Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle.

Systemic control uses the vertebrate hosts of zoonotic pathogens as "Trojan horses," killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.

Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival.

Leishmania donovani is the main cause of visceral leishmaniasis (VL) in East Africa. Differences between northern Ethiopia/Sudan (NE) and southern Ethiopia (SE) in ecology, vectors, and patient sensitivity to drug treatment have been described, however the relationship between differences in parasite genotype between these two foci and phenotype is unknown. Whole genomic sequencing (WGS) was carried out for 41 L. donovani strains and clones from VL and VL/HIV co-infected patients in NE (n = 28) and SE (n = 13). Chromosome aneuploidy was observed in all parasites examined with each isolate exhibiting a unique karyotype. Differences in chromosome ploidy or karyotype were not correlated with the geographic origin of the parasites. However, correlation between single nucleotide polymorphism (SNP) and geographic origin was seen for 38/41 isolates, separating the NE and SE parasites into two large groups. SNP restricted to NE and SE groups were associated with genes involved in viability and parasite resistance to drugs. Unique copy number variation (CNV) were also associated with NE and SE parasites, respectively. One striking example is the folate transporter (FT) family genes (LdBPK_100390, LdBPK_100400 and LdBPK_100410) on chromosome 10 that are single copy in all 13 SE isolates, but either double copy or higher in 39/41 NE isolates (copy number 2-4). High copy number (= 4) was also found for one Sudanese strain examined. This was confirmed by quantitative polymerase chain reaction for LdBPK_100400, the L. donovani FT1 transporter homolog. Good correlation (p = 0.005) between FT copy number and resistance to methotrexate (0.5 mg/ml MTX) was also observed with the haploid SE strains examined showing higher viability than the NE strains at this concentration. Our results emphasize the advantages of whole genome analysis to shed light on vital parasite processes in Leishmania.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

First detection of Leishmania donovani in sand flies from Cameroon and its epidemiological implications.

OBJECTIVES: A sound knowledge of the vector-host-parasite transmission dynamics is a prerequisite for adequate control measures of vector-borne diseases. To achieve this, an entomological investigation was conducted in the cutaneous leishmaniasis (CL) endemic focus of Mokolo District, northern Cameroon to identify the insect vector(s) of the disease. METHODS: Phlebotomine sand flies were collected in and around Mokolo using New Standard CDC Miniature Light Traps. Individual sand flies were used for morphological species identification, and the remainder of the body for DNA analysis. Sand flies were demonstrated to harbour Leishmania spp. parasites using ITS1 PCR. Mitochondrial vertebrate-specific Cytochrome b -PCR was used to identify blood meals ingested by female sand flies. PCR amplicons were sequenced for Leishmania and blood sources discrimination. RESULTS: This study revealed the presence of Leishmania donovani complex DNA (n = 1) in Phlebotomus duboscqi and of lizard-borne Leishmania tarentolae-like DNA (n = 3) in Sergentomyia spp. in 79 sand fly specimens from Mokolo district. CONCLUSIONS: The causative agent of CL could not be detected in potential vectors. Instead, we found evidence for visceral leishmaniasis (VL) parasites in Phlebotomus duboscqi as well as enzootic reptile parasites in the Mokolo area. We recommend that an epidemiological survey be carried out in the area to evaluate the prevalence and eventually describe the clinical manifestations of VL in the human population. Political instability in neighbouring countries and the resulting refugee migration are likely explanations for the emergence of VL in Mokolo.

Estimation of infection prevalence and sensitivity in a stratified two-stage sampling design employing highly specific diagnostic tests when there is no gold standard.

