Host preference and human blood index of Phlebotomus orientalis, an exophilic sand fly vector of visceral leishmaniasis in eastern Sudan.

Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani, transmitted by Phlebotomus orientalis, is a serious systemic disease that causes high morbidity and mortality rates in Sudan and other parts of East Africa and the world. Despite progress in understanding the epidemiology of the disease in East Africa, little is known about the host preference of P. orientalis in kala azar endemic villages of Sudan, which have some of the highest VL incidence rates in the world. The present study used host choice experiments and blood-meal identification approaches to determine the host preference of P. orientalis in kala azar endemic villages in Gedarif state, eastern Sudan. In the host choice experiment, tent traps were used to compare the attractiveness of cows, donkeys, sheep and goats for host-seeking P. orientalis. In the blood-meal identification study, blood-fed P. orientalis females, captured inside houses and peri-domestic habitats, were subjected to molecular typing using cytochrome b gene (cyt b) amplification and sequence analysis. Cows and donkeys were the most attractive to blood-seeking P. orientalis, followed by goats. Similarly, the blood-meal analysis of P. orientalis showed that the vector preferentially feeds on cows, followed by donkeys, humans and goats. The human blood index of P. orientalis was 19.4% (42/216), indicating a high zoophilic habit of the vector, both inside and outside the houses. Although the order of host preference varied by location, it was clear that cows are the most preferred host of P. orientalis in the area. Results are discussed in relation to the role of domestic/livestock animals in VL zoopotentiation and zooprophylaxis. Inference is made on the potential impact of insecticide treatment of cows in control of the vector and the transmission of VL in Sudan and other parts of East Africa.

Understanding the risk perception of visceral leishmaniasis exposure and the acceptability of sandfly protection measures among migrant workers in the lowlands of Northwest Ethiopia: a health belief model perspective.

BACKGROUND: Visceral leishmaniasis (VL) is the leading cause of health concerns among Ethiopian migrant workers. Understanding risk perception and health-protective behavior are significant challenges in the prevention and eradication of the disease. As a result, studies are required to assess these important epidemiological factors, which will provide guidance on how to assist migrant workers in taking preventive measures against VL. METHOD: We conducted qualitative research among migrant workers on seasonal agricultural farms in Northwest Ethiopia between June and November 2019 to assess their perception of the risk of contracting VL and their willingness to use protective measures against the disease. Seventeen focus group discussions and 16 key informant interviews were conducted to study migrant workers' risk perception in relation to sandfly bite exposure and use of sandfly control measures. For analysis, all interviews were recorded, transcribed, and translated. ATLASti was used to perform qualitative content analysis on the data. RESULT: Migrant workers are fearful of VL because of previous exposure and the disease's prevalence in the area. They believe, however, that VL is a minor illness that is easily treated. While Insecticide Treated Nets (ITNs) are widely accepted as a protective measure, there are still reservations about using them due to the seasonality of the transmission, difficulties in hanging them on farm areas, and a preference for alternative traditional practices. Regardless of perceived self-efficacy, the central cues were the message delivered by the health workers and an increase in sandfly bite irritation. Based on the findings, three levels of intervention modalities are suggested: 1) increasing pre-arrival awareness through outdoor media (posters, stickers, billboards), 2) encouraging proper use of protective measures upon arrival at farm camps, and 3) informing departing workers on disease recognition and best practices for health-seeking continuous use of protective measures at home. CONCLUSION: This finding suggests that VL prevention interventions should focus on individuals' perceptions in order to promote consistent use of protective measures. The findings are highly useful in planning effective interventions against VL.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

A genotypically distinct, melanic variant of Anopheles arabiensis in Sudan is associated with arid environments.

