogs in the control group. Significance was calculated for the proportion of live fed sand fly counts from treated versus control group dogs. Results: Comparison of the survival proportions between treated and control groups showed that fluralaner insecticidal efficacy was highly significant in both trials (P < 0.001 or P < 0.01 in different assessments) through to day 56. In the first trial, efficacy reached 100% on days 1 and 28, and 99.1% on day 56; in the second trial, the insecticidal efficacy was 98.5, 100 and 85.9%, respectively on the same days. On day 84, efficacy was in the range of 53–57% (P < 0.05) in the first trial and 0% in the second trial. Conclusion: A single oral fluralaner administration to dogs under laboratory conditions results in strong and reproducible insecticidal efficacy against P. perniciosus for at least 8 weeks. [ABSTRACT FROM AUTHOR]

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Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography‐mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p‐cymene and the benzenoid m‐cresol, p‐cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose–response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five‐component blends, referred to as Blend‐f and Blend‐m for females and males respectively, were formulated and tested in dose–response assays against 1‐octen‐3‐ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant‐derived VOCs can be exploited for sand fly management. [ABSTRACT FROM AUTHOR]

Background: Among neglected zoonotic diseases, leishmaniases caused by Leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. Vector management plays a crucial role in reducing the burden of vector-borne diseases by WHO’s global plan. The objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from Esfahan Province, central Iran, to the recommended insecticides by WHO. Methods: Sand flies were collected by mouth aspirator in Matin Abad desert Eco-resort and were tested using WHO adult mosquito test kit against Dichlorodiphenyltrichloroethane (DDT) 4%, Deltamethrin 0.05%, Malathion 5% and Propoxur 0.1%. The number of knockdown sand flies were recorded during exposure time in ten minutes interval for DDT and Deltamethrin and they were allowed to recover for 24 hours. Knockdown Time50 (KD50) and KD90 were generated for them using Probit software. They were mounted and identified by valid keys. Results: Among the tested insecticides against female Phlebotomus papatasi, DDT, Deltamethrin, and Malathion recorded the highest mortality rate of 100%, followed by Propoxur with 92.2% mortality for a one-hour exposure. For DDT, KD50 and KD90 were calculated 21.87 and 42.93 and for Deltamethrin, they were 23.74 and 56.50 minutes respectively. Total sand flies exposed with DDT and Deltamethrin shed their leg(s). Conclusion: It is concluded that Ph. papatasi from central Iran is susceptible to DDT, Deltamethrin, Malathion, and Propoxur. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a parasitic disease, that in the Iberian peninsula is caused by the protozoan Leishmania infantum and transmitted mainly by the phlebotomine sand fly vector Phlebotomus perniciosus. The main reservoir of the parasite is the dog, although it also affects other animals, including humans. The spatial distribution of the vector in Torrelodones (Madrid, Spain) has been analyzed using geographic information systems (GIS). The presence of Sergentomyia minuta (54.4%) and P. perniciosus (45.6%) has been confirmed. The highest vector density was recorded near the animal shelter. A statistical analysis of the association between sand flies density and macro- and microclimatic variables shows a significant negative correlation between vector density and high precipitation and temperature values. These data will allow the development of containment protocols for phlebotomine sand fly populations, elaborating multifactorial control plans based on vector control systems and divulgation through environmental education projects. [ABSTRACT FROM AUTHOR]

Abstract Background Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. Methods In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. Results Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. Conclusions Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

