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Ed W Cupp1, Charles D Mackenzie2, Thomas R Unnasch31Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA; 2Department of Pathobiology, Michigan State University, East Lansing, MI, USA; 3Department of Global Health, University of South Florida, Tampa, FL, USAAbstract: Ivermectin (registered for human use as Mectizan®) was donated by Merck & Co Inc in 1987 for the treatment and control of human onchocerciasis ("river blindness"). This philanthropic gesture has had a remarkable effect in reducing the incidence and prevalence of this serious ocular and dermatological disease, while changing health system support for millions of people worldwide. Over 800 million doses have been given to more than 80 million people for onchocerciasis during the past 23 years. As a result, onchocerciasis has been significantly reduced in more than 25 countries, transmission has been interrupted in foci in at least 10 countries, and the disease is no longer seen in children in many formerly endemic foci. Recent communications have suggested that the drug's efficacy as the major therapeutic agent for these control and elimination programs may be threatened, but alternative interpretations for suboptimal response/resistance suggest otherwise. Current research needs and control methods by which the public health community in endemic countries may respond to resistance, should it occur in their area, are discussed, along with the continuing importance of this anthelmintic as the mainstay in onchocerciasis control programs.Keywords: Ivermectin, Onchocerca volvulus, river blindness, resistance, African Programme for Onchocerciasis Control, Onchocerciasis Elimination Program for the Americas

Onchocerciasis, caused by infection with Onchocerca volvulus, has been targeted for elimination by 2030. Currently, onchocerciasis elimination programs rely primarily on mass distribution of ivermectin. However, ivermectin alone may not be sufficient to achieve elimination in some circumstances, and additional tools may be needed. Vector control has been used as a tool to control onchocerciasis, but vector control using insecticides is expensive and ecologically detrimental. Community-directed removal of the trailing vegetation black fly larval attachment sites (slash and clear) has been shown to dramatically reduce vector biting densities. Here, we report studies to optimize the slash and clear process. Conducting slash and clear interventions at Simulium damnosum sensu stricto breeding sites located within 2 km of afflicted communities resulted in a 95% reduction in vector biting. Extending slash and clear further than 2 km resulted in no further decrease. A single intervention conducted at the first half of the rainy season resulted in a 97% reduction in biting rate, whereas an intervention conducted at the end of the rainy season resulted in a 94% reduction. Vector numbers in any of the intervention villages did not fully recover by the start of the following rainy season. These results suggest that slash and clear may offer an inexpensive and effective way to augment ivermectin distribution in the effort to eliminate onchocerciasis in Africa.

Human landing collections (HLCs) have been the standard method for the collection of black flies that serve as vectors for Onchocerca volvulus , the causative agent of onchocerciasis or river blindness. However, HLCs are inefficient and may expose collectors to vector-borne pathogens. The Esperanza window trap (EWT) has been shown to be a potential alternative to HLCs for the collection of Simulium damnosum , the principal vector of O. volvulus in Africa. To improve the performance of the EWT, sweat from individuals highly attractive or less attractive to S. damnosum sensu stricto was examined by gas chromatography and mass spectroscopy. Twelve compounds were identified which were solely present or present in increased amounts in the sweat of the highly attractive individuals. Two of these compounds (naphthalene and tert-hexadecyl mercaptan) were found to be attractive to S. damnosum s.s. in behavioral assays. Traps baited with these compounds outperformed those baited with the current standard bait of worn socks. Using these newly identified compounds as baits will make the EWT more efficient in collecting vector black flies and may enhance the potential utility of the EWT as a local vector control measure.

Attention is increasingly focusing on how best to accelerate progress toward meeting the WHO's 2030 goals for neglected tropical diseases (NTDs). For river blindness, a major NTD targeted for elimination, there is a long history of using vector control to suppress transmission, but traditional larvicide-based approaches are limited in their utility. One innovative and sustainable approach, "slash and clear", involves clearing vegetation from breeding areas, and recent field trials indicate that this technique very effectively reduces the biting density of Simulium damnosum s.s. In this study, we use a Bayesian data-driven mathematical modeling approach to investigate the potential impact of this intervention on human onchocerciasis infection. We developed a novel "slash and clear" model describing the effect of the intervention on seasonal black fly biting rates and coupled this with our population dynamics model of Onchocerca volvulus transmission. Our results indicate that supplementing annual drug treatments with "slash and clear" can significantly accelerate the achievement of onchocerciasis elimination. The efficacy of the intervention is not very sensitive to the timing of implementation, and the impact is meaningful even if vegetation is cleared only once per year. As such, this community-driven technique will represent an important option for achieving and sustaining O. volvulus elimination.

