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BackgroundSticky 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 forPhlebotomus orientalis, the principal vector of visceral leishmaniasis in East Africa, and propose an explanation for the highly male-biased collection of sticky traps.MethodsA number of field experiments were carried out in March–June 2016–2019, in Gedarif state, eastern Sudan. In the first experiment, we compared numbers ofP. orientaliscaught 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 ofP. orientaliscaptured 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.ResultsPhlebotomus orientalisshowed significant attraction to white, yellow and transparent traps, with negligible numbers caught on the black and the red traps. Similarly, significantly higher numbers ofP. orientaliswere 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.ConclusionsWe demonstrate that, forP. 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 ofP. orientalisbehaviour and have important implications for optimization of sticky trap design for vector surveillance purposes.

Background: Leishmaniasis is the top health problem among migrant workers in Ethiopia. Poor disease risk perception and inappropriate health-seeking behaviour are significant challenges to prevent and eliminate the disease. Therefore, there is a lack of guidance on supporting migrant workers in taking actions to prevent Leishmaniasis.Method: Between June and November 2019, we conducted qualitative research among migrant workers at risk of Leishmaniasis in Ethiopia. Seventeen Focus Group Discussions and 16 Key informant interviews were conducted to study risk perception in relation to exposure to sandfly bites and the use of sandfly control measures amongst migrant workers. All interviews were recorded, transcribed, and translated for analysis. Data were analyzed by qualitative content analysis using ATLASti. Result: Migrant workers feel susceptible to Leishmaniasis due to their past exposure, and the disease is prevalent in the area. However, they feel that Leishmaniasis is a mild illness that is easy to cure. While there is a general acceptance of Insecticide Treated Net (ITN) as a protective measure, hesitations remain in the actual behaviour of utilizing it due to the alternative use of the traditional practice, seasonality, and the inconvenience to hang in the farmland areas. The central cues were the message reminders from health workers and rose in sandfly bite irritation regardless of the high perceived self-efficacy. Based on the findings, three-level intervention modalities are recommended: 1) awareness creation throughout of door media (posters, stickers, billboards) at pre-arrival, 2) the arrival level aims to stimulate the use of protective measures to be initiated and maintained, and 3) the aim at the departure level is to inform about the recognition of late signs and symptoms and the use of the protective measure to be continued at home. Conclusion: This finding proposes that leishmaniasis prevention interventions should focus on individuals' perceptions to promote consistent use of protective measures. Results empowered the researchers to foster comprehension of the content required for effective intervention.

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., Author summary Phlebotomus orientalis is the predominant vector of visceral leishmaniasis (VL, kala azar) in Sudan and other countries of East Africa, where the disease causes high morbidity and mortality. This sylvatic sand fly species is abundant in wild habitats characterized by presence of black cotton soil and vegetation dominated by Balanites aegyptiaca and/or Acacia seyal trees. In villages, the vector bites people in the household yard and in nearby peri-domestic locations, exhibiting limited indoor resting behaviour. The marked exophagic and exophilic behaviours of P. orientalis represent a profound challenge for VL control by excluding indoor residual spraying of insecticides (IRS) and compromising the efficacy of insecticide-impregnated bednets (ITNs). In this study, we evaluated the entomological efficacy of residual pyrethroid applied outdoors to household boundary fences and the exterior walls of household huts (outdoor residual insecticide spraying, ODRS), to reduce the abundance of P. orientalis inside and outside houses. We also evaluated the entomological impact of a restricted outdoor residual insecticide spraying (RODRS), whereby insecticide was applied only to the boundary fence. The study was carried out in June 2016-June 2017 in Jebel-Algana and Umsalala villages, Gedarif state, eastern Sudan, which are highly endemic for VL. The results showed that a single ODRS application of 20mg a.i. /m2 2.8% deltamethrin provided average reductions of 83%-99% in outdoor and peridomestic P. orientalis sand fly numbers relative to unsprayed control clusters. RODRS reduced outdoor and peridomestic P. orientalis by 60%-88%. The average cost of ODRS and RODRS per household were $5.76 and $3.48, respectively. The costs of these community-based control measures were substantially lower than the costs of LLINs, which is the only evidence-based tool used to protect against VL in the area. Future studies should evaluate the impact of ODRS/RODRS transmission of VL incidence in endemic villages and in seasonal agricultural farms.

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.

Visceral leishmaniasis (VL), known as Kala-azar in India, is a parasite transmitted by the bite of the sand fly vector Phlebotomus argentipes. Published information on the species indicates it is a poor flyer, mainly hopping and gliding. This study describes the vector as more arboreal than previously documented. Data collected indicate the ability of P. argentipes and Sergentomyia spp to attain vertical heights in Palmyra palm trees Borassus flabellifer up to 18.4 m above ground level. To determine if sand flies were either climbing the tree trunk to rest in the canopy or flying, sticky traps were set around the tree trunk and checked for captures overnight. CDC traps set in the palm tree canopy resulted in the capture of 5,067 sand flies, 3,990 of which were P. argentipes. Traps were set during daylight hours to determine if sand flies remained and rested in the canopy. A total of 128 sand flies were trapped over 29 trap days in the palm trees. With the CDC traps, 130 P. argentipes and no Sergentomyia spp were captured. The converse was true for the sticky traps set around tree trunks 3 m below the CDC traps. Of the 105 sand flies collected, only one was P. argentipes and 104 were Sergentomyia spp. As reported elsewhere, this indicates Sergentomyia spp tend to climb and hop, wheareas P. argentipes are capable of longer and more sustained flight. Data presented herein suggest that P. argentipes is more exophylic and exophagic than previously reported. These findings have implications for sand fly control.

A literature review is provided on the state of knowledge of the ecology and control of the sand fly vectors of Leishmania donovani in East Africa, with a special emphasis on Phlebotomus orientalis. Visceral leishmaniasis caused by L. donovani is a major health problem in several areas in East Africa. Studies conducted in the past 70 years identified P. orientalis Parrot and P. martini Parrot as the principal vectors of L. donovani in Sudan, Ethiopia and Kenya and P. celiae Minter as the secondary vector of the parasite in one focus in Ethiopia. Findings on sand fly fauna and other circumstantial evidence indicate that P. martini is also responsible for transmission of L. donovani in VL endemic foci of Somalia and Uganda. Several studies showed that P. orientalis occupy distinct habitat characterized by black cotton soil and Acacia seyal-Balanites aegyptiaca vegetation, whereas P. martini and P. celiae are associated with termite mounds. Little knowledge exists on effective control measures of sand fly vectors of L. donovani in East Africa. However, recent evidence showed that use of insecticide impregnated bednets and insect repellents may reduce exposure to the bites of P. orientalis.

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.

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.

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.

The ecology of Anopheles arabiensis and its relationship to malaria transmission was investigated in two villages in eastern Sudan. Seasonal malaria case incidence was compared with the number of vectors detected and with climatic variables. Following the end of the short rainy season in October the number of A. arabiensis detected dropped gradually until February when neither outdoor human bait trapping nor indoor spray catches revealed any mosquitoes. Vectors re-appeared in June as humidity rose with the onset of rain. Despite the apparent absence of the vector at the height of the long, hot dry season between February and May, sporadic asymptomatic malaria infections were detected in the two villages. The low endemicity of malaria in the area was reflected by the relatively low total September-December parasite and sporozoite rates (15 and 1.4%, respectively) measured in the villages. The entomological inoculation rate (EIR) was estimated to be around two to three infective bites per person per year, although heterogeneity in the transmission indices of malaria between the two villages was observed. The implications of these patterns of anopheline population dynamics for the epidemiology and control of malaria in eastern Sudan are considered.

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. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-492) contains supplementary material, which is available to authorized users.

Background Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN- γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. Methodology/Principal findings Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. Conclusion/Significance The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection., Author Summary Sand flies transmit Leishmania parasites during bloodfeeding. Salivary molecules are deposited alongside parasites and can reshape the host's immune response to infection. Exposure to uninfected sand fly bites or immunization with salivary molecules protects the host against Leishmania infection. Here we show that mice exposed to bites of uninfected Phlebotomus duboscqi sand flies are protected against P. duboscqi-transmitted L. major and characterize the formerly unknown early cellular infiltrate at the bite site following L.major vector-transmission. The kinetics and nature of the inflammatory response at the bite site of exposed mice were notably different from those of naïve mice showing an amplified expression of cytokines and chemokines after parasite transmission. The transcripts reflected a faster and more robust infiltrate of immune cells to the bite site of exposed mice composed of neutrophils, macrophages, monocytes, NK cells and CD4+ T cells. In addition, there was an increased influx of activated IFN-γ producing CD4+ T cells and Granzyme B-producing mature NK cells in exposed animals. These findings suggest that the observed robust and persistent proinflammatory response in exposed animals restrict parasite multiplication.

A study of malaria prevalence and transmission was carried out in Khartoum, the capital of Sudan. The sentinel sites were El manshia, an urban area on the Blue Nile and Ed dekheinat, a lower-income peri-urban area bordering the White Nile. Anopheles arabiensis, the only malaria vector encountered, was present throughout the year although vector density varied seasonally. Plasmodium falciparum was the only species found in El manshia. In Ed dekheinat P. falciparum, Plasmodium ovale and Plasmodium vivax constituted 84.9, 8.2 and 6.9% of the cases, respectively. Plasmodium ovale appears to have recently spread into Khartoum since it has not previously been reported there. We conclude that focal transmission of malaria in the districts bordering both Niles has become established and that the reservoir of human infections has increased in recent years leading to increased risk of malaria epidemics, particularly in the aftermath of seasonal flooding.

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., Author Summary Leishmania are pathogenic protozoa characterized by substantial diversity in the sand fly species that can transmit them, in the mammalian species that can serve as their reservoir hosts, and in the disease forms and severity of the clinical outcomes they can produce in humans. The possibility that this diversity has arisen, at least in part, by a process involving genetic exchange was recently given experimental support by the recovery of hybrid parasites from sand flies co-infected with two strains of Leishmania major. Here, we demonstrate the sexual competency of L. major strains originating across the full geographic range of this parasite species, and in both natural and unnatural sand fly vectors. Our genotype analyses of a large number of hybrid clones confirmed that they inherited both parental alleles at the majority of chromosomal marker loci analyzed, consistent with a meiotic process, while kinetoplast DNA was inherited from only one parent. Surprisingly, a few nuclear loci were sometimes inherited from only one parent, suggesting loss of heterozygosity. The early timing of hybrid recovery suggests that nectomonad promastigotes are the most likely mating competent stage of the parasite. These studies provide the strongest evidence to date that sex is a component of the natural reproductive strategy of L. major.

Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500 ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4(+) T cells. Two weeks after challenge, early induction of IFN-γ specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-γ in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-γ-producing CD4(+) T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly-transmitted Leishmania somewhat mimicking "leishmanization"-induced protective immunity but without its associated lesions.

Background Cutaneous Leishmaniasis (CL) is endemic in Sudan. It is caused by Leishmania major parasites and transmitted by Phlebotomus papatasi sandflies. Recently, uncommon clinical manifestations of CL have been reported. Moreover, L. donovani parasites that cause Visceral Leishmaniasis (VL) have been isolated from CL lesions of some patients who contracted the disease in Khartoum State, Central Sudan with no history of travelling to VL endemic sites on south-eastern Sudan. Because different clinical manifestations and the parasite behaviour could be related to genetic differentiation, or even sub-structuring within sandfly vector populations, a population genetic study was conducted on P. papatasi populations collected from different localities in Khartoum State known for their uncommon CL cases and characterized by contrasting environmental conditions. Methods A set of seven microsatellite loci was used to investigate the population structure of P. papatasi samples collected from different localities in Khartoum State, Central Sudan. Populations from Kassala State, Eastern Sudan and Egypt were also included in the analyses as outgroups. The level of genetic diversity and genetic differentiation among natural populations of P. papatasi was determined using FST statistics and Bayesian assignments. Results Genetic analyses revealed significant genetic differentiation (FST) between the Sudanese and the Egyptian populations. Within the Sudanese P. papatasi populations, one population from Gerif West, Khartoum State, exhibited significant genetic differentiation from all other populations including those collected as near as 22 km. Conclusion The significant genetic differentiation of Gerif West P. papatasi population from other Sudanese populations may have important implication for the epidemiology of leishmaniasis in Khartoum State and needs to be further investigated. Primarily, it could be linked to the unique location of Gerif West which is confined by the River Nile and its tributaries that may act as a natural barrier for gene flow between this site and the other rural sites. The observed high migration rates and lack of genetic differentiation among the other P. papatasi populations could be attributed to the continuous human and cattle movement between these localities.

