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.

Improving houses in the Bolivian Chaco increases effectiveness of residual insecticide spraying against infestation with Triatoma infestans, vector of Chagas disease.

OBJECTIVE: Failure to control domestic Triatoma infestans in the Chaco is attributed to vulnerable adobe construction, which provides vector refuges and diminishes insecticide contact. We conducted a pilot to test the impact of housing improvement plus indoor residual spraying (IRS) on house infestation and vector abundance in a rural community in the Bolivian Chaco. METHODS: The intervention included three arms: housing improvement + IRS [HI], assisted IRS [AS] in which the team helped to clear the house pre-IRS and routine IRS [RS]. HI used locally available materials, traditional construction techniques and community participation. Vector parameters were assessed by Timed Manual Capture for 2 person-hours per house at baseline and medians of 114, 173, 314, 389 and 445 days post-IRS-1. A second IRS round was applied at a median of 314 days post-IRS-1. RESULTS: Post-intervention infestation indices and abundance fell in all three arms. The mean odds of infestation was 0.29 (95% CL 0.124, 0.684) in the HI relative to the RS arm. No difference was observed between AS and RS. Vector abundance was reduced by a mean 44% (24.8, 58.0) in HI compared to RS, with no difference between AS and RS. Median delivered insecticide concentrations per house were lower than the target of 50 mg/m2 in >90% of houses in all arms. CONCLUSION: Housing improvement using local materials and community participation is a promising strategy to improve IRS effectiveness in the Bolivian Chaco. A larger trial is needed to quantify the impact on reinfestation over time.

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control.

Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions.

Performance of a Noninvasive Test for Detecting Mycobacterium bovis Shedding in European Badger (Meles meles) Populations.

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.

Assessing reservoir host status in leishmaniasis with special reference to the infectiousness of Leishmania (Viannia) braziliensis infections in wild rodents.

Molecular methods have been responsible for a notable increase in the detection of Leishmaniinae infections in wild animals. Determining their infectiousness is of paramount importance in evaluating their epidemiological significance. One of the most efficient ways of determining infectiousness for vector borne diseases is xenodiagnosis with the appropriate vector. However, this is logistically very difficult to accomplish in the field, and an ideal solution is to find a molecular surrogate for xenodiagnosis. In this review we discuss different approaches to the problem by focusing on the infectiousness of Leishmania (Viannia) braziliensis in rodents under laboratory and field conditions. Comparisons with similar studies for other Leishmania species emphasizes that there are pivotal differences in the infectiousness and the importance of asymptomatic infections in different hosts. Potentially the most promising surrogate is the real time quantitative PCR (qPCR). However, its success depends on choosing a tissue that relates to the vector's feeding location and the parasite's tissue tropism. This requires detailed knowledge of the infection of each species in its wild hosts. We conclude that for L. (V.) braziliensis infections in wild rodents the tissue of choice for a molecular xenodiagnostic test, based on the qPCR is blood, providing that a significant number of samples must be examined.

Incidence of Human and Free-Ranging Wild Rodent Infections with Leishmania (Viannia) braziliensis, Aetiological Agent of Cutaneous Leishmaniasis.

BACKGROUND: Human and wild rodent infection rates with Leishmania (Viannia) braziliensis are needed to differentiate transmission pathways in anthropogenically altered habitats. METHODS: Human participants in northeast Brazil were tested by the leishmanin skin test (LST) and inspected for lesions/scars characteristic of American clinical leishmaniasis (ACL). Molecular (PCR/qPCR) test records of free-ranging rodents were available from a concurrent capture-mark-recapture study. Force of Infection (λ) and recovery (ρ) rates were estimated from cross-sectional and longitudinal datasets. RESULTS: Cumulative prevalences of human LST+ves and ACL scar+ves were 0.343-0.563 (n = 503 participants) and 0.122-0.475 (n = 503), respectively. Active ACL lesions were not detected. Annual rates of LST conversions were λ = 0.03-0.15 and ρ = 0.02-0.07. The probability of infection was independent of sex and associated with increasing age in addition to the period of exposure. Rodents (n = 596 individuals of 6 species) showed high rates of exclusively asymptomatic infection (λ = 0.222/month) and potential infectiousness to the sand fly vector. Spatially concurrent rodent and household human infection prevalences were correlated. CONCLUSIONS: Human exposure to L. (V.) braziliensis continues to be high despite the substantial drop in reported ACL cases in recent years. Spill-over transmission risk to humans from rodents in peridomestic habitats is likely supported by a rodent infection/transmission corridor linking houses, plantations, and the Atlantic Forest.