Objective:In Turkey, leishmaniasis occurs in two clinical forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). CL has been reported mainly from south-eastern Anatolia and the eastern part of the Mediterranean region, whereas VL is sporadic in almost all geographical regions of Turkey. Both clinical forms of the disease have been recorded in Kayseri province for decades, but no study has been conducted on the sand fly fauna in this area. Therefore, we determine the species composition and population dynamics of sand flies prevalent in Kayseri province and identify possible vector species.Methods:Data related to the recent locations of CL and VL cases were obtained from the Ministry of Health, and sand flies were collected in 14 localities of different districts using CDC light traps. The specimens were transferred to the laboratory in 70% ethanol, and morphological identification to the species level was performed using previously written keys.Results:A total of 1,636 sand fly specimens were collected. Morphological identification revealed eight species of the genus Phlebotomus (P. perfiliewi s.l., P. halepensis, P. simici, P. major s.l., P. papatasi, P. tobbi, P. sergenti and P. mascittii) and one species of the genus Sergentomyia (S. dentata). Among all specimens, members of subgenus Adlerius (54.58%) formed the dominant group, followed by the subgenus Larroussius (43.76%).Conclusion:Our results showed that P. halepensis, belonging to subgenus Adlerius, and P. perfiliewi, belonging to the subgenus, are probable vectors of cutaneous and VL in the province, respectively.

Aim Phlebotominae sandflies are primary vectors of phleboviruses, causing the sandfly fever disease. The aim of this study was to detect and report the presence of flaviviruses in Phlebotominae sandflies captured in Bosnia and Herzegovina. Methods After a microscopic and morphometric analysis, the final identification of collected Phlebotomus specimens was confirmed by PCR, using a hemi-nested polymerase chain reaction on extracted and reversely transcribed RNA. Results We obtained a 155 nt long fragment of the viral non-structural protein 5 (NS5) gene (GenBank accession no. MN090154). The acquired nucleotide sequence, provisionally named as Drežnica, showed a maximum of 70-80% identity in 70-88% (110-137 nucleotides) of the query coverage with several Anopheles, Sabethes, Calbertado and Culex flaviviruses. Maximum likelihood phylogenetic analysis showed that the new flavivirus Drežnica clusters together with the flavivirus isolated from Culiseta annulata mosquitos. Conclusion We report the presence of flavivirus in Phlebotominae sandflies, captured in Drežnica, Herzegovina for the first time. The next phase of research will be directed towards virus cultivation, obtaining a longer or complete virus sequence and clarifying the medical and epidemiological importance of the Drežnica virus.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Background: Sand fly females require a blood meal to develop eggs. The size of the blood meal is crucial for fecundity and affects the dose of pathogens acquired by females when feeding on infected hosts or during experimental membrane-feeding., Methods: Under standard laboratory conditions, we compared blood meal volumes taken by females of ten sand fly species from four genera: Phlebotomus, Lutzomyia, Migonomyia, and Sergentomyia. The amount of ingested blood was determined using a haemoglobin assay. Additionally, we weighed unfed sand flies to calculate the ratio between body weight and blood meal weight., Results: The mean blood meal volume ingested by sand fly females ranged from 0.47 to 1.01 µl. Five species, Phlebotomus papatasi, P. duboscqi, Lutzomyia longipalpis, Sergentomyia minuta, and S. schwetzi, consumed about double the blood meal size compared to Migonomyia migonei. The mean body weight of females ranged from 0.183 mg in S. minuta to 0.369 mg in P. duboscqi. In males, the mean body weight ranged from 0.106 mg in M. migonei to 0.242 mg in P. duboscqi. Males were always lighter than females, with the male-to-female weight ratio ranging from 75% (in Phlebotomus argentipes) to 52% (in Phlebotomus tobbi)., Conclusions: Females of most species took a blood meal 2.25-3.05 times their body weight. Notably, the relatively tiny females of P. argentipes consumed blood meals 3.34 times their body weight. The highest (Mbl/Mf) ratios were found in both Sergentomyia species studied; females of S. minuta and S. schwetzi took blood meals 4.5-5 times their body weight. This parameter is substantially higher than that reported for mosquitoes and biting midges., (© 2024. The Author(s).)

The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania, has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum, Leishmania major, or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it., Methods: Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the 'Servicios Veterinarios de Sil' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection., Results: A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum., Conclusions: This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease., (© 2024. The Author(s).)