BACKGROUND: Anopheles arabiensis, an important malaria vector in Sudan and other countries in sub-Saharan Africa, exhibits considerable ecological and behavioural plasticity allowing it to survive in the harsh conditions of arid regions. It has been shown that adult populations of An. arabiensis in the semi-desert habitat of western Khartoum State survive through the long dry season in a state of partial aestivation, characterized by limited feeding activity and a degree of arrested ovarian development. Anopheles arabiensis in these sites occurs in two phenotypic forms. One is large and heavily melanized, the other has the typical characteristics of An. arabiensis as found elsewhere in Africa. The extent of genetic variation in these forms was examined in widely separated locations in Sudan, including Kassala, Gedaref and the Northern States between 1998 and 1999 and 2004 and 2006. METHODS: Each mosquito specimen was identified using standard morphological keys and a species-specific PCR test. Sequence variation in a 660 bp fragment of the mtDNA ND5 coding region was examined and the extent of genetic divergence between the forms was estimated from FST values using DNASP version 4.9. TCS 1.13 software was used to determine the genealogical relationships and to reflect clustering among mtDNA haplotypes. RESULTS: The melanic and normal forms were found in sympatry in Kassala, Gedaref and Khartoum states, with the melanic form commonest in the hottest and most arid areas. Both forms were encountered in the periods of study: 1998-1999, and 2004-2006. Only ten specimens of An. arabiensis were collected from the Northern State in February 2006, all of which were of the normal form.Based on the ND5 analysis, there was a marked subdivision between the normal and melanic forms (FST = 0.59). Furthermore, the melanic form showed more genetic variability, as measured by haplotype diversity (0.95) compared with the normal form (0.57), suggesting larger effective population. CONCLUSIONS: This is the first demonstration of correspondent phenotypic and genetic structuring in An. arabiensis. The high level of genetic differentiation shown by the mtDNA ND5 locus suggests that the two forms may represent separate species. It is hypothesized that the melanic form is better adapted to hot and arid environments.

Infection of Leishmania donovani in Phlebotomus orientalis Sand Flies at Different Microhabitats of a Kala-Azar Endemic Village in Eastern Sudan.

A study was carried out to compare the infection rates of Leishmania donovani in Phlebotomus orientalis sandflies at different microhabitats of a VL endemic village in Gedarif state, Sudan. DNA extracts of 1078 P. orientalis sand fly females sampled by CDC light traps from indoor, outdoor, peri-domestic, and sylvatic sites, in three transmission seasons, March-June 2016-18, in Helat-Belo village, were subjected to independent PCR amplifications targeting Leishmania kDNA and the cpb gene followed by ITS1 region sequencing. Leishmania kDNA was detected in 1.4% of the 1078 P. orientalis females captured in the area. Two of these specimens showed a characteristic 741 bp band of L. donovani after cpb gene amplification. The DNA sequence of the ITS1 region of the parasites matched the ITS1 L. donovani genotype F. There were no signficant differences between rates of infection of L. donovani in P. orientalis captured at different sites. Blood meals found in infected flies origninated from human (5 specimens), cattle (4 specimens) and donkey (2 specimens). The finding of fresh cow and donkey blood in the infected flies suggests the possible role of these animals in the zoopotentiation and/or zooprophylaxis against VL. The study provides important information for VL transmission models and control programs in East Africa.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed ß-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Genetic Polymorphism and Phylogenetics of Aedes aegypti from Sudan Based on ND4 Mitochondrial Gene Variations.

This study investigated the genetic differences between Aedes aegypti subspecies (Aedes aegypti aegypti (Aaa) and Aedes aegypti formosus (Aaf)) from Sudan using the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene marker. Nineteen distinct haplotypes of the ND4 were identified in female Aedes aegypti mosquitoes from the study sites. The phylogenetic relationship of the 19 ND4 haplotypes was demonstrated in a median-joining haplotype network tree with Aaa and Aaf populations found to share three haplotypes. The genetic variance (Pairwise FST values) was estimated and found to range from 0.000 to 0.811. Isolation by distance test revealed that geographical distance was correlated to genetic variation (coefficient value (r) = 0.43). The Polar maximum likelihood tree showed the phylogenetic relationship of 91 female Aaa and Aaf from the study sites, with most of the Aaf haplotypes clustered in one group while most of the Aaa haplotypes gathered in another group, but there was an admixture of the subspecies in both clusters, especially the Aaa cluster. The Spatial Analysis of Molecular Variance (SAMOVA) test revealed that the eight populations clustered into two phylogeographic groups/clusters of the two subspecies populations. The 2 Aedes aegypti subspecies seemed not to be totally separated geographically with gene flow among the populations.

Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania.

Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-gamma at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG(2) antibody levels and significant IFN-gamma production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-gamma and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.

Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model.

Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.

Distribution and Genetic Diversity of Aedes aegypti Subspecies across the Sahelian Belt in Sudan.

Aedes aegypti is the most important arboviral disease vector worldwide. In Africa, it exists as two morphologically distinct forms, often referred to as subspecies, Aaa and Aaf. There is a dearth of information on the distribution and genetic diversity of these two forms in Sudan and other African Sahelian region countries. This study aimed to explore the distribution and genetic diversity of Aedes aegypti subspecies using morphology and Cytochrome oxidase-1 mitochondrial marker in a large Sahelian zone in Sudan. An extensive cross-sectional survey of Aedes aegypti in Sudan was performed. Samples collected from eight locations were morphologically identified, subjected to DNA extraction, amplification, sequencing, and analyses. We classified four populations as Aaa and the other four as Aaf. Out of 140 sequence samples, forty-six distinct haplotypes were characterized. The haplotype and nucleotide diversity of the collected samples were 0.377-0.947 and 0.002-0.01, respectively. Isolation by distance was significantly evident (r = 0.586, p = 0.005). The SAMOVA test indicated that all Aaf populations are structured in one group, while the Aaa clustered into two groups. AMOVA showed 53.53% genetic differences within populations and 39.22% among groups. Phylogenetic relationships indicated two clusters in which the two subspecies were structured. Thus, the haplotype network consisted of three clusters.

Understanding sand fly sampling methods: sticky traps are attraction-based and not interceptive sampling tools of Phlebotomus orientalis.

BACKGROUND: Sticky traps are generally viewed as interceptive sand fly sampling methods; although no previous experimental evidence has supported this assumption. In this study, we tested this assumption experimentally for Phlebotomus orientalis, the principal vector of visceral leishmaniasis in East Africa, and propose an explanation for the highly male-biased collection of sticky traps. METHODS: A number of field experiments were carried out in March-June 2016-2019, in Gedarif state, eastern Sudan. In the first experiment, we compared numbers of P. orientalis caught on sticky traps made of black, red, transparent, white, yellow, green and blue A4 size papers set simultaneously at different lunar light conditions. In the second and third experiments, we compared numbers of P. orientalis captured on sticky traps placed side-by-side horizontally or vertically on the ground, or horizontally on a 15 cm height stool. We also witnessed mating behaviour of sand flies following their landing on un-sticky papers placed on the ground. RESULTS: Phlebotomus orientalis showed significant attraction to white, yellow and transparent traps, with negligible numbers caught on the black and the red traps. Similarly, significantly higher numbers of P. orientalis were attracted to the horizontal traps, resulting in an 8-fold increase in sand fly trapping efficacy as compared to the vertical traps. Placing the traps on the stools resulted in significant reduction in this attraction. In contrast to the sticky traps that captured only very few females; we found that when male sand flies land on un-sticky white paper they successfully lure females and copulate with them. CONCLUSIONS: We demonstrate that, for P. orientalis, sticky traps are more attractant-based than interception-based sampling tools. Further, our findings support the notion that males of this sand fly species likely utilize the bright surface of the trap papers to perform mating rituals that attract the females for copulation. However, pre-mature death in the sticky oil hampers the completion of these rituals, and thus results in failure to attract the females. These findings inform our understanding of P. orientalis behaviour and have important implications for optimization of sticky trap design for vector surveillance purposes.

Impact of Education on Knowledge and Practice of Kala Azar Preventive Measures among Seasonal and Migrant Agricultural Workers in Northwest Ethiopia.

Kala azar occurs among seasonal and migrant agricultural workers in northwest Ethiopia and accounts for almost 60% of the disease burden in the country. We conducted a quantitative study on the level of knowledge and practice of this vulnerable group in relation to kala azar transmission and acceptability of its vector control tools. A total of 403 workers were randomly selected from eight farms using a purposive sampling technique. Knowledge and practice scores were calculated based on 12 and 9 core questions, respectively. Binary logistic regression was used to identify factors associated with knowledge and practice. A large gap in knowledge of the disease and the vector was evident with 61.8%, 24.6%, and 13.6% of the workers having poor, moderate, and good levels of knowledge scores, respectively. Similarly, 95% of the seasonal workers reported poor level of use of protective measures against the bite of the sand fly vector. Good level of knowledge about kala azar and its sand fly vector was statistically associated with formal education (adjusted odds ratio [AOR] = 2.11; 95% CI = 1.17, 3.80; P < 0.05) and previous exposure to health education (AOR = 4.72; 95% CI = 1.99, 11.16; P < 0.001). Despite poor current knowledge and practice, a large proportion of the study participants showed interest in using vector control tools if made available, with 78% of the seasonal and migrant workers expressing some willingness to pay for different measures that can protect against sand fly bites. Therefore, we strongly recommend that comprehensive health education and vector control programs should be provided to these workers.