High levels of infectiousness of asymptomatic Leishmania (Viannia) braziliensis infections in wild rodents highlights their importance in the epidemiology of American Tegumentary Leishmaniasis in Brazil.

BACKGROUND: The epidemiological significance of wildlife infections with aetiological agents causing human infectious diseases is largely determined by their infection status, contact potential with humans (via vectors for vector-borne diseases), and their infectiousness to maintain onward transmission. This study quantified these parameters in wild and synanthropic naturally infected rodent populations in an endemic region of tegumentary leishmaniasis in northeast Brazil. METHODS: Capture-mark-recapture (CMR) of rodents was conducted over 27 months in domestic/peri domestic environs, household plantations and nearby Atlantic Forest (9,920 single trap nights). Rodent clinical samples (blood and ear tissue) were tested for infection by conventional PCR and quantitative PCR (qPCR) for Leishmania (Viannia) braziliensis, and xenodiagnosis to measure infectiousness to the local sand fly vector. RESULTS: A total 603 individuals of 8 rodent species were (re)captured on 1,051 occasions. The most abundant species were Nectomys squamipes (245 individuals, 41% of the total catch), Rattus rattus (148, 25%), and Necromys lasiurus (83, 14%). All species were captured in greater relative frequencies in plantations; R. rattus was the only species captured in all three habitats including in and around houses. Four species, comprising 22.6% of individuals captured at least twice, were geolocated in more than one habitat type; 78.6% were infected with L. (V.) braziliensis, facilitating inter-species and inter-habitat transmission. Species specific period prevalence ranged between 0%-62% being significantly higher in N. squamipes (54-62%) and Hollochillus sciureus (43-47%). Xenodiagnosis was performed on 41 occasions exposing 1,879 Nyssomyia whitmani sand flies to five rodent species (37 individuals). Similar mean levels of infectiousness amongst the more common rodent species were observed. Longitudinal xenodiagnosis of the N. squamipes population revealed a persistent level of infectiousness over 13 months follow-up, infecting a median 48% (IQR: 30.1%-64.2%) of exposed blood-fed vectors. The proportion of exposed flies infected was greater in the low compared to in the high seasonal period of vector abundance. L. (V.) braziliensis parasite loads in rodent blood quantified by qPCR were similar across rodent species but did not represent a reliable quantitative marker of infectiousness to sand flies. The standardised risk of rodent infection in plantations was 70.3% relative to 11.3% and 18.4% in peri domestic and forest habitats respectively. R. rattus was the only exception to this trend indicating greatest risk in the peri domestic environment. CONCLUSIONS: The results support the view that a collective assemblage of wild and synanthropic rodent species is an important wild reservoir of L. (V.) braziliensis in this region, with N. squamipes and R. rattus probably playing a key role in transmission within and between habitat types and rodent species. Rodents, and by implication humans, are at risk of infection in all sampled habitats, but more so in homestead plantations. These conclusions are based on one of the longest CMR study of small rodents in an American Tegumentary Leishmaniasis (ATL) foci.

Demographic patterns of human antibody levels to Simulium damnosum s.l. saliva in onchocerciasis-endemic areas: An indicator of exposure to vector bites.

BACKGROUND: In onchocerciasis endemic areas in Africa, heterogenous biting rates by blackfly vectors on humans are assumed to partially explain age- and sex-dependent infection patterns with Onchocerca volvulus. To underpin these assumptions and further improve predictions made by onchocerciasis transmission models, demographic patterns in antibody responses to salivary antigens of Simulium damnosum s.l. are evaluated as a measure of blackfly exposure. METHODOLOGY/PRINCIPAL FINDINGS: Recently developed IgG and IgM anti-saliva immunoassays for S. damnosum s.l. were applied to blood samples collected from residents in four onchocerciasis endemic villages in Ghana. Demographic patterns in antibody levels according to village, sex and age were explored by fitting generalized linear models. Antibody levels varied between villages but showed consistent patterns with age and sex. Both IgG and IgM responses declined with increasing age. IgG responses were generally lower in males than in females and exhibited a steeper decline in adult males than in adult females. No sex-specific difference was observed in IgM responses. CONCLUSIONS/SIGNIFICANCE: The decline in age-specific antibody patterns suggested development of immunotolerance or desensitization to blackfly saliva antigen in response to persistent exposure. The variation between sexes, and between adults and youngsters may reflect differences in behaviour influencing cumulative exposure. These measures of antibody acquisition and decay could be incorporated into onchocerciasis transmission models towards informing onchocerciasis control, elimination, and surveillance.

Modelling Sand Fly Lutzomyia longipalpis Attraction to Host Odour: Synthetic Sex-Aggregation Pheromone Dominates the Response.

Zoontic visceral leishmaniasis (ZVL) due to Leishmania infantum is a potentially fatal protozoan parasitic disease of humans and dogs. In the Americas, dogs are the reservoir and the sand fly, Lutzomyia longipalpis, the principal vector. A synthetic version of the male sand fly produced sex-aggregation pheromone attracts both female and male conspecifics to co-located insecticide, reducing both reservoir infection and vector abundance. However the effect of the synthetic pheromone on the vector's "choice" of host (human, animal reservoir, or dead-end host) for blood feeding in the presence of the pheromone is less well understood. In this study, we developed a modelling framework to allow us to predict the relative attractiveness of the synthetic pheromone and potential alterations in host choice. Our analysis indicates that the synthetic pheromone can attract 53% (95% CIs: 39%-86%) of host-seeking female Lu. longipalpis and thus it out-competes competing host odours. Importantly, the results suggest that the synthetic pheromone can lure vectors away from humans and dogs, such that when co-located with insecticide, it provides protection against transmission leading to human and canine ZVL.

Indoor residual spraying practices against Triatoma infestans in the Bolivian Chaco: contributing factors to suboptimal insecticide delivery to treated households.

BACKGROUND: Indoor residual spraying (IRS) of insecticides is a key method to reduce vector transmission of Trypanosoma cruzi, causing Chagas disease in a large part of South America. However, the successes of IRS in the Gran Chaco region straddling Bolivia, Argentina, and Paraguay, have not equalled those in other Southern Cone countries. AIMS: This study evaluated routine IRS practices and insecticide quality control in a typical endemic community in the Bolivian Chaco. METHODS: Alpha-cypermethrin active ingredient (a.i.) captured onto filter papers fitted to sprayed wall surfaces, and in prepared spray tank solutions, were measured using an adapted Insecticide Quantification Kit (IQK™) validated against HPLC quantification methods. The data were analysed by mixed-effects negative binomial regression models to examine the delivered insecticide a.i. concentrations on filter papers in relation to the sprayed wall heights, spray coverage rates (surface area / spray time [m2/min]), and observed/expected spray rate ratios. Variations between health workers and householders' compliance to empty houses for IRS delivery were also evaluated. Sedimentation rates of alpha-cypermethrin a.i. post-mixing of prepared spray tanks were quantified in the laboratory. RESULTS: Substantial variations were observed in the alpha-cypermethrin a.i. concentrations delivered; only 10.4% (50/480) of filter papers and 8.8% (5/57) of houses received the target concentration of 50 mg ± 20% a.i./m2. The delivered concentrations were not related to those in the matched spray tank solutions. The sedimentation of alpha-cypermethrin a.i. in the surface solution of prepared spray tanks was rapid post-mixing, resulting in a linear 3.3% loss of a.i. content per minute and 49% loss after 15 min. Only 7.5% (6/80) of houses were sprayed at the WHO recommended rate of 19 m2/min (± 10%), whereas 77.5% (62/80) were sprayed at a lower than expected rate. The median a.i. concentration delivered to houses was not significantly associated with the observed spray coverage rate. Householder compliance did not significantly influence either the spray coverage rates or the median alpha-cypermethrin a.i. concentrations delivered to houses. CONCLUSIONS: Suboptimal delivery of IRS is partially attributable to the insecticide physical characteristics and the need for revision of insecticide delivery methods, which includes training of IRS teams and community education to encourage compliance. The IQK™ is a necessary field-friendly tool to improve IRS quality and to facilitate health worker training and decision-making by Chagas disease vector control managers.