Background: Sand flies are principal vectors of the protozoan parasites Leishmania spp. and are widely distributed in all warmer regions of the world, including the Mediterranean parts of Europe. In Central European countries, the sand fly fauna is still under investigation. Phlebotomus mascittii, a suspected but unproven vector of Leishmania infantum, is regarded as the most widely distributed species in Europe. However, many aspects of its biology and ecology remain poorly known. The aim of this study was to provide new data on the biology and ecology of Ph. mascittii in Austria to better understand its current distribution and potential dispersal. Methods: Sand flies were collected by CDC light traps at four localities in Austria for 11 (2018) and 15 weeks (2019) during the active sand fly season. Climatic parameters (temperature, relative humidity, barometric pressure and wind speed) were retrospectively obtained for the trapping periods. Sand flies were identified by a combined approach (morphology, DNA barcoding, MALDI-TOF protein profiling), and blood meals of engorged females were analysed by DNA sequencing and MALDI-TOF mass spectrometry. Results: In total, 450 individuals of Ph. mascittii were caught. Activity was observed to start at the beginning of June and end at the end of August with peaks in mid-July at three locations and early August at one location. Increased activity was associated with relatively high temperatures and humidity. Also, more individuals were caught on nights with low barometric pressure. Analysis of five identified blood meals revealed chicken (Gallus gallus) and equine (Equus spp.) hosts. Sand fly abundance was generally associated with availability of hosts. Conclusion: This study reports unexpectedly high numbers of Ph. mascittii at selected Austrian localities and provides the first detailed analysis of its ecology to date. Temperature and humidity were shown to be good predictors for sand fly activity. Blood meal analyses support the assumption that Ph. mascittii feeds on mammals as well as birds. The study significantly contributes to understanding the ecology of this sand fly species in Central Europe and facilitates prospective entomological surveys. [ABSTRACT FROM AUTHOR]

[Display omitted] • The Brazilian Leishmania major -like isolates are in fact L. major. • These isolates were imported from the Old World by at least two independent events. • All L. major isolates (previous named L. major -like) presented aneuploidies. • The infective isolate presented an increased gene copy number of known virulence factors. Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named " L. major -like", and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major -like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major -like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phlebotomus argentipes is a sole vector of Leishmania donovani in the Indian subcontinent. • 40% of humans in the study area have IgG antibodies against P. argentipes saliva. • A correlation was found between IgG responses against P. argentipes saliva and rPagSP06. • rPagSP06 is a valid antigen to measure human exposure to P. argentipes. Phlebotomus argentipes is a predominant vector of Leishmania donovani , the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes , sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti- P. argentipes saliva IgG responses. The anti-rPagSP06 and anti- P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti- P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area. [ABSTRACT FROM AUTHOR]

Background The aim was to investigate an emerging focus of cutaneous leishmaniasis (CL) in Gampaha district, Western Sri Lanka. Methods Sociodemographics of CL patients in Mirigama, Gampaha district, were compared with controls using a structured questionnaire. Clinical data and geographic positioning system coordinates of patients were recorded. Sandflies collections were performed in four selected sites using indoor hand collection (162 units) and cattle-baited net traps (n=3). Results Of 73 patients (mean age 43.6 y; SD 20), 58.9% were males. Lesions consisted of nodules (52.1%) and ulcers (47.9%). Single lesions were seen in 69.9% of patients, while 30.1% had multiple lesions. Except for one mucosal lesion, all the others were cutaneous. Treatment consisted of sodium stibogluconate and cryotherapy (46.6%), sodium stibogluconate (45.2%) or cryotherapy (8.2%). Multiple lesions were more likely to receive combination therapy (p=0.013). The majority of those employed (81.1%; 27/33) were engaged in outdoor work. Male gender, low education attainment and poor disease awareness were significant risk factors for CL (p<0.05). Four households reported multiple cases. Phlebotomus argentipes was detected in all cattle-baited net traps and 35.3% of indoor hand collection units. Conclusions CL is emerging in Gamapha district with Mirigama as the epicentre. Case distribution indicates local transmission, with P. argentipes as the probable vector. [ABSTRACT FROM AUTHOR]

Background: Phlebotomine sand flies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a first update on their occurrence, species composition and bloodmeal sources after 50 years. Methods: During 5 seasons (2013–2017), 58 localities from 20 regions were surveyed for presence of sand flies using CDC light traps and manual aspirators. Species identification was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profiling). In engorged females, host blood was identified by three molecular techniques (RFLP, cytb sequencing and MALDI-TOF peptide mass mapping). Population structure of most abundant species was studied by cox1 haplotyping; phylogenetic analyses of ITS2 and cox1 genetic markers were used to resolve relationships of other detected species. Results: In total, 793 sand flies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identified by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfiliewi and Phlebotomus sp. (Adlerius), the first being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only five haplotypes of cox1 detected. Distinct morphological and molecular characters of Phlebotomus sp. (Adlerius) specimens suggest the presence of a yet undescribed species. Conclusions: Our study revealed the presence of stable sand fly populations of three species in Moldova that represent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied. [ABSTRACT FROM AUTHOR]

The sand fly, Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae), is the main vector of Leishmania major Yakimoff and Schokhor, 1914, the causative agent of zoonotic cutaneous leishmaniasis North Africa, the Middle East, and North Sinai. The purpose of this study was to determine the effect of fungi on P. papatasi larvae, pupae, and adults using light microscopic analysis, scanning electron microscope (SEM), and histopathological studies. Specifically, larvae, pupae, and adult P. papatasi were infected with the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. Scanning electron microscope and histopathological methods were used to investigate the destructive impact of the fungi on the external and internal structures of P. papatasi. The results revealed propagation of the conidia on the cuticles of all P. papatasi life stages, including on the compound eyes, leg setae, thorax, wings, and abdomen of the adults. Histological sections of the control and treated larvae, pupae, and adults showed many alterations and malformations in the body and tissues of all life stages after 72 h. These results demonstrated that B. bassiana was more effective than M. anisopliae as a biological control of phlebotomine sand flies. Further studies to determine the best methods for delivery and application in the diverse ecological settings of the various leishmaniasis vectors are recommended. [ABSTRACT FROM AUTHOR]

The late Bronze Age eruption of the Thera volcano was among the largest eruptions of the Holocene era. This catastrophic event might perish all organisms from the ancient Santorini and could seriously impact the sand fly fauna of the Aegean islands. To investigate these effects, the survival possibility of the sand fly fauna in the Santorini islands and the biogeographic investigation of the sand fly fauna of eleven Aegean islands were conducted. It was found that only the south and east slopes of the massifs of Thira could provide refuge for sand fly populations. The expression-based heat map of the Jaccard coefficient matrix data showed that the Santorini islands and their neighbouring Anafi, Folegandros had clearly different z -score patterns compared to the other islands. It could be a late sign of the devastating effect of the Minoan eruption and/or the consequence of the distance of these islands from the mainland. Neither the glacial seashore patterns nor the geographic-climatic conditions can explain the present sand fly fauna of the Aegean Archipelago. If the sand fly populations of ancient Santorini survived the Minoan cataclysm, it could indicate that the environmental tolerance and the resilience of the sand fly populations can be high, and local geological and geomorphological conditions can play a greater role in the survival of sand fly species than previously assumed. [ABSTRACT FROM AUTHOR]

Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on FST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging. [ABSTRACT FROM AUTHOR]