Background: Onchocerciasis, or river blindness, has historically been an important cause of blindness, skin disease and economic disruption in Africa and the Americas. It is caused by the filarial parasite Onchocerca volvulus, which is transmitted by black flies in the genus Simulium. Over the past decade, several international programs have been formed to control, or more recently eliminate onchocerciasis, using mass drug administration (MDA) of ivermectin. However, in many areas of Africa (particularly those which are endemic for the eyeworm, Loa loa, or where vector densities are very high) ivermectin MDA alone will not be sufficient to achieve elimination. In these situations, additional interventions may be necessary., Methodology/principal Findings: The Esperanza Window trap (EWT), a simple trap originally developed to replace human landing collections for entomological surveillance of O. volvulus transmission was optimized, resulting in a 17-fold improvement in trap performance. The optimized trap was tested in trials in schools and in agricultural fields to determine if it could reduce vector biting locally. The traps resulted in a 90% reduction in biting in the school setting. In the field setting, results varied. In one location, the traps reduced biting by roughly 50%, while in a separate trial, the traps did not significantly reduce the biting rate. Examination of the two settings suggested that trap placement may be critical to their success., Conclusions/significance: These results suggest that the optimized EWT might be capable of reducing local vector black fly biting in areas commonly frequented by residents. Together with other recently developed methods of community directed vector control, the traps may augment ivermectin MDA, bringing the goal of onchocerciasis elimination within reach in much of Africa., Competing Interests: The authors have declared that no competing interests exist.

Background: Onchocerciasis a neglected tropical disease that historically has been a major cause of morbidity and an obstacle to economic development in the developing world. It is caused by infection with Onchocerca volvulus, which is transmitted by black flies of the genus Simulium. The discovery of the potent effect of Mectizan (ivermectin) on O. volvulus microfilariae and the decision by its manufacturer to donate the drug for onchocerciasis spurred the implementation of international programs to control and, more recently, eliminate this scourge. These programs rely primarily on mass distribution of ivermectin (MDA) to the afflicted populations. However, MDA alone will not be sufficient to eliminate onchocerciasis where transmission is intense and where ivermectin MDA is precluded by co-endemicity with Loa loa. Vector control will likely be required as a supplemental intervention in these situations., Methodology/principal Findings: Because biting by the black fly vectors is often a major nuisance in onchocerciasis afflicted communities, we hypothesized that community members might be mobilized to clear the breeding sites of the vegetation that represents the primary black fly larvae attachment point. We evaluated the effect of such a community based "slash and clear" intervention in multiple communities in Northern Uganda. Slash and Clear resulted in 89-99% declines in vector biting rates. The effect lasted up to 120 days post intervention., Conclusions/significance: Slash and clear might represent an effective, inexpensive, community- based tool to supplement ivermectin distribution as a contributory method to eliminate onchocerciasis and prevent recrudescence., Competing Interests: The authors have declared that no competing interests exist.

Background: The Esperanza Window Trap (EWT) baited with CO2 and human sweat compounds is attractive to Simulium ochraceum s.l., the primary vector of Onchocerca volvulus in the historically largest endemic foci in México and Guatemala., Methodology/principal Findings: The ability of the EWT to locally reduce numbers of questing S. ochraceum s.l. was evaluated in two formerly onchocerciasis endemic communities in Southern México. At each community, two EWTs were placed in or near a school or household and flies were collected sequentially for a total of 10 days. Black fly collections were then carried out for an additional 10 days in the absence of the EWTs. Flies were also collected outside the dwellings to control for variations in the local fly populations. When the EWTs were present, there was a significant reduction in the human biting rate at both the household and school locations at collection sites, with a greater effect observed in the schools., Conclusions/significance: These results indicate that the EWTs not only have potential as a black fly monitoring tool but may be used for reducing personal exposure to fly bites in Mesoamerica.

Many blackfly species (Diptera: Simuliidae) are economically important insect pests, both as nuisance biters and as vectors of pathogens of medical and veterinary relevance. Among the important blackfly pest species in North America is Simulium vittatum Zetterstedt sensu lato. The objective of this study was to identify compounds excreted by mammalian hosts that are attractive to host-seeking S. vittatum females. The attractiveness of putative compounds to colonized S. vittatum was tested through electrophysiological (electroantennography; n = 58 compounds) and behavioural (Y-tube assays; n = 7 compounds in three concentrations) bioassays. Five compounds were significantly attractive to host-seeking S. vittatum females: 1-octen-3-ol; 2-heptanone; acetophenone; 1-octanol, and naphthalene. These candidate compounds might be useful as attractants in traps that could be developed for use in alternative or complementary management tactics in programmes to suppress nuisance blackfly populations, or for the collection of samples in which to study the transmission ecology of pathogens transmitted by blackflies of the S. vittatum complex., (© 2016 The Royal Entomological Society.)

Background: Mexico is one of the six countries formerly endemic for onchocerciasis in Latin America. Transmission has been interrupted in the three endemic foci of that country and mass drug distribution has ceased. Three years after mass drug distribution ended, post-treatment surveillance (PTS) surveys were undