Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

A next-generation (DNA) sequencing (NGS)-based method for identifying the sources of sugar meals in mosquito vectors of West Nile virus in Israel.

Mosquitoes of the genus Culex comprise important vectors of pathogenic arboviruses in our region, including West Nile and Rift Valley Fever viruses. To improve our understanding of the epidemiology and transmission dynamics of arboviruses, we need to study the behavior and ecology of their vectors. The feeding patterns of the vector mosquitoes can be very useful in determining how and where to focus control efforts. For example, determining the preferred blood hosts of the females can assist in the implementation of potentially efficacious strategies for focused control of mosquito females. Determining the plants from which both sexes derive their sugar meals can comprise the initial step towards the formulation of efficient lures for trapping mosquitoes. In the past, plant meal identification was based mainly on chemical detection of fructose and microscopical observations of cellulose particles in mosquito guts. More recent studies have utilized DNA barcoding capable of identifying plant food sources. In the current study, we identify multiple plant species from which large numbers of mosquitoes obtained their sugar meals in one experimental procedure. We employed next generation DNA sequencing to sequence the chloroplast specific plant genes atpB and rbcL.

Leishmania infection in cats and dogs housed together in an animal shelter reveals a higher parasite load in infected dogs despite a greater seroprevalence among cats.

BACKGROUND: An outbreak of leishmaniosis was studied in cats and dogs housed together with no separation in an animal shelter in Israel. METHODS: The study included recording of clinical signs, serology for Leishmania infection by ELISA, PCR of blood for Leishmania DNA by ITS1 HRM and kDNA PCR, parasite quantification, and trapping of sand flies around the shelter. RESULTS: Thirty-seven % (22/60) of the dogs and 75% (50/67) of the cats were seropositive to L. infantum with a significantly higher seropositivity rate in the cat population (χ2 = 42.160, P < 0.0001). Twenty-five percent (15/60) of the dogs were positive for Leishmania by blood PCR, 12% by the Leishmania ITS1 HRM PCR and 22% by kDNA PCR. Of the cats, 16% (11/67) were positive by kDNA PCR and none by ITS1 HRM PCR. All the PCR-positive animals were infected by L. infantum verified by DNA sequencing and there was no significant difference between the PCR-positivity in the dog and cat populations. Altogether, 43% (26/60) of the dogs and 79% (53/67) of the cats were positive by serology or PCR for L. infantum. The average Leishmania parasite load in the blood of PCR-positive dogs (42,967 parasites/ml) was significantly higher than in PCR-positive cats (1259 parasites/ml) (t(12) = 2.33, P = 0.037). Dogs that were positive by the Leishmania ITS1 HRM PCR and kDNA PCR had significantly higher parasite loads than dogs positive only by the kDNA PCR (t(11) = - 3.186580, P < 0.009). No significant effect was found for FIV seropositivity on Leishmania infection in the cats (χ2 = 0.506, P = 0.777). A higher percentage of Leishmania-positive dogs showed clinical signs compatible with leishmaniosis compared to Leishmania-positive cats (100 vs 52.8%, χ2 =15.242, P < 0.0001). Phlebotomus perfiliewi, a proven vector of L. infantum, comprised 92% of trapped sand flies. CONCLUSIONS: Comparisons of populations of cats and dogs exposed to sand flies and L. infantum under the same conditions indicated that although a high rate of exposure was detected in cats as manifested by a significantly greater degree of seropositivity, dogs had significantly higher blood parasite loads, and were likely to be more infectious to sand flies than cats.

Molecular characterization of six Hyalomma species using mitochondrial markers.

Hyalomma species (Acari: Ixodidae) are vectors of several human and animal pathogens. However, due to their similar morphological properties, classification of related Hyalomma species is often challenging. Here we describe a combined approach for molecular characterization of six Hyalomma species: H. aegyptium, H. dromedarii, H. excavatum, H. impeltatum, H. marginatum and H. turanicum. This procedure was developed using a combination of PCR amplification of four molecular markers, followed by sequencing and species-specific restriction analysis. Segments from the following genes were used as markers: 12S rRNA, 16S rRNA, Cytochrome C oxidase subunit 1 (COX1), and Cytochrome B (CytB). Phylogenetic analysis based on the amplified sequences was consistent with the morphology-based classification. It revealed relative close proximity of H. excavatum, H. marginatum and H. turanicum, and close proximity of H. aegyptium and H. dromedarii to each other. H. impeltatum was examined using the COX1 and CytB markers, and in both cases was located on a separate clade from the other five species. Digestion of the amplified products using specific restriction enzymes enabled clear distinction between the six species. This report is the first to describe CytB marker sequences of the studied species, and the first to describe COX1 marker sequences of H. aegyptium, H. excavatum, H. impeltatum and H. turanicum. The information obtained in this study may therefore be useful for future combined morphological-molecular Hyalomma characterization.