Outdoor Residual Insecticide Spraying (ODRS), a New Approach for the Control of the Exophilic Vectors of Human Visceral Leishmaniasis: Phlebotomus orientalis in East Africa.

Visceral Leishmaniasis (VL) due to Leishmania donovani is a neglected protozoan parasitic disease in humans, which is usually fatal if untreated. Phlebotomus orientalis, the predominant VL vector in East Africa, is a highly exophilic/exophagic species that poses a major challenge to current Integrated Vector Management (IVM). Here we report results of pilot studies conducted in rural villages in Gedarif state, Sudan, to evaluate outdoor residual spraying of 20mg active ingredient (a.i.) /m2 deltamethrin insecticide applied to the characteristic household compound boundary reed fence and to the outside of household buildings (Outdoor Residual Insecticide Spraying, ODRS), and as an alternative, spraying restricted to the boundary fence only (Restricted Outdoor Residual Insecticide Spraying, RODRS). Four to six clusters of 20 households were assigned to insecticide treatments or control in three experiments. Changes in sand fly numbers were monitored over 2,033 trap-nights over 43-76 days follow-up in four sentinel houses per cluster relative to unsprayed control clusters. Sand fly numbers were monitored by sticky traps placed on the ground on the inside ("outdoor") and the outside ("peridomestic") of the boundary fence, and by CDC light traps suspended outdoors in the household compound. The effects of ODRS on sand fly numbers inside sleeping huts were monitored by insecticide knockdown. After a single application, ODRS reduced P. orientalis abundance by 83%-99% in outdoor and peridomestic trap locations. ODRS also reduced numbers of P. orientalis found resting inside sleeping huts. RODRS reduced outdoor and peridomestic P. orientalis by 60%-88%. By direct comparison, RODRS was 58%-100% as effective as ODRS depending on the trapping method. These impacts were immediate on intervention and persisted during follow-up, representing a large fraction of the P. orientalis activity season. Relative costs of ODRS and RODRS delivery were $5.76 and $3.48 per household, respectively. The study demonstrates the feasibility and high entomological efficacy of ODRS and RODRS, and the expected low costs relative to current IVM practises. These methods represent novel sand fly vector control tools against predominantly exophilic/exophagic sand fly vectors, aimed to lower VL burdens in Sudan, with potential application in other endemic regions in East Africa.

Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE2/IL-10 sequential pathway.

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen-induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge-induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP-1alpha, TNF-alpha, and leukotriene B4 (LTB4). Furthermore, SGE treatment enhanced the production of anti-inflammatory mediators, IL-10 and PGE2. SGE treatments failed to inhibit neutrophil migration and MIP-1alpha and LTB4 production in IL-10-/- mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE-induced IL-10 production, and PGE2 release triggered by SGE remained increased in IL-10-/- mice, suggesting that prostanoids are acting through an IL-10-dependent mechanism. SGE treatments in vivo reduced the OVA-induced lymphoproliferation of spleen-derived cells. Further, the in vitro incubation of bone marrow-derived dendritic cells (DC) with SGE inhibited the proliferation of CD4+T cells from OVA-immunized mice, which was reversed by indomethacin and anti-IL-10 antibody treatments. Supporting these results, SGE induced the production of PGE2 and IL-10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC-membrane expression of MHC-II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation-induced neutrophil migration by an autocrine DC sequential production of PGE2/IL-10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.

The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies.

BACKGROUND: In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. RESULTS: Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein. CONCLUSION: This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.

Impact of aerial spraying of pyrethrin insecticide on Culex pipiens and Culex tarsalis (Diptera: Culicidae) abundance and West Nile virus infection rates in an urban/suburban area of Sacramento County, California.

