Alon Warburg

Outbreak of Cutaneous Leishmaniasis in Northern Israel

This study describes a new focus of cutaneous leishmaniasis (CL) due to Leishmania tropica, in the Galilee region of northern Israel. Thirty-three cases from 4 villages (northern part) and from the city of Tiberias (southern part) have been clinically diagnosed since 1996. Parasites from 13 patients and from 6 sand flies were characterized by isoenzyme electrophoresis, 2 immunological methods, and 3 polymerase chain reaction (PCR)-based methods. Isolates from the northern part were antigenically similar to Leishmania major and were different from other L. tropica isolates, including those from the southern part of the focus. They belonged to a newly reported zymodeme and were separable from all known Israeli L. tropica isolates, by use of 2 different PCR-based methods. Five (5.2%) of 97 Phlebotomus (Adlerius) arabicus and 2 (1.2%) of 162 Phlebotomus (Paraphlebotomus) sergenti females from the northern part of the focus were found to be infected with L. tropica. Three of 29 hyraxes (Procavia capensis) were positive for Leishmania ribosomal DNA. Thus, the northern part of this emerging focus of CL in Israel is distinct from all known L. tropica foci. P. arabicus is the main vector, and it transmits parasites that are different from other L. tropica isolates, with respect to antigenic, molecular, and biochemical parameters.

Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel.

TOC summary for table of contents: Infection with Leishmania tropica is emerging because of encroachment of rock hyraxes and transmission by multiple vector species.

Leishmaniasis in the Jordan Valley II. Sandflies and transmission in the central endemic area

Cutaneous leishmaniasis in the Jordan Valley is maintained within the close association of the rodent Psammomys obesus and sandfly Phlebotomus papatasi, which appear to be the exclusive host and vector species. The incidence of the disease was similar to the distribution of Psammomys colonies in the region, i.e., the plains of light stoneless soil. An infection rate of 93% was recorded in the very common P. obesus. Other potential host species, except for Mus musculus, were scarce and no infection with Leishmania was found in them. The only Phlebotomus species caught in significant numbers was Ph. papatasi and this was also the only species harbouring leishmanial parasites, up to 56% in one sample. All Leishmania isolates from Psammomys and from Ph. papatasi were identical to those from local human cases. The density of Ph. papatasi populations in uncultivated areas was correlated with soil conditions favouring high humidity in Psammomys burrows. A very low rate of engorged females among the Sergentomyia species collected suggests that the common species, S. antennata and S. africana asiatica, are highly autogenous.

Leishmania tropica in Rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis.

Cutaneous leishmaniasis, caused by Leishmania tropica, has recently emerged in urban and rural foci of central and northern Israel, and constitutes a major public health concern. Rock hyraxes (Procavia capensis), the suspected natural reservoir, were trapped in the cutaneous leishmaniasis urban focus of Maale Adumim in central Israel and evaluated for L. tropica infection by real-time kinetoplast DNA (kDNA) polymerase chain reaction (PCR) and serology. Real-time PCR on blood and computerized western blot serology analysis was positive for L. tropica in 58% and 80%, respectively, of the hyraxes tested. Phylogenetic analysis of the ribosomal internal transcribed spacer 1 region indicated that similar genotypes were present in humans and hyraxes from the same habitat. The high rates of infection and exposure to L. tropica among hyraxes supports their involvement in the transmission cycle of this parasite, and their potential role as a reservoir for human disease.

The ecology of cutaneous leishmaniasis in Nizzana, Israel: infection patterns in the reservoir host, and epidemiological implications

We conducted an extensive interdisciplinary study in an emerging focus of cutaneous leishmaniasis in the Western Negev Desert of Israel between July 1998 and February 2000. The aims of the this study were to determine (1) the reservoir hosts, (2) the distribution of the pathogen within the host range, (3) the associations of host, vector, and pathogen within defined habitats, (4) the demographic distribution of the pathogen within the host populations, and (5) to apply the newly acquired epizootiological data to explain morbidity patterns in humans. Fourteen square (60 m width) sampling plots were delimited in three types of habitats each with a different kind of substrate: loess, sand, and sand-loess ecotone. Rodents and sand flies were trapped and several environmental variables were measured. Leishmania infections in rodents were detected microscopically in stained smears of ear tissue and by a Leishmania-specific polymerase chain reaction. Results indicate that, contrary to previous reports, Psammomys obesus and not Meriones crassus is the main reservoir host in the region. Additional rodents (12 Gerbillus dasyurus and two M. crassus) were also found positive for Leishmania DNA. Prevalence of Leishmania infections amongst P. obesus was highest in loess habitats (65%), intermediate in the sandy-loess ecotone (20%), and 0% in the sandy habitats. Psammomys obesus individuals in the loess habitat of the Nizzana ruins were larger, on average (probably older), than those in the sandy habitat of the Mt. Keren junction. Sand fly density was positively correlated to soil moisture being higher in the relatively humid plots of Nizzana ruins and much lower in the drier sandy soil of Mt. Keren. Elucidation of fundamental ecological factors affecting this disease has helped explain an apparent discrepancy between the distribution of the disease in the zoonotic system and among humans.