Epidemiologic and phylogenetic analysis of the 2018 West Nile virus (WNV) outbreak in Israel demonstrates human infection of WNV lineage I.

As at 12 November 2018, an outbreak of West Nile virus (WNV) was responsible for 139 WNV infection cases in Israel. Here, we characterise the epidemiology of the outbreak and demonstrate that only WNV lineage I was circulating in mosquitoes and responsible for WNV infection in humans. This suggests that the concurrence of the outbreak in Israel with WNV outbreaks in several European countries is not due to a common, more virulent WNV genotype.

High Endemicity and Distinct Phylogenetic Characteristics of Sindbis Virus in Israel.

Sindbis virus (SINV) is a mosquito-borne Alphavirus responsible for outbreaks of SINV disease, mainly in north Europe. SINV has been isolated from mosquitoes in Israel since the 1980s but SINV disease outbreaks have never been recorded. To gain better understanding of the kinetics of SINV circulation in Israel, 3008 mosquito pools, collected 2004-2006 and 2013-2015, were tested for SINV and phylogenetic analysis was conducted on partially sequenced SINV-positive pools. Results indicate possible expansion of SINV circulation across Israel in 2013-2015 compared to 2004-2006 with 6.35% (191 pools) of total pools positive for SINV RNA. Phylogenetic analysis showed all sequenced Israeli SINV strains belong to genotype I and form, together with SINV sequences from Saudi Arabia, a distinct Middle Eastern cluster. With high endemicity of SINV and as a major crossroads for bird migration between Africa and Eurasia, Israel provides valuable information on SINV dynamics and pathogenicity.

Spatial distribution of West Nile virus in humans and mosquitoes in Israel, 2000-2014.

OBJECTIVES: Israel has a long history of West Nile virus (WNV) morbidity, and the rate of detection of WNV in mosquitoes has been high since 2000. The aim of this study was to integrate several WNV datasets in order to gain an insight into the geographical distribution of WNV in Israel. METHODS: Three choropleth maps were generated showing WNV human morbidity, WNV prevalence in mosquitoes, and the results of a nationwide serological survey, based on the division of Israel into 15 sub-districts. RESULTS: The maps show a high endemicity of WNV in Israel. In respect to the morbidity map, the population residing in the central part of the country and in Arava Region is at higher risk of developing the disease than the population of the rest of Israel. Interestingly, high prevalence rates of both WNV serology and WNV-infected mosquitoes were detected in Arava Region, but lower prevalence rates were detected in most areas of the coastal region, suggesting that other factors might also be important in the development of symptomatic WNV infections. CONCLUSIONS: These results underline the high prevalence of WNV in Israel and point to specific risk areas for WNV infections across the country.

Usutu Virus RNA in Mosquitoes, Israel, 2014-2015.

We identified Usutu virus (USUV) RNA in 6 pools of mosquitoes trapped in northern Israel during 2014-2015. These Israeli strains were most similar to strains identified in Senegal and Germany, which further elucidates common ancestry and evolutionary dynamics of USUV. Our findings suggest that human infection with USUV might occur in Israel.

Distribution and Dispersal of Phlebotomus papatasi (Diptera: Psychodidae) in a Zoonotic Cutaneous Leishmaniasis Focus, the Northern Negev, Israel.

BACKGROUND: Zoonotic cutaneous leishmaniasis has long been endemic in Israel. In recent years reported incidence of cutaneous leishmaniasis increased and endemic transmission is being observed in a growing number of communities in regions previously considered free of the disease. Here we report the results of an intensive sand fly study carried out in a new endemic focus of Leishmania major. The main objective was to establish a method and to generate a data set to determine the exposure risk, sand fly populations' dynamics and evaluate the efficacy of an attempt to create "cordon sanitaire" devoid of active jird burrows around the residential area. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were trapped in three fixed reference sites and an additional 52 varying sites. To mark sand flies in the field, sugar solutions containing different food dyes were sprayed on vegetation in five sites. The catch was counted, identified, Leishmania DNA was detected in pooled female samples and the presence of marked specimens was noted. Phlebotomus papatasi, the vector of L. major in the region was the sole Phlebotomus species in the catch. Leishmania major DNA was detected in ~10% of the pooled samples and the highest risk of transmission was in September. Only a few specimens were collected in the residential area while sand fly numbers often exceeded 1,000 per catch in the agricultural fields. The maximal travel distance recorded was 1.91km for females and 1.51km for males. The calculated mean distance traveled (MDT) was 0.75km. CONCLUSIONS: The overall results indicate the presence of dense and mobile sand fly populations in the study area. There seem to be numerous scattered sand fly microsites suitable for development and resting in the agricultural fields. Sand flies apparently moved in all directions, and reached the residential area from the surrounding agricultural fields. The travel distance noted in the current work, supported previous findings that P. papatasi like P. ariasi, can have a relatively long flight range and does not always stay near breeding sites. Following the results, the width of the "cordon sanitaire" in which actions against the reservoir rodents were planned, was extended into the depth of the agricultural fields.

Mosquito Surveillance for 15 Years Reveals High Genetic Diversity Among West Nile Viruses in Israel.

West Nile Virus (WNV) is endemic in Israel and has been the cause of several outbreaks in recent years. In 2000, a countrywide mosquito survey was established to monitor WNV activity and characterize viral genotypes in Israel. We analyzed data from 7135 pools containing 277 186 mosquitoes collected over the past 15 years and, here, report partial sequences of WNV genomes obtained from 102 of the 336 positive mosquito pools. Phylogenetic analysis demonstrated that cluster 4 and the Mediterranean and Eastern European subtypes of cluster 2 within WNV lineage 1 circulated in Israel, as did WNV lineage 2, highlighting a high genetic diversity of WNV genotypes in our region. As a major crossroads for bird migration between Africa and Eurasia and with a long history of human infection, Israel serves as a resource hub for WNV in Africa and Eurasia and provides valuable information on WNV circulation in these regions.

Zoonotic disease in a peripheral population: persistence and transmission of Leishmania major in a putative sink-source system in the Negev Highlands, Israel.

Populations at the edge of their geographic distributions are referred to as peripheral populations. Very little attention has been given to this topic in the context of persistence of infectious disease in natural populations. In this study, we examined this question using zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major in the Negev Desert of Israel as a model system. Here, we suggest that the regional persistence of Phlebotomus papatasi populations and L. major transmission in the Sede Boqer region could be explained through processes akin to sink-source and/or mainland-island metapopulation dynamics. Given its potentially enzootically superior ecological conditions, we hypothesize that the Zin Valley ecotope constitutes the "mainland" or the "source" patch for the Sede Boqer area where L. major transmission is persistent and resistant to local extinctions (die-outs) whereas the local sand fly populations on the Zin Plateau ("island patch" or "sink patch") are more prone to local extinctions. Between 2007 and 2008, we trapped sand flies and sand rats in the two areas and compared sand fly abundance and L. major infection prevalence in both. In both 2007 and 2008, sand fly abundance was high and continuous in the Zin Wadi but low and discontinuous in the Zin Plateau. Infection prevalence of sand rats was significantly higher in the Wadi (13%) compared with the Zin Plateau (3%). Minimum infection rate in sand flies did not differ significantly between the two areas. Overall, our results are consistent with the premise that the Zin Valley population is relatively robust in terms of L. major transmission, whereas transmission is potentially more tenuous in the plateau. Understanding the biotic and abiotic processes enabling the persistence of L. major and other vector-borne diseases in peripheral disease foci is important for predicting the effect of anthropogenic land use and climate change.

Molecular identification of bloodmeals from sand flies and mosquitoes collected in Israel.

In Israel, sand flies are the vectors of Leishmania Ross and mosquitoes are the vectors of West Nile Virus. In the Judean Desert and Tiberias, the sand fly Phlebotomus sergenti Parrot is the vector of Leishmania tropica (Wright) and the rock hyrax (Procavia capensis Pallas) is considered the main reservoir animal. The main vectors of West Nile Virus are Culex pipiens L. and Culex perexiguus Theobald. Bloodmeals of engorged field-caught female sand flies and mosquitoes are an important source for defining host preferences. Recent progress in DNA molecular techniques has enabled the accurate identification of blood sources within the arthropod gut. In this study, we applied molecular approach for species-specific identification based on polymerase chain reaction and nucleotide sequence analysis of polymorphic regions along two mitochondrial genes, 12S and 16S rRNA. The research was carried out on 261 engorged female sand flies collected in the Judean Desert and Tiberias and 50 engorged female mosquitoes collected in Tel-Aviv and Arava. Species identification of bloodmeals was successful in 92% of the samples. Rock hyrax was the most abundant host in bloodmeals of P. sergenti, while human blood was found in only seven (3%) females. L. tropica DNA was detected in three P. sergenti females from Tiberias that contained rock hyrax blood. Avian sequences were detected in 67% (10 of 15) of the identified bloodmeals from Cx. perexiguus and in 10% (3 of 29) of the identified meals from Cx. pipiens. Human sequences were found in 14% of the identified bloodmeals from Cx. pipiens. The successful analysis of the majority of the bloodmeals performed on wild sand flies and mosquitoes suggests that bloodmeal identification can be applied as one of the routine procedures in vector surveillance programs.