In response to an epidemic amplification of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), the Sacramento and Yolo Mosquito and Vector Control District (SYMVCD) sprayed ultralow-volume (ULV) formulations of pyrethrin insecticide (Evergreen EC 60-6: 6% pyrethrin insecticide, 60% piperonylbutoxide; MGK, Minneapolis, MN, applied as 0.003 kg/ha [0.0025 lb/acre] ) over 218 km2 in north Sacramento and 243.5 km2 in south Sacramento on three consecutive evenings in August 2005. We evaluated the impact of this intervention in north Sacramento on the abundance and WNV infection rates of Culex pipiens L. and Culex tarsalis Coquillett. Mortality rates of caged Cx. tarsalis sentinels ranged from 0% under dense canopy to 100% in open fields. A comparison of weekly geometric mean mosquito abundance in CO2-baited traps in sprayed and unsprayed areas before and after treatment indicated a 75.0 and 48.7% reduction in the abundance of Cx. pipiens and Cx. tarsalis, respectively. This reduction was statistically significant for Cx. pipiens, the primary vector of WNV, with highest abundance in this urban area, but not for Cx. tarsalis, which is more associated with rural areas. The infection rates of WNV in Cx. pipiens and Cx. tarsalis collected from the spray zone were 8.2 and 4.3 per 1,000 female mosquitoes in the 2 wk before and the 2 wk after applications of insecticide, respectively. In comparison, WNV infection rates in Cx. pipiens and Cx. tarsalis collected at same time interval in the unsprayed zone were 2.0 and 8.7 per 1,000, respectively. Based on the reduction in vector abundance and its effects on number of infective bites received by human population, we concluded that the aerial application ofpyrethrin insecticide reduced the transmission intensity of WNV and decreased the risk of human infection.

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.

Effects of vegetation control on mosquitoes in seasonal freshwater wetlands.

Wetland restorations benefit wildlife, but wetland vegetation may promote mosquitoes. We studied the effects of removing joint grass (Paspalum distichum L.) on the abundance and distribution of immature mosquitoes in seasonal freshwater wetlands in California. In 4 >4-ha plots and one 29.8-ha field, joint grass was removed via herbicide and disking. Four untreated plots and one 4.1-ha field were controls. Fields were sampled every 7-14 days in early autumn by taking 75 dips along levees, plus a 25-dip circular transect 6 m from field edges. Untreated plots had 7 times more larvae and 20 times more pupae than did vegetation-removal plots. Similar densities of mosquitoes were found along edges and within fields.

Eco-epidemiology of visceral leishmaniasis in Ethiopia.

Visceral leishmaniasis (VL, Kala-azar) is one of the growing public health challenges in Ethiopia with over 3.2 million people at risk and estimated up to 4000 new cases per year. Historically, VL was known as the diseases of the lowlanders; in the lower and upper Kola agro-ecological zones of Ethiopia. The 2005-07 out breaks in highlands of Libo Kemkem and Fogera, in the Woina Degas, that affected thousands and claimed the life of hundreds misdiagnosed as drug resistance malaria marked that VL is no more the problem of the lowlanders. The Kola (lower and upper) and the Woina Dega are the most productive agroecological zones, supporting both the ongoing and planned expansions of large or small scale agriculture and/or agriculture based industries. Thus, the (re)emergence of VL is not only a public health and social problem but also have a direct implication on the country's economy and further development. Thus is high time for its control and/or elimination. Yet, the available data seem incomplete to plan for a cost-effective and efficient VL control strategy: there is a need to update data on vector behaviour in specific ecosystems and the roles of domestic animals need to be ascertained. The effectiveness and social acceptability of available vector control tools need be evaluated. There is a need for identifying animal reservoir(s), or establish the absence of zoonosis in Ethiopia. The planning of prevention of (re)emergence and spread of VL to areas adjacent to endemic foci need be supported with information from spatio-temporal mapping. In affected communities, available data showed that their knowledge about VL is generally very low. Thus, well designed studies to identify risk factors, as well as better tools for social mobilization with the understanding of their knowledge, aptitude and practice towards VL are necessary.

Risk mapping of visceral leishmaniasis: the role of local variation in rainfall and altitude on the presence and incidence of kala-azar in eastern Sudan.

Visceral leishmaniasis (VL) is a vector-borne disease highly influenced by environmental factors. A model was developed for mapping the distribution and incidence of VL in Gedaref State, eastern Sudan, in relation to different environmental factors. Geographical information systems (GIS) were used to extract and map regression results for environmental variables of 190 villages in Gedaref State, including rainfall, vegetation status, soil type, altitude, distance from river, topography, wetness indexes, and average rainfall estimates. VL incidence in each village was calculated from hospital records. By use of logistic and linear multivariate regression analyses, models were developed to determine which environmental factors explain variability in VL presence and incidence. We found that average rainfall and the altitude were the best predictors of VL incidence. The resulting models were mapped by GIS software predicting both VL presence or absence and incidence at any locality in Gedaref State. The results are discussed in relation to VL control.