Background Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE) with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Methods Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA) were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge. Results Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4+CD62LlowCCR7low effector memory T cells pre- and post-sand fly challenge. Conclusions This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection., Author Summary Leishmaniasis is a neglected disease caused by the Leishmania parasite and transmitted by the bite of an infective sand fly. Despite the importance of this disease there is no vaccine available for humans. Studies have shown that vector-transmitted infections are more virulent, promoting parasite establishment and abrogating protection observed against needle-injected parasites in vaccinated mice. KSAC and L110f, derived from Leishmania-based polyproteins, protected mice against the needle-injected parasites. Here, we tested the two molecules for their capacity to protect mice against cutaneous leishmaniasis transmitted by an infective sand fly. Our results show that KSAC, but not L110f, confers protection against Leishmania transmitted by sand fly bites where protection was correlated to a strong immune response to Leishmania antigens by memory T cells before and after sand fly transmission of the parasite. This is the first report of a Leishmania-based vaccine that confers protection against a virulent sand fly challenge. Our results support the importance of screening Leishmania vaccine candidates using infective sand flies before moving forward with the costly steps of vaccine development.

The closely related sandfly species of the subgenus Phlebotomus namely, Phlebotomus papatasi (Scopoli, 1786), Phlebotomus duboscqi Neveu-Lemair, 1906 and Phlebotomus bergeroti Parrot, 1934 (Diptera: Psychodidae), are major vectors of Leishmania major (Kinetoplastida: Trypanosomatidae), the causative agent of cutaneous leishmaniasis in the Old World. Although allopatric in most of their distribution, the three species exist sympatrically in many places in central and eastern Sudan. Males of the three species can be distinguished using morphological characters; however, females are much harder to identify, thus complicating epidemiological studies. We carried out a morphometric and a molecular study to determine reliable morphological features and develop a polymerase chain reaction (PCR) assay for distinguishing females of these species. Males and females from each species were collected from sites in Sudan, East Africa and from one site in Mali, West Africa. Males were analysed morphologically and 20 characters and 10 character ratios were used in a stepwise discriminant analysis. This led to the identification of four characters with high discriminant loading scores sufficient for accurate male species identification. Male DNA was then used for the development of a PCR-based species diagnostic based on the second internal transcribed spacer (ITS2) of the ribosomal DNA. A set of four primers was developed to generate fragment sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Both the morphometric and the molecular findings of this study have important applications for studies of the epidemiology of cutaneous leishmaniasis.

Oviposition preferences of Lutzomyia longipalpis Lutz & Neiva for surfaces containing frass (colony remains), larval rearing medium, and rabbit feces were investigated in the laboratory. In oviposition choice chambers, significantly more eggs were laid on sites containing frass, larval rearing medium, or rabbit feces than on untreated control sites. Experiments using unwashed and washed materials indicated, for the first time, the presence of chemical oviposition attractants or stimulants or both in larval medium (consisting of equal weight proportions of rabbit feces, potting compost, sand, and Daphnia ). In an olfactometer, aroma from rabbit feces strongly attracted ovipositing females, and significantly higher numbers of eggs were laid in the olfactometer test chamber containing the rabbit feces than in the untreated control chamber. In comparisons of oviposition responses to hexane and distilled water extracts of rabbit feces, only the water extract was attractive. Similarly, the distilled water extract of rabbit feces proved to be an oviposition stimulant. Females kept individually with filter papers impregnated with the extract of feces oviposited earlier and survived longer than females kept with untreated papers.