West Nile fever in Israel: the reemergence of an endemic disease.

BACKGROUND: As a crossroads for bird migration between Africa and Eurasia and with its long history of human infection, Israel has been a major focus of attention during the continuing global spread of West Nile fever (WNF). This article reviews the background and reemergence of WNF in Israel; the recent epidemiology of WNF among Israelis; and the disease-control strategies being used to combat the disease. METHODS: Employing the comprehensive base of case data that are reported to the Ministry of Health, an epidemiological record was constructed that details the incidence and distribution of WNF cases in Israel in recent years. RESULTS: After decades of small, intermittent outbreaks, nearly 1400 cases of WNF were reported in Israel between 2000 and 2012. Incidence was consistently highest in the coastal cities, among elderly patients, and in the late summer months and early autumn. A broad range of control measures to prevent human infection has been implemented, and attention has been given to issues such as the protection of the national blood bank and the occurrence of long-term sequelae. CONCLUSIONS: The reemergence of WNF in Israel is likely the result of a combination of factors including past immunity to the virus among the human population, a marked increase in awareness of WNF among physicians, and more frequent requests for the laboratory testing of suspected cases. In the absence of effective vaccine to protect humans from WNF, the best disease-control strategies include intensive vector-control measures, the continued development of techniques to forecast outbreaks, and effective public education programs that are targeted toward the high-risk elderly population.

Barkedji virus, a novel mosquito-borne flavivirus identified in Culex perexiguus mosquitoes, Israel, 2011.

Barkedji virus, named after the area of its first identification in Senegal, is a newly discovered flavivirus (FV), for which we propose the abbreviation BJV. In the present study, we report the first-time detection of BJV in Culex perexiguus mosquitoes in Israel in 2011 and determination of its almost complete polyprotein gene sequence. We characterized the BJV genome and defined putative mature proteins, conserved structural elements and potential enzyme motifs along the polyprotein precursor. By comparing polyproteins and individual proteins of BJV with several other FVs, a distant relationship of BJV to Nounane virus (NOUV), a recently described African FV, is demonstrated. Phylogenetic analysis of 55 selected flaviviral polyprotein gene sequences exhibits two major clusters, one made up of the classical three clades of FVs: mosquito-borne, tick-borne and those without known vectors. The other cluster exclusively contains so-called 'insect-specific' FVs, which do not replicate in vertebrate cells. Based on our phylogenetic analysis, BJV is related to other members of the mosquito-borne clade with yet unknown vertebrate hosts, such as NOUV, Donggang virus, Chaoyang virus and Lammi virus. However, with a maximum identity of only 54 % to NOUV, BJV represents a distinct new virus species.

A sharp increase in the natural abundance of sand flies in Kfar Adummim, Israel.

Monitoring sand flies in the cutaneous leishmaniasis foci Kfar Adummim and Ma'ale Adummim from May to October generally yielded several hundred specimens per CO(2) baited trap. In the summer of 2009, a sharp rise in the number of sand flies trapped was recorded in Kfar Adummim, while numbers were similar to previous years in Ma'ale Adummim; approximately 4,000 specimens compared to about 400, with maximal catches of about 16,500 specimens in Kfar Adummim. We postulate that the sharp increase in sand fly numbers is directly related to the intensive construction conducted which enhanced sand fly breeding habitats.

Evaluation of a metofluthrin fan vaporizer device against phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in the Judean Desert, Israel.

The OFF! Clip-On fan vaporizer device releasing metofluthrin was evaluated against phletobomine sand flies in the Judean Desert, Israel, in October, 2009. A total of 76,400 sand flies was collected, with male flies representing 98.3%Phlebotomus sergenti and 1.7%P. papatasi. Females comprised 43.0% of the total catch and included 6.7% blood-fed females. Similar proportions of flies were collected in both suction and sticky traps. In trials with unbaited suction traps, similar numbers of sand flies were collected in traps with a metofluthrin device, blank device, or no device (i.e., suction only). In suction traps baited with CO(2) , higher numbers of P. sergenti males and blood-fed females were collected in traps with a blank device compared to traps with a metofluthrin device. In sticky traps baited with CO(2) , there were no significant differences between catches in traps with a metofluthrin device, blank device, or no device. The results suggest metofluthrin from the device is not repellent against sand flies in a field environment despite showing insecticidal activity against flies collected in suction traps.