Phlebotomine Sand Flies (Diptera: Psychodidae) of the Palestinian West Bank: Potential Vectors of Leishmaniasis

Abstract Two forms of leishmaniasis are endemic to the Jenin district in the northern region of the West Bank. Visceral leishmaniasis (VL), caused by Leishmania infantum, mainly affects infants. Cutaneous leishmaniasis (CL) affects a broader age group and is probably caused by L. tropica. Although the Jenin district is the most important focus of leishmaniasis in the West Bank, the sand fly fauna of the area has never been studied in a systematic manner. We collected base-line data on sand fly species, their distribution, and their feeding preferences to facilitate risk assessments for contracting leishmaniasis. Light traps, sticky traps, insecticide knockdown collections, aspirator, and human-landing collections were used. A total of 4,082 sand flies was collected in foci of confidence limits and/or VL between June and December 1998. Nine Phlebotomus species representing seven subgenera were identified: P. (Larroussius) perfiliewi transcaucasicus Perfil’ev, P. (La.) tobbi Adler & Theodor, P. (La.) mascitti canaaniticus Adler & Theodor, P. (La.) mascitti mascitti Grassi, P. (La.) syriacus Adler & Theodor, P. (Phlebotomus) papatasi Scopoli, P. (Synphlebotomus) s.p., P. (Paraphlebotomus) sergenti Parrot, P. (Par.) jacusieli Theodor, P. (Adlerius) halepensis Theodor. Two other Phlebotomus subspecies, P. (La.) major major Annandale, P. (La.) neglectus Tonnoir, require confirmation. In addition, four species of the closely related genus, Sergentomyia were also found: S. (Sergentomyia) theodori Parrot, S. (S.) fallax Parrot, S. (Sintonius) tiberiadis Adler, Theodor & Lourie, S. (Sin.) christophersi Sinton. Among five species of sand flies collected on human bait, P. papatasi constituted ≈90% followed by P. major syriacus (8%) and P. mascitti (2%). Sand fly human-biting activity occurred through the night and it was highest between 2400 and 0300 hours. P. papatasi. P. perfiliewi, P, major and P.tobbi were the more endophilic species constituting 93% of all flies caught indoors. Seven Phlebotomus spp. constitute potential vectors of leishmaniasis but the most probable ones are as follows: P. papatasi the main human-biting species, a recognized vector of L. major (CL), P. sergenti, L. tropica (CL) and P. syriacus, L. infantum (VL).

Recent advances in phlebotomine sand fly research related to leishmaniasis control

Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.

Anthropogenic disturbances enhance occurrence of cutaneous leishmaniasis in Israel deserts: Patterns and mechanisms

A continuous and gradual increase in the incidence of cutaneous leishman- iasis (CL) has been reported in southern Israel over the last 20 years. The goal of our research was to determine if and how anthropogenic disturbances enhance the occurrence of the disease. To assess the effect of anthropogenic disturbances, we selected twelve 60 3 60 m plots, six in disturbed and six in undisturbed habitats at each of five study sites in southern Israel. We trapped rodents and sand flies, determined Leishmania majorinfection prevalence in rodents, and measured various environmental parameters. Infection prevalence in the reservoir host, the rodent Psammomys obesus, was significantly higher in disturbed habitats than in undisturbed ones. Infection prevalence was positively correlated with vector (Phlebotomus papatasi) density but not with host density. P. papatasi density was positively correlated with soil moisture. Soil in disturbed habitats had significantly more moisture, and plants were significantly more lush than in undisturbed habitats. P. obesus density was positively correlated with plant lushness. These results suggest that an important impact of anthropogenic disturbance, the addition of water, improves the conditions for vector breeding and promotes larger host populations by improving the quality of their food. These effects, in turn, should enhance disease transmission risk to humans.

Effect of sand fly saliva on Leishmania uptake by murine macrophages

Leishmania promastigotes are introduced into the skin by blood-sucking phlebotomine sand flies. In the vertebrate host, promastigotes invade macrophages, transform into amastigotes and multiply intracellularly. Sand fly saliva was shown to enhance the development of cutaneous leishmaniasis lesions by inhibiting some immune functions of the host macrophages. This study demonstrates that sand fly saliva promotes parasite survival and proliferation. First, macrophages gravitated towards increasing concentrations of sand fly saliva in vitro. Secondly, saliva increased the percentage of macrophages that became infected with Leishmania promastigotes and exacerbated the parasite load in these cells. Thus, during natural transmission, saliva probably reduces the exposure of promastigotes to the immune system by attracting macrophages to the parasite inoculation site and by accelerating the entry of promastigotes into macrophages. Saliva may also enhance lesion development by shortening the generation time of dividing intracellular amastigotes.

Identification of Blood Meals Imbibed by Phlebotomine Sand Flies Using Cytochrome b PCR and Reverse Line Blotting

Blood meal identification is important for determining the host preferences and the vectorial capacity of hematophagous arthropods. In the past, mostly serological techniques using host-specific antibodies were used, but in recent years more sensitive and accurate polymerase chain reaction (PCR)-based molecular approaches for identifying blood meals have been developed. Here, a vertebrate-specific PCR is combined with reverse line blot analysis for identifying blood meals ingested by female phlebotomine sand fly vectors of leishmaniasis. Species-specific oligonucleotides were covalently linked to nylon membranes, and biotinylated PCR products of the mitochondrial cytochrome b gene were used as probes in a hybridization reaction revealed using colorimetric or enhanced chemiluminescent detection systems. This combination identified blood meals up to 96 hours after ingestion containing minimal amounts of DNA (>0.1 pg). The specific probes discriminated between putative host species in several study areas. The source of blood was identified in 68 of 89 wild-caught sand flies tested (76%). Mixed blood meals were identified in 15 (17%) of those. The advantages and limitations of this method are discussed.