In this work, we describe a two-stage sampling design to estimate the infection prevalence in a population. In the first stage, an imperfect diagnostic test was performed on a random sample of the population. In the second stage, a different imperfect test was performed in a stratified random sample of the first sample. To estimate infection prevalence, we assumed conditional independence between the diagnostic tests and develop method of moments estimators based on expectations of the proportions of people with positive and negative results on both tests that are functions of the tests' sensitivity, specificity, and the infection prevalence. A closed-form solution of the estimating equations was obtained assuming a specificity of 100% for both tests. We applied our method to estimate the infection prevalence of visceral leishmaniasis according to two quantitative polymerase chain reaction tests performed on blood samples taken from 4756 patients in northern Ethiopia. The sensitivities of the tests were also estimated, as well as the standard errors of all estimates, using a parametric bootstrap. We also examined the impact of departures from our assumptions of 100% specificity and conditional independence on the estimated prevalence.

Sero-prevalence of Leishmania donovani infection in labour migrants and entomological risk factors in extra-domestic habitats of Kafta-Humera lowlands - kala-azar endemic areas in the northwest Ethiopia.

BACKGROUND: Visceral leishmaniasis (VL) or kala-azar cases in seasonal labour migrants from highland areas are addressed to travel history to the Metema-Humera lowlands, northwestern Ethiopia. Factors that affect the incidence of VL in extra-domestic habitats were not evaluated. The aim of this study was to evaluate sero-prevalence of Leishmania donovani infection in randomly selected labour migrant workers and entomological risk factors which might affect the incidence of kala-azar. METHODS: Sero-prevalence of L. donovani infection in labour migrants was obtained from Direct Agglutination Test (DAT) using blood samples. Logistic regression analysis was used to correlate the possible risk factors with L. donovani infection. The season for L. donovani infection or Phlebotomus orientalis bite was estimated from the study of population dynamic of P. orientalis in areas where the blood was sampled. RESULT: A total of 7, 443 P. orientalis (1,748 female and 5,695 male) were collected from agricultural fields and thickets of Acacia seyal using 461 CDC light traps. The highest mean number of P. orientalis/trap in the thickets of A. seyal and agricultural fields were 46.9 and 43.9 in March and April respectively. The mean P. orientalis/trap for November - May dry season in agricultural fields (11.39) and thickets of A. seyal (25.30) were higher compared to 0.66 in fields and 3.92 in thickets during June - August weeding season. Of the total 359 labour migrants screened using DAT, 45 (12.5%) were DAT-positive (≥1:800) for L. donovani infections. Very high titers (1:12800) were found in 3 (0.8%) individuals who had the risk of kala-azar development. Statistically significant p-values and odd ratio (OR) for staying in the areas both in the weeding and harvesting seasons (p = 0.035; OR = 2.83) and sleeping in the agricultural fields (p = 0.01; OR = 15.096) were positively correlated with L. donovani infection. Night harvest (p = 0.028; OR = 0.133) and knowledge about sign or symptoms (p = 0.042; OR = 0.383) were negatively associated with this infection. CONCLUSIONS: Sleeping in open agricultural fields was related with L. donovani infections in labour migrants during June-August weeding season.

Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

BACKGROUND: Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. METHODS: The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. RESULTS: Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. CONCLUSIONS: Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

The evolution of borneol repellency in culicine mosquitoes.

Insects have developed remarkable adaptations to effectively interact with plant secondary metabolites and utilize them as cues to identify suitable hosts. Consequently, humans have used aromatic plants for centuries to repel mosquitoes. The repellent effects of plant volatile compounds are mediated through olfactory structures present in the antennae, and maxillary palps of mosquitoes. Mosquito maxillary palps contain capitate-peg sensilla, which house three olfactory sensory neurons, of which two are mainly tuned to either carbon dioxide or octenol - two animal host odorants. However, the third neuron, which expresses the OR49 receptor, has remained without a known ecologically-relevant odorant since its initial discovery. In this study, we used odorant mixtures and terpenoid-rich Cannabis essential oils to investigate the activation of OR49. Our results demonstrate that two monoterpenoids, borneol and camphor, selectively activate OR49, and OR9-expressing neurons, as well as the MD3 glomerulus in the antennal lobe. We confirm that borneol repels female mosquitoes, and knocking out the gene encoding the OR49 receptor suppresses the response of the corresponding olfactory sensory neuron. Importantly, this molecular mechanism of action is conserved across culicine mosquito species, underscoring its significance in their olfactory systems.

Identification of bloodmeals in wild caught blood fed Phlebotomus argentipes (Diptera: Psychodidae) using cytochrome b PCR and reverse line blotting in Bihar, India.

Identification of the source of bloodmeals in vectors plays an important role in epidemiological studies by determining the host preferences of wild sand flies in natural habitat. The anthropophilic index is a crucial component in human leishmaniasis. Bloodmeal analysis can identify the reservoir hosts of vector borne diseases. The amplification of the mitochondrial cytochrome b gene, followed by reverse line blot analysis, helps to identify the bloodmeal ingested by the wild caught sand flies. In the current study, blood fed sand flies were collected from three different villages in Bihar, India, by using Centers for Disease Control mini traps with incandescent light. Traps were placed in five different sites in the villages. Whole genome DNA was extracted from the blood fed sand flies and was amplified for the cytochrome b region, followed by reverse line blot analysis. In total, 442 blood fed sand flies were analyzed out of which 288 (65%) were positive to cytochrome b polymerase chain reaction. Humans, cattle, buffalo, and goats were the major bloodmeals identified, followed by chickens. In some of the blood fed sand flies, multiple bloodmeals were identified. In the current study, sand flies mostly fed on humans, followed by cattle, buffalo, and goats. In this regard, it is necessary to also consider cattle, buffalo, and goats when addressing vector control in Bihar, India.

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.

Effects of larval rearing substrates on some life-table parameters of Lutzomyia longipalpis sand flies.

Sand flies are the insects responsible for transmitting Leishmania parasites, the causative agents of leishmaniasis in humans. However, the effects of sand fly breeding sites on their biology and ecology remain poorly understood. Herein, we studied how larval nutrition associated with putative breeding sites of the sand fly Lutzomyia longipalpis affects their oviposition, development, microbiome, and susceptibility to Leishmania by rearing L. longipalpis on substrates collected from an endemic area for leishmaniasis in Brazil. The results showed that female L. longipalpis select the oviposition site based on its potential to promote larval maturation and while composting cashew leaf litter hindered the development, larvae reared on chicken feces developed rapidly. Typical gut microbial profiles were found in larvae reared upon cashew leaf litter. Adult females from larvae reared on substrate collected in chicken coops were infected with Leishmania infantum, indicating that they were highly susceptible to the parasite. In conclusion, the larval breeding sites can exert an important role in the epidemiology of leishmaniasis.

Ecology of Leishmaniasis in an urbanized landscape: Relationship of sand fly densities, and Leishmania tropica infection rates with reservoir host colonies.

Urbanization may influence the transmission of leishmaniasis, which is as a serious public health issue in Palestine. Semi urban environments can provide suitable habitats for the reservoir host species and the vector sand flies to create favorable condition for disease transmission. This study was aimed to evaluating the effect of distance from hyrax (reservoir host) colonies on sand fly (vector) abundance and its relationship to Leishmania infection within a semi urban landscape. In the Aleskan neighborhood the town of Tubas, sand flies were collected in July and September of 2016. Five trapping transects were set running from a rocky area containing hyrax dwelling habitats into an area of inhabited houses. 1051 Phlebotomus sand flies from 9 species were captured. The numbers of Phlebotomus sergenti, the vector species, correlated negatively with the distance from hyrax. Infected, blood engorged female sand flies were captured closer to hyrax colonies than infected, non-fed females. The risk of disease transmission to humans increased with a high density of reservoirs, closer proximity of reservoir hosts, and high densities of sand flies near houses. These results must be taken into account when implementing future interventions to reduce CL in urban environments.

The spatial and temporal distribution, species composition, and host preference of phlebotomine sand flies in the Bethlehem District of Palestine.

In the Palestinian West Bank, leishmaniasis is emerging as a serious public health issue with incidence increasing over time, especially in the western and the northern parts. This study was aimed to evaluating the effect of altitude on sand fly density, temporal and spatial distribution, species composition, and host preference within and between three villages in the Bethlehem District. The three villages occur along an elevation cline, ranging from the disease-free area of Kisan (KIS; 732-782 m ASL), down to the endemic areas of Arab Ar-Rashaiyda (AAR; 522-568 m ASL), and Al'Azazma (AZA; 473-510 m ASL) in the Bethlehem District (southeastern West Bank). Sand flies were trapped monthly from May through October in 2013 in traps located north, south, east, and west of each village. The abundance of sand flies differed among the three villages, with Arab Ar-Rashaiyda (AAR) > Al 'Azazma (AZA) >Kisan (KIS). The sex ratio was even in AAR and AZA, and female biased in KIS. Both male and female Phlebotomus sand fly densities varied with elevation, with greatest densities found at intermediate elevations (AAR). Elevation correlated negatively with the proportion of blood-fed Phlebotomus females. Male Phlebotomus species composition differed among sites, with all the species found in this study present in AZA, while some species were absent from AAR and KIS. The host blood species engorged by P. sergenti were grouped in four categories: human (45.5%), livestock (25%), avian (19.9%), and dog (9.6%). This study provided information about the spatial and temporal distribution, age and species composition, and host preference of sand fly vectors that influence disease transmission. This information will allow us to better target sand flies, to control the disease, and to monitor risk prone areas.

Development of a next generation DNA sequencing-based multi detection assay for detecting and identifying Leishmania parasites, blood sources, plant meals and intestinal microbiome in phlebotomine sand flies.

Leishmaniasis is a disease caused by Leishmania parasites transmitted by phlebotomine sand flies (Diptera: Psychodidae). Human infections with different Leishmania species cause characteristic clinical manifestations; cutaneous or visceral leishmaniasis. Here we describe the development and application of a Miseq Next GenerationSequencing (NGS)-based Multi Detection Assay (MDA) designed to characterize metagenomics parameters pertinent to the sand fly vectors which may affect their vectorial capacity for Leishmania. For this purpose, we developed a MDA by which, DNA fragments were amplified through polymerase chain reactions (PCR) and then sequenced by MiSeq/NGS. PCR amplification was achieved using some published and some new primers designed specifically for identifying Leishmania spp. (ITS1), sand fly spp. (cytochrome oxidase I), vertebrate blood (Cytochrome b), plant DNA ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL), and prokaryotic micobiome (16 s rRNA). This MDA/NGS analysis was performed on two species of wild-caught sand flies that transmit different Leishmania spp. in two ecologically distinct, but geographically neighboring locations. The results were analyzed to identify, quantitate and correlate the measured parameters in order to assess their putative importance in the transmission dynamics of leishmaniasis.

A molecular analysis of sand fly blood meals in a visceral leishmaniasis endemic region of northwestern Ethiopia reveals a complex host-vector system.