Given the importance that the evolution of cooperation bears in evolutionary biology and the social sciences, extensive theoretical work has focused on identifying conditions that promote cooperation among individuals. In insects, cooperative or altruistic interactions typically occur amongst social insects and are thus explained by kin selection. Here we provide evidence that in Lutzomia longipalpis, a small biting fly and an important vector of leishmaniasis in the New World, cooperative blood-feeding in groups of non-kin individuals results in a strong decrease in saliva expenditure. Feeding in groups also strongly affected the time taken to initiate a bloodmeal and its duration and ultimately resulted in greater fecundity. The benefits of feeding aggregations were particularly strong when flies fed on older hosts pre-exposed to sand fly bites, suggesting that flies feeding in groups may be better able to overcome their stronger immune response. These results demonstrate that, in L. longipalpis, feeding cooperatively maximizes the effects of salivary components injected into hosts to facilitate blood intake and to counteract the host immune defences. As a result, cooperating sand flies enjoy enormous fitness gains. This constitutes, to our knowledge, the first functional explanation for feeding aggregations in this species and potentially in other hematophagous insects and a rare example of cooperation amongst individuals of a non-social insects species. The evolution of cooperative group feeding in sand flies may have important implications for the epidemiology of leishmaniasis., Author Summary Understanding the processes that promote cooperation amongst animals in nature is a fundamental question in evolutionary biology with ramifications in the social sciences. In insects, cooperative or altruistic interactions are usually observed amongst genetically related social insects (kin selection). Here we provide evidence that in Lutzomia longipalpis, a small biting fly and an important vector of disease in the New World, cooperative blood-feeding occurs in groups of non-kin individuals. Groups of 20 flies and single flies were fed on hamster hosts and we compared their salivary gland usage as well as the time taken to initiate a bloodmeal, its duration, and the number of eggs they produced. Our results show that flies feeding in aggregations benefit from decreased saliva expenditure and greatly enhanced blood intake and egg production. These effects were particularly strong on older hamsters pre-exposed to sand fly bites, suggesting that group-feeding flies may better overcome their stronger immune response. These experiments demonstrate that, in L. longipalpis, feeding cooperatively maximizes the effects of saliva injected into hosts to facilitate blood intake and to counteract the host immune defences, resulting in much increased fecundity. This constitutes the first explanation for the function of feeding aggregations in hematophagous insects and a fascinating example of cooperation amongst individuals in a non-social organism.

Response of female Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) to an oviposition attractant and/or stimulant associated with conspecific eggs was investigated in the laboratory. Females of two populations laid significantly higher mean number of eggs on sites with 160 eggs already present than on bare control sites. This response was lost when eggs placed on test sites were previously washed in organic solvents and distilled water. Age of eggs placed on test sites, 1–6 days after being laid, did not seem to affect the oviposition response of the females. Comparing effects of different numbers of eggs, positive responses were obtained when 80, 160 or 320 eggs were placed on test sites. No significant differences between mean egg numbers laid on test sites and controls were detected when batches of only 20 or 40 eggs were used.

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.

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 100,000Leishmania infantum chagasiparasites together withLutzomyia longipalpissaliva 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 ofLu. longipalpisresulted 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-γ/TGF-β 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-γ was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-γ 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-Leishmaniaimmunity. 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.

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-1α, TNF-α, 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-1α 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.

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.