Community deployment of a synthetic pheromone of the sand fly Lutzomyia longipalpis co-located with insecticide reduces vector abundance in treated and neighbouring untreated houses: Implications for control of Leishmania infantum.

BACKGROUND: The rising incidence of visceral leishmaniasis due to Leishmania infantum requires novel methods to control transmission by the sand fly vector. Indoor residual spraying of insecticide (IRS) against these largely exophilic / exophagic vectors may not be the most effective method. A synthetic copy of the male sex-aggregation pheromone of the key vector species Lutzomyia longipalpis in the Americas, was co-located with residual pyrethroid insecticide, and tested for its effects on vector abundance, hence potential transmission, in a Brazilian community study. METHODS: Houses within eight defined semi-urban blocks in an endemic municipality in Brazil were randomised to synthetic pheromone + insecticide or to placebo treatments. A similar number of houses located >100m from each block were placebo treated and considered as "True Controls" (thus, analysed as three trial arms). Insecticide was sprayed on a 2.6m2 surface area of the property boundary or outbuilding wall, co-located within one metre of 50mg synthetic pheromone in controlled-release dispensers. Vector numbers captured in nearby CDC light traps were recorded at monthly intervals over 3 months post intervention. Recruited sentinel houses under True Control and pheromone + insecticide treatments were similarly monitored at 7-9 day intervals. The intervention effects were estimated by mixed effects negative binomial models compared to the True Control group. RESULTS: Dose-response field assays using 50mg of the synthetic pheromone captured a mean 4.8 (95% C.L.: 3.91, 5.80) to 6.3 (95% C.L.: 3.24, 12.11) times more vectors (female Lu. longipalpis) than using 10mg of synthetic pheromone. The intervention reduced household female vector abundance by 59% (C.L.: 48.7, 66.7%) (IRR = 0.41) estimated by the cross-sectional community study, and by 70% (C.L.: 56.7%, 78.8%) estimated by the longitudinal sentinel study. Similar reductions in male Lu. longipalpis were observed. Beneficial spill-over intervention effects were also observed at nearby untreated households with a mean reduction of 24% (95% C.L.: 0.050%, 39.8%) in female vectors. The spill-over effect in untreated houses was 44% (95% C.L.: 29.7%, 56.1%) as effective as the intervention in pheromone-treated houses. Ownership of chickens increased the intervention effects in both treated and untreated houses, attributed to the suspected synergistic attraction of the synthetic pheromone and chicken kairomones. The variation in IRR between study blocks was not associated with inter-household distances, household densities, or coverage (proportion of total households treated). CONCLUSIONS: The study confirms the entomological efficacy of the lure-and-kill method to reduce the abundance of this important sand fly vector in treated and untreated homesteads. The outcomes were achieved by low coverage and using only 1-2% of the quantity of insecticide as normally required for IRS, indicating the potential cost-effectiveness of this method. Implications for programmatic deployment of this vector control method are discussed.

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas.

BACKGROUND: Simulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC-MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC-MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase. CONCLUSIONS/SIGNIFICANCE: This study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.

Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for Diagnosis of canine Leishmania infection.

There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P

Synthetic sex-aggregation pheromone of Lutzomyia longipalpis, the South American sand fly vector of Leishmania infantum, attracts males and females over long-distance.

BACKGROUND: In South America the sand fly Lutzomyia longipalpis is the predominant vector of Leishmania infantum, the parasite that causes canine and human visceral leishmaniasis. Co-location of synthetic male sex-aggregation pheromone with an insecticide provided protection against canine seroconversion, parasite infection, reduced tissue parasite loads, and female sand fly densities at households. Optimising the sex-aggregation pheromone + insecticide intervention requires information on the distance over which female and male Lu. longipalpis would be attracted to the synthetic pheromone in the field. METHODOLOGY/PRINCIPAL FINDINGS: Wild Lu. longipalpis were collected at two peridomestic study sites in Governador Valadares (Minas Gerais, Brazil). Sand flies were marked with coloured fluorescent powder using an improved protocol and then released into an existing domestic chicken shed at two independent sites, followed by recapture at synthetic-pheromone host-odour baited traps placed up to 30 metres distant from the release point. In total 1704 wild-caught Lu. longipalpis were released into the two chicken sheds. Overall 4.3% of the marked flies were recaptured in the pheromone baited experimental chicken sheds compared to no marked flies recaptured in the control sheds. At the first site, 14 specimens (10.4% of the marked and released specimens) were recaptured at 10m, 36 (14.8%) at 20m, and 15 (3.4%) at 30m. At the second site, lower recapture rates were recorded; 8 marked specimens (1.3%) were recaptured at 5 and 10m and no marked specimens were recaptured at 15m. Approximately 7x more marked males than females were recaptured although males were only 2x as common as females in the released population. 52% of the marked Lu. longipalpis were collected during the first night of sampling, 32% on the second night, and 16% on the third night. CONCLUSIONS/SIGNIFICANCE: The study established that both male and female sand flies can be attracted to the synthetic sex-aggregation pheromone in the presence of host odour over distances up to at least 30m in the field depending on local environmental and meterological conditions.

Significant reduction in abundance of peridomestic mosquitoes (Culicidae) and Culicoides midges (Ceratopogonidae) after chemical intervention in western São Paulo, Brazil.

BACKGROUND: We assessed the impact of two sand fly insecticide interventions (insecticide spraying and insecticide-impregnated dog collars) on the peridomestic abundance and distribution of mosquitoes (Culicidae) and biting midges (Ceratopogonidae) in western São Paulo (Brazil) in a long-term (42-month) evaluation. Both of these dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil. METHODS: The interventions in the 3-arm stratified randomised control trial were: pheromone + insecticide (PI) (chicken roosts were sprayed with microencapsulated lambda-cyhalothrin; pheromone lure has no effect on the Diptera pests studied here); dog-collars (DC) (dogs fitted with deltamethrin-impregnated collars); and control (C) (unexposed to pyrethroids) were extended by 12 months. During that time, adult mosquitoes and midges were sampled along 280 households at three household locations (inside human dwellings, dog sleeping sites and chicken roosts). RESULTS: We collected 3145 culicids (9 genera, 87.6% Culex spp.) distributed relatively uniformly across all 3 arms: 41.9% at chicken roosts; 37.7% inside houses; and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.9%) or houses (2.4%). Mosquitoes and Culicoides were most abundant during the hot and rainy season. Increased daytime temperature was marginally associated with increased mosquito abundance (Z = 1.97, P = 0.049) and Culicoides abundance (Z = 1.71, P = 0.087). There was no significant association with daily average rainfall for either group. Household-level mosquito and midge numbers were both significantly reduced by the PI intervention 56% [incidence rate ratio, IRR = 0.54 (95% CI: 0.30-0.97), P ≤ 0.05] and 53% [IRR = 0.47 (95% CI: 0.26-0.85), P ≤ 0.05], respectively, compared to the control intervention. The abundance of both dipteran groups at dog sleeping sites was largely unaffected by the PI and DC interventions. The PI intervention significantly reduced abundance of mosquitoes inside houses (41%) and at chicken roosting sites (48%) and reduced midge abundance by 51% in chicken roosting sites. CONCLUSIONS: Sprayed insecticide at chicken roosting sites reduced the abundance of mosquitoes and midges at the peridomestic level while dog collars had no effect on numbers for any group.

