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.

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.

Sensitive Molecular Diagnostics for Cutaneous Leishmaniasis.

BACKGROUND: Rapid diagnosis of cutaneous leishmaniasis (CL) and identification of Leishmania species is highly important for the disease management. In Israel, CL is caused mainly by Leishmania major and Leishmania tropica species. METHODS: We established an easy to handle point of care lesion-swabbing, combined with a highly sensitive multiplex real time PCR (multiplex qPCR) for accurate and rapid diagnosis of Leishmania species. RESULTS: Using three probes: one general for: Leishmania species, and two specific for L major, and L tropica, we screened 1783 clinical samples collected during two years. Leishmania species was found in 1086 individuals, 1008 L major, and 70 L tropica. Eight samples positive for Leishmania species only, were further tested using a second set of multiplex qPCR developed, and were found positive for Leishmania braziliensis and Leishmania infantum/donovani (2 and 6 samples, concomitantly). CONCLUSIONS: Taken together, the test enabled diagnostics and better treatment of Leishmania infections from the Old World (1078 samples) and the New World (8 samples), and the subtyping of the dominant strains in the region, as well as in returning travelers'.

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.

External Ophthalmomyiasis Caused by a Rare Infesting Larva, Sarcophaga argyrostoma.

Purpose. External ophthalmomyiasis (EO) is caused by infesting larvae belonging to various species of flies. Most documented cases result from sheep (Oestrus ovis) and Russian (Rhinoestrus purpureus) botfly larvae, but we recently discovered a rare case of EO caused by flesh fly (Sarcophaga argyrostoma) larvae. Here, we report the case of a patient with EO who had been hospitalized and sedated for 1 week because of unrelated pneumonia. Methods. Case report. Results. A total of 32 larvae were removed from the adnexae of both eyes. Larvae identification was confirmed through DNA analysis. Treatment with topical tobramycin resulted in complete resolution of EO. Conclusion. EO can be caused by S. argyrostoma, and the elderly and debilitated may require extra ocular protection against flies during sedation.

Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus.

A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program-one susceptible (S), one resistant (R-that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance.

Expression of stress-response proteins upon whitefly-mediated inoculation of Tomato yellow leaf curl virus in susceptible and resistant tomato plants.

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as a TYLCV resistance source, one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response with stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related (PR) proteins. The two biotic stresses-non-viruliferous whitefly feeding and virus infection with viruliferous insects--led to a slow decline in abundance of MAPKs, HSPs, and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PR proteins, beta-1,3-glucanase, and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied, followed by a decline; the virus-susceptible and -resistant tomato lines behaved similarly in response to the fungus.

Reproductive development and phenotypic differences in garlic are associated with expression and splicing of LEAFY homologue gaLFY.

Modern garlic (Allium sativum L.) cultivars are sterile and propagated only vegetatively. The recent discovery of fertile genotypes in Central Asia and the restoration of flowering and fertility by environmental manipulations open the way for in-depth florogenetic, genetic, and molecular research in garlic. In the present work, two bolting garlic accessions were employed: #3026, developing normal flowers and seeds, and #2509, in which flowers abort at the early stages of development. Morphological studies showed transition of the apical meristems from the vegetative to the reproductive stage and inflorescence initiation in both genotypes. Low temperatures promote transition of the apex and stem elongation, but have no effect on the phenotypic expression of the inflorescence development. The initial stages of reproductive development in non-flowering #2509 plants were followed by abortion of floral primordia at the differentiation stage. A search for genes involved in the control of flowering in garlic resulted in identification of the garlic LEAFY/FLO homologue, gaLFY. Further comparative analyses of gene expression revealed two gaLFY transcripts, differing in 64 nucleotides, with clear splicing borders. The short variant transcript was identified in both genotypes throughout all development stages, whereas the long variant appears in the flowering genotype #3026 only during reproductive development. The phenotypic differences in garlic, with regard to flowering, may be associated with the efficacy of the splicing process.