Background: In a countrywide study aiming to update the knowledge on diversity of sand fly species in Romania, a sand fly population was observed in an isolated system of cave microhabitats. The caves are located in the protected area of Canaraua Fetii, Dobrogea region, southeastern Romania. The highest sand fly diversity was recorded in this area between 1968 and 1970. This work presents a study conducted to estimate the seasonal variation of the sand fly species in correlation with the particular environmental factors of the isolated system of cave microhabitats. Methods: Sand flies were collected between May and October 2020 from one trapping site of interest in Canaraua Fetii. The trapping site consisted of a cave entrance. CDC miniature light traps and sticky traps were used to collect insects from the exterior walls of the cave entrance. Species identification of collected sand flies was done using morphological keys. Statistical analysis of the trapping and climatic data was performed. Results: From all collected sand flies, 99.7% (818/822) were Phlebotomus neglectus, 0.1% (1/822) Ph. balcanicus and 0.2% (2/822) Sergentomyia minuta. Sand fly activity was first observed on 2 July and last on 24 September. A monomodal abundance trend was present, with the peak activity between 16 and 17 July. The analysis of the climatic data showed correlations between the total number of captured sand flies and both average temperature and average relative humidity. The total number of collected specimens was statistically higher when CDC miniature light traps were used compared to sticky traps. The number of females on the sticky traps was significantly higher than the number of males on the same trap type. Compared with the sticky traps, significantly more males were collected by CDC miniature light traps. This is the first record of Se. minuta in Romania after 50 years of no records (despite the trapping effort of the last 5 years in the country). Also, Ph. sergenti, previously present in this location, was not found. Conclusions: In the investigated natural habitat, the diversity of the sand fly species appears to have changed, with the predominance of Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the two predominant species in 1968–1970). A monomodal abundance trend was observed as in other regions of the country. The sand fly activity in this particular cave microhabitat appears to be longer than in other regions in Romania. Longer sand fly activity increases the zoonotic risk of various pathogenic species' transmission, with an impact on public health, as sand flies are important insect vectors. [ABSTRACT FROM AUTHOR]

Background: Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. Methods: In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. Results: The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well. Conclusions: Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples. [ABSTRACT FROM AUTHOR]

Background: An entomological study was conducted in the Canaraua Fetii Special Protection Area in the Dobrogea region, South-Eastern Romania. Four sand fly species were recorded at this location between 1968 and 1970: Phlebotomus neglectus, Ph. balcanicus, Ph. sergenti and Sergentomyia minuta. The most abundant sand fly species recorded at that time were Ph. balcanicus and Se. minuta. In the context of a countrywide study to update the sand fly species diversity, we surveyed the same area, recording also a previously unknown Ph. (Transphlebotomus) sp., for which we provide a formal description here. Methods: Sand flies were collected between July and August in 2018 and 2019 in three sites from Canaraua Fetii, Dobrogea region, Romania. The general aspect of the landscape is of a canyon (vertical, narrow walls and deep valleys). Species identification was done using both morphological and molecular analyses. Results: Out of 645 collected sand flies, 644 (99.8%) were morphologically identified as Ph. neglectus, while one female specimen (0.2%) was assigned to a previously unknown species, belonging to the subgenus Transphlebotomus. The morphological and molecular examination of this specimen showed that it is a previously unknown species which we formally describe here as Phlebotomus (Transphlebotomus) simonahalepae n. sp. Also, Ph. balcanicus, Ph. sergenti, and Se. minuta (previously recorded in this location) were not present. Conclusions: The study revealed for the first time the presence of sand flies of the subgenus Transphlebotomus in Romania. Moreover, a new species, Ph. simonahalepae n. sp., was described based on a female specimen, raising the number of species in this subgenus to six. In the investigated natural habitat, the predominant species was Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the predominant species in 1968–1970). [ABSTRACT FROM AUTHOR]