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic “cutoff” value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite., Author Summary Many infectious diseases are initiated when pathogenic organisms are deposited into the skin of the human host by the bite of an insect. In the case of the parasite Leishmania, the causative agent of Leishmaniasis, factors associated with the bite of the infected sand fly vector influence infection outcome, suggesting that this is the most relevant means to initiate disease in experimental hosts. However, transmission frequency, and the dose of parasites delivered when transmission occurs, can vary enormously both within and between populations of experimentally infected flies. This variability represents a major obstacle to the widespread use of sand flies to study Leishmaniasis. We identified a parameter of Leishmania infection in the sand fly vector that predicts the degree of transmission competence within a group of experimentally infected flies. We also identified environmental and biological factors that influence transmission frequency. This information will make experiments relying on infected sand fly challenge more manageable, thereby increasing the likelihood that infected sand fly challenge, rather than needle challenge, will be used in future experimentation. Lastly, we demonstrated that the number of organisms acquired by the sand fly can influence subsequent sand fly infection intensity, and that this infection intensity has implications for disease outcome.

Background The study aims to determine the role of domestic dogs in transmission of visceral leishmaniasis in eastern Sudan. A cross-sectional survey was conducted in 10 villages along the River Rahad in eastern Sudan to elucidate the role of domestic dogs (Canis familiaris, Linnaeus, 1758) as a reservoir host of Leishmania donovani. In this study, 87 dogs were screened for infection by Leishmania donovani. Blood and lymph node samples were taken from 87 and 33 dogs respectively and subsequently screened by the Polymerase Chain Reaction (PCR) and Direct Agglutination Test (DAT) test. Additional lymph node smears were processed for microscopy and parasite culture. Host preference of the visceral leishmaniasis (VL) vector in the area, Phlebotomus orientalis, and other sandflies for the Nile rat (Arvicanthis niloticus, É. Geoffrey, 1803), the genet (Genetta genetta, Linnaeus, 1758), the mongoose (Herpeistes ichneumon, Linnaeus, 1758), and the domestic dog were determined by counting numbers of sand flies attracted to CDC traps that were baited by these animals. Results DAT on blood samples detected anti-Leishmania antibodies in 6 samples (6.9%). Two out of 87 (2.3%) blood samples tested were PCR positive, giving an amplification product of 560 bp. The two positive samples by PCR were also positive by DAT. However, none of the 33 lymph nodes aspirates were Leishmania positive when screened by microscopy, culture and genus-specific PCR. The dog-baited trap significantly attracted the highest number of P. orientalis and sand fly species (P < 0.001). This was followed by the Egyptian mongoose baited trap and less frequently by the genet baited trap. Conclusion It is concluded that the results obtained from host attraction studies indicate that dog is more attractive for P. orientalis than Egyptian mongoose, common genet and Nile rat.

Background Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani is a major health problem in Sudan and other East African countries. In this region the only proven vectors of L. donovani are Phlebotomus orientalis in eastern Sudan, Ethiopia and Upper Nile areas of Southern Sudan and Phlebotomus martini in Ethiopia, Kenya and Southern Sudan. In this report, we present the first evidence that Phlebotomus rodhaini may also play a role in maintaining transmission of L. donovani between animal reservoir hosts in eastern Sudan. The study was conducted in a zoonotic focus of visceral leishmaniasis in Dinder National Park, eastern Sudan, where previous work showed high infection rates of L. donovani in P. orientalis. Sand flies, captured by CDC traps were dissected and examined for infection with Leishmania parasites. Parasite isolates were subjected to L. donovani specific PCR. Field experiments were also carried out to compare efficiency of rodent baited and un-baited CDC traps in collection of P. rodhaini and determine its man-biting rate. Results Three female P. rodhaini were found infected with Leishmania parasites in an astonishingly small number of flies captured in three separate field trips. Two of these isolates were typed by molecular methods as L. donovani, while the third isolate was inoculated into a hamster that was subsequently lost. Although P. rodhaini is generally considered a rare species, results obtained in this study indicate that it can readily be captured by rodent-baited traps. Results of human landing collection showed that it rarely bites humans in the area. Conclusion It is concluded that P. rodhaini is a possible vector of L. donovani between animal reservoir hosts but is not responsible for infecting humans. It is suggested that the role of P. rodhaini in transmission of L. donovani in other zoonotic foci of visceral leishmaniasis in Africa should be re-examined.