The importance of vector control for the control and elimination of vector-borne diseases.

Vector-borne diseases (VBDs) such as malaria, dengue, and leishmaniasis exert a huge burden of morbidity and mortality worldwide, particularly affecting the poorest of the poor. The principal method by which these diseases are controlled is through vector control, which has a long and distinguished history. Vector control, to a greater extent than drugs or vaccines, has been responsible for shrinking the map of many VBDs. Here, we describe the history of vector control programmes worldwide from the late 1800s to date. Pre 1940, vector control relied on a thorough understanding of vector ecology and epidemiology, and implementation of environmental management tailored to the ecology and behaviour of local vector species. This complex understanding was replaced by a simplified dependency on a handful of insecticide-based tools, particularly for malaria control, without an adequate understanding of entomology and epidemiology and without proper monitoring and evaluation. With the rising threat from insecticide-resistant vectors, global environmental change, and the need to incorporate more vector control interventions to eliminate these diseases, we advocate for continued investment in evidence-based vector control. There is a need to return to vector control approaches based on a thorough knowledge of the determinants of pathogen transmission, which utilise a range of insecticide and non-insecticide-based approaches in a locally tailored manner for more effective and sustainable vector control.

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.

Vaccination against canine leishmaniasis in Brazil.

Prevention of canine Leishmania infantum infection is critical to management of visceral leishmaniasis in people living in endemic areas of Brazil. A bill (PL 1738/11), currently under consideration, proposes to establish a national vaccination policy against canine leishmaniasis in Brazil. However, there is no solid scientific evidence supporting the idea that this could reduce transmission from infected vaccinated dogs to sand flies to a level that would significantly reduce the risk of L. infantum infection or visceral leishmaniasis in humans. Thus, we advocate that insecticide-impregnated collars should the first line protective measure for public health purposes and that vaccines are applied on a case-by-case, optional basis for individual dog protection.

Increasing incidence of zoonotic visceral leishmaniasis on Crete, Greece.

To determine whether the incidence of canine leishmaniasis has increased on Crete, Greece, we fitted infection models to serodiagnostic records of 8,848 dog samples for 1990-2006. Models predicted that seroprevalence has increased 2.4% (95% confidence interval 1.61%-3.51%) per year and that incidence has increased 2.2- to 3.8-fold over this 17-year period.

Selection of appropriate serological tests to measure the incidence of natural Leishmania infantum infection during DNA/MVA prime/boost canine vaccine trials.

In response to the increasing need for field trials of experimental DNA vaccines against zoonotic visceral leishmaniasis in dogs, our aim was to validate the use of ELISA protocols which will be suitable for detection of natural infection in vaccinated dogs. We have previously demonstrated that DNA/modified vaccinia virus Ankara (MVA) vaccine expressing tryparedoxin peroxidase (TRYP) induced high titres of TRYP antigen-specific IgG in immunized dogs. Here we report our findings that seroconversion to an unrelated diagnostic antigen rK39 did not occur in vaccinated dogs, and that responses to crude Leishmania infantum promastigote antigen (CLA) were weak and short-lived. This is in contrast to strong responses to both antigens shown in naturally infected dogs. To select an appropriate serological test for measurement of infection incidence, we also tested longitudinal samples from an immunologically well-characterized cohort of naturally infected dogs. The sensitivity of CLA ELISA was superior to that of rK39 in early stage infection (from 2 months before, to 2 months after the first detection of infection by PCR or parasitological culture), and more sensitive than rK39 in cross-sectional sampling (81.0% vs 61.9%). We conclude that CLA ELISA will provide sensitive estimates of L. infantum infection incidence in DNA/MVA vaccinated dogs, though optimal testing would include rK39, or a similar recombinant antigen, to improve overall specificity.