BACKGROUND: Visceral leishmaniasis (VL, or "kala-azar") is a major cause of disability and death, especially in East Africa. Its vectors, sand flies (Diptera: Psychodidae: Phlebotominae), are poorly controlled and guarded against in these regions, owing in part to a lack of understanding about their feeding behavior. METHODS: A total of 746 freshly fed female sand flies were collected in five population centers in Kafta Humera (northwestern Ethiopia), where VL is endemic. Flies were collected from habitats that ranged from inside houses to open fields, using light traps and sticky traps. Sources of sand fly blood meals were identified using enzyme-linked immunosorbent assays (ELISA) and DNA amplification with reverse-line blot analysis (PCR-RLB); 632 specimens were screened using ELISA, 408 of which had identifiable blood meals, and 114 were screened using PCR-RLB, 53 of which yielded identifications. Fly species determinations were based on morphology, and those specimens subjected to PCR-RLB were also screened for Leishmania parasites using conventional PCR to amplify the nuclear marker ITS1 (internal transcribed spacer 1) with Leishmania-specific primers. RESULTS: More than three-fourths of all sand flies collected were Phlebotomus orientalis, and the remaining portion was comprised of nine other species. Nearly two-thirds of P. orientalis specimens were collected at village peripheries. The most common blood source for all flies was donkey (33.9% of all identifications), followed by cow (24.2%), human (17.6%), dog (11.8%), and goat or sheep (8.6%); mixtures of blood meals from different sources were found in 28.2% of all flies screened. Unidentified blood meals, presumably from wildlife, not domestic animals, were significantly higher in farm fields. Leishmania parasites were not detected in any of the 114 flies screened, not surprising given an expected infection rate of 1-5 out of 1,000. Meals that included a mixture of human and cow blood were significantly more frequent relative to all cow meals than human blood meals were to non-cow meals, suggesting a zoopotentiative interaction between cows and humans in this system. CONCLUSIONS: Habitat and host preferences of sand fly vectors in Kafta Humera confirmed the finding of previous reports that the main vector in the region, Phlebotomus orientalis, is a highly opportunistic feeder that prefers large animals and is most commonly found at village peripheries. These results were similar to those of a previous study conducted in a nearby region (Tahtay Adiabo), except for the role of cattle on the prevalence of human blood meals. Preliminary examinations of blood meal data from different settings point to the need for additional surveys and field experiments to understand the role of livestock on biting risks.

Inventory and taxonomy of phlebotomine sand flies of the Mokolo leishmaniasis focus, northern Cameroon, with description of new Sergentomyia taxa (Diptera: Psychodidae).

Leishmaniasis is endemic in northern Cameroon. However, the sand fly vectors have not been incriminated. A sand fly species inventory was generated by integrating a number of techniques. Miniature light traps were used for collecting sand flies in a variety of ecotopes found across the area, and a morphological and molecular identification approach for taxonomic confirmation was undertaken. In a pilot survey conducted in September 2012, we captured 687 sand flies, 259 of which were morphologically identified to species level. They represent 14 species of the genera Sergentomyia and Grassomyia. No Phlebotomus spp. were found. A second series of collections was carried out during 2013 in five different environmental setups: two urban, two peri-urban/rural and one sylvatic; 14,036 sand flies (6665 males and 7371 females) were collected. A total of 5926 females and 98 males were morphologically identified to species level, representing 19 species of the genera Sergentomyia, Grassomyia and Phlebotomus, including Ph. duboscqi, a known vector of cutaneous leishmaniasis in the region. Two new taxa were found and are described: Sergentomyia (Sintonius) thomsoni mandarai ssp. nov. and Se. coronula sp. nov. Our study is the first to report the following species in Cameroon: Se. (Sin.) thomsoni (as ssp. nov. mandarai), Se. (Ser.) cincta, Se. (Sin.) affinis ssp. vorax, Se. (Sin.) adami, Se. (Sin.) herollandi, and Se. (Sin.) christophersi. In addition, some morphologically atypical Sergentomyia specimens (combination of Ser. x Sin. traits) were recorded. A checklist of 32 species reports from Cameroon is presented.

The structure of the female sand fly (Phlebotomus papatasi) alimentary canal.

In the sand fly vector, Leishmania parasites are confined to the alimentary canal. During much of their development, promastigotes are attached to the wall of the gut via their flagella. In this context, the surface of the different regions of the sand fly alimentary tract lumen warrants scientific attention. In this paper, the various regions are described, for the first time using scanning electron microscopy. The cibarium and the pharynx, which function as pumping organs, are lined with cuticle. Parts of the cibarium and the pharynx bear different types of cuticular spines and appendages. The midgut is lined with microvillar epithelium, which secretes the peritrophic matrix following a blood meal. The wider proximal part of the hindgut (= pylorus) is lined with transverse cuticular ridges with tentacle-like appendages. Leishmania major promastigotes were found to anchor themselves in the midgut and the stomodaeal valve via their flagella. The possible roles of the different internal structures and their importance for the development of Leishmania parasites are discussed.

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Some aspects of entomological determinants of Phlebotomus orientalis in highland and lowland visceral leishmaniasis foci in northwestern Ethiopia.

Visceral leishmaniasis (VL) is one of the major public health problems in northwest Ethiopia, mainly in Libo-Kemkem and Metema districts, where Phlebotomus orientalis is the most probable vector of the disease. The aim of this study was to determine the physiological age, host preference and vectorial potential of P. orientalis in the highland and lowland foci of the region. Sand flies were collected using CDC light traps between May 2011 and April 2012 in Libo-Kemkem and October 2012 and September 2013 in Metema from household compounds, farm field and mixed forest. Females belonging to Phlebotomus were dissected for physiological age determination and Leishmania detection and isolation. Leishmania infections in sand flies were investigated using molecular methods. Freshly fed Phlebotomus females were tested to identify blood meal sources using PCR-RLB and ELISA. A total of 1149 (936 from Libo-Kemkem and 213 from Metema) blood unfed female P. orientalis were dissected for age determination. The parity rate was 45.6% and 66.2% in Libo-Kemkem and Metema, respectively. None of 798 female P. orientalis dissected (578 from Libo-Kemkem and 220 from Metema) was infected with Leishmania parasites. A total of 347 P. orientalis specimens collected from Libo-Kemkem were processed using PCR, of which 10 (2.8%) specimens were found with DNA of Leishmania spp. Of a total 491 freshly fed female P. orientalis analyzed for blood meal origins by RLB-PCR and ELISA, 57.6% (67.8% from Libo-Kemkem and 49.8% from Metema) were found to contain bovine blood while 4.9% (3.7% from Libo-Kemkem and 5.7% from Metema) were of human blood. In conclusion, the present study showed parity difference between the two populations of P. orientalis and that both populations have strong zoophilic behavior. Based on the presented evidences, the species is strongly implicated as a vector of kala-azar in both areas. Therefore, vector control should be a component of a strategy to manage visceral leishmaniasis in both study areas.

A fine scale eco-epidemiological study on endemic visceral leishmaniasis in north ethiopian villages.

Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani that affects almost half a million people annually. In Northern Ethiopia, VL is common in migrant agricultural laborers returning from the lowland sesame fields of Metema and Humera. Recent VL foci have emerged in resident rural populations near the town. In the current study, we evaluate multilevel entomological, epidemiological and ecological factors associated with infection and disease through fine-scale eco-epidemiological analyses in three villages. Satellite images showed that villages constructed in or close to vertisols, were likely to become endemic for VL. Vertisols or black-cotton soil, are characterized by high contents of smectitic clay minerals, which swell when hydrated and shrink upon desiccation, causing extensive deep cracking during the dry season. The population densities of Phlebotomus orientalis, the vector, were negatively correlated with distance from vertisols and persons living close to vertisols were more likely to be bitten by sand flies, as evidenced by sero-positivity to Ph. orientalis saliva. Apparent (albeit non-significant) clustering of VL cases and abundant asymptomatic infections close to vertisols, suggest anthroponotic transmission around houses located close to vertisols. Comparable rates of male and female volunteers, mostly under 15 years of age, were infected with L. donovani but a significantly higher proportion of males succumbed to VL indicating a physiological gender-linked male susceptibility. Our data suggest that the abundant infected persons with high parasitemias who remain asymptomatic, may serve as reservoir hosts for anthroponotic transmission inside villages. Only limited insights on the transmission dynamics of L. donovani were gained by the study of environmental factors such as presence of animals, house structure and vegetation cover.