[Personal protection measures against blood-sucking insects and ticks].

Blood-sucking arthropods are major vectors of various pathogens like viruses, bacteria, protozoa and nematodes. Preventing exposure to the vector is imperative especially when vaccine and prophylactic treatments are not available. Personal protection measures (PPM) are essential and often the only means available when dealing with blood-sucking disease transmitting arthropods. Awareness of the risk in the specific areas of travel is the first step to be taken before and while traveling. PPM include preventive personal behavior, suitable clothing, application of insect repellents to the skin, the use of space repellents, impregnation of clothing, camping gear and bed nets and, when necessary, ground spraying of insecticides. The registered and recommended active ingredients for skin application are Deet, picaridin (icaridin), p-menthane-3,8-diol (PMD) and IR3535. Volatile pyrethrins are used as space repellents while pyrethroids, especially permethrin, are employed for impregnation and for ground spraying. It is recommended to purchase only products registered in Israel or other developed countries. These products should have a detailed label specifying the concentration of the active ingredient, application instructions and the duration of protection.

Comparison of mosquito control programs in seven urban sites in Africa, the Middle East, and the Americas

Mosquito control programs at seven urban sites in Kenya, Egypt, Israel, Costa Rica, and Trinidad are described and compared. Site-specific urban and disease characteristics, organizational diagrams, and strengths, weaknesses, obstacles and threats (SWOT) analysis tools are used to provide a descriptive assessment of each mosquito control program, and provide a comparison of the factors affecting mosquito abatement. The information for SWOT analysis is collected from surveys, focus-group discussions, and personal communication. SWOT analysis identified various issues affecting the efficiency and sustainability of mosquito control operations. The main outcome of our work was the description and comparison of mosquito control operations within the context of each study site's biological, social, political, management, and economic conditions. The issues identified in this study ranged from lack of inter-sector collaboration to operational issues of mosquito control efforts. A lack of sustainable funding for mosquito control was a common problem for most sites. Many unique problems were also identified, which included lack of mosquito surveillance, lack of law enforcement, and negative consequences of human behavior. Identifying common virtues and shortcomings of mosquito control operations is useful in identifying "best practices" for mosquito control operations, thus leading to better control of mosquito biting and mosquito-borne disease transmission.

Comparison of mosquito control programs in seven urban sites in Africa, the Middle East, and the Americas.

Mosquito control programs at seven urban sites in Kenya, Egypt, Israel, Costa Rica, and Trinidad are described and compared. Site-specific urban and disease characteristics, organizational diagrams, and strengths, weaknesses, obstacles and threats (SWOT) analysis tools are used to provide a descriptive assessment of each mosquito control program, and provide a comparison of the factors affecting mosquito abatement. The information for SWOT analysis is collected from surveys, focus-group discussions, and personal communication. SWOT analysis identified various issues affecting the efficiency and sustainability of mosquito control operations. The main outcome of our work was the description and comparison of mosquito control operations within the context of each study site's biological, social, political, management, and economic conditions. The issues identified in this study ranged from lack of inter-sector collaboration to operational issues of mosquito control efforts. A lack of sustainable funding for mosquito control was a common problem for most sites. Many unique problems were also identified, which included lack of mosquito surveillance, lack of law enforcement, and negative consequences of human behavior. Identifying common virtues and shortcomings of mosquito control operations is useful in identifying "best practices" for mosquito control operations, thus leading to better control of mosquito biting and mosquito-borne disease transmission.

Attempts to control sand flies by insecticide-sprayed strips along the periphery of a village.

The customary method for controlling leishmaniasis and sand fly bites in Israel is the spraying of large quantities of residual insecticides on walls of houses and neighboring surfaces. However, the high summer temperatures, strong radiation, and dust limit the efficacy of the method. The sand fly vectors of Leishmania appeared to come uphill to the village of Kfar Adumim. Therefore, to block their path and improve control, beta-cyfluthrine or DDT were repeatedly sprayed on a strip of cloth that was stretched on the ground along and near the peripheral houses. The efficacy of the treatments was examined by comparing the proportions of sand flies that had been caught before and after the treatments, below and above the belt. The capture near the houses of sand flies that had been marked by colored sugar baits below the protective belt indicated that they were moving uphill and crossing the strip. The catches showed a gradual uphill decrease, up to the houses, in the number of sand flies. However, the proportions of sand flies caught in the different sites were similar before and after insecticide treatments showing that the belt was ineffective. Laboratory tests of pieces of the sprayed strip and exposure of sand flies to similar pieces indicated that the lack of effect was not caused by deficiency of insecticides.