Leishmanioses are vector-borne diseases, some of zoonotic concern, transmitted by phlebotomine sand flies (Diptera, Psychodidae). Reports of reptile-associated Leishmania tarentolae in humans, and of Leishmania infantum in Sergentomyia minuta sand flies prompted the development of an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) to detect and differentiate these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n = 188) and Se. minuta (n = 171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n = 4) and Tarentola mauritanica (n = 3). The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from Leishmania spp. and spiked DNA samples from lizards, was 2.3 x 10-7 ng/2 µl for L. infantum and 2.1 x 10-7 ng/2 µl for L. tarentolae. The dqPCR detected up to 4.3 x 10-6 ng/2 µl of L. infantum and up to 4.9 x 10-7 ng/2 µl of L. tarentolae. Of the 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantum and L. tarentolae, respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive for L. tarentolae. This new dqPCR may improve the diagnosis of L. infantum and L. tarentolae and aid to assess the role of lizards as reservoirs and of Se. minuta as vector, for these Leishmania spp. [ABSTRACT FROM AUTHOR]

There is a need for standardizing sand fly sampling methodology and guidance on trap positioning for quantitative sand fly studies. We investigated differences in sand fly density with 'sticky' interception and CO2‐light attraction traps, in relation to trap distance to the ground and the presence or absence of a continuous or discontinuous (wire mesh) vertical surface adjacent to the trap. The study, conducted in a dog kennel in southeast Spain, lasted 48 days and collected 692 Phlebotomus papatasi, P. perniciosus, P. ariasi and Sergentomyia minuta specimens. There were no significant differences between species with respect to trap position. Overall, density in sticky traps was highest closest to the ground and next to the continuous vertical surface, followed sequentially by traps similarly placed adjacent to the wire mesh and those hanging from a rope across the kennel yard. In contrast, density in CO2‐light traps was highest in traps hanging from the rope near the ground, followed by those next to the continuous vertical surface. The overall negative relationship between sand fly density and ground distance was not significant for CO2‐light traps next to the continuous vertical surface. Modelling also suggested that sand flies do not use the wire mesh to move vertically. [ABSTRACT FROM AUTHOR]

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using "sticky" interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis is an ancient endemic disease in Iran and continues to be a growing health threat to community development and the environment. This paper explains how to use the facilities of health centers for developing a laboratory network on vectors and reservoir hosts of cutaneous leishmaniasis in Iran. Methods: A literature search was performed of the relevant multiple databases to include studies on vectors and reservoirs of cutaneous leishmaniasis in Iran. A team of experienced experts was performed. After holding several meetings, field visits and organizing workshops, the activities of laboratories were determined at three levels. Results: Entomological studies showed that 5 species of the genus Phlebotomus and 10 species of the genus Sergentomyia are active in the south, 4 species of the genus Phlebotomus and one species of the genus Sergentomyia in the central part and 5 species of the genus Phlebotomus and 2 species of the genus Sergentomyia in the north east. Reservoir hosts were identified in the study areas. The tasks of laboratories were regulated at different levels. Conclusion: The Iranian Ministry of Health and Medical Education should prioritize the employment of capable persons in the field of Medical Entomology and Vector Control. The survival of this laboratory network depends on hiring and employing interested and persistent people. The universities of Medical Sciences that have the facilities to set up this network will be a very effective partner in the control of the disease in high risk areas. The results can be used in neighboring countries. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly. Methods: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans. Results: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal. Conclusions: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect. [ABSTRACT FROM AUTHOR]

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. 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.

Leishmaniases are caused by protozoan parasites of the genus Leishmania transmitted by females blood-feeding phlebotomine insects (Diptera: Psychodidae). In Tunisia, cutaneous and visceral leishmaniases are of public health concern. In Tunisia, 17 species of phlebotomine sand flies are described. Here we investigate natural infection in Tunisian mixed foci regions of leishmaniases. We trap female sandflies during the Leishmania transmission season in the country's central-eastern and northern parts. We investigate Leishmania infection using PCR-RFLP targeting the ITS1 ribosomal DNA, followed by enzymatic digestion with HaeIII; then, we identify sand flies using molecular methodologies. We confirm the presence of Phlebotomus papatasi and Phlebotomus perniciosus infected by L. major and L. infantum parasites in Tunisia.

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. [ABSTRACT FROM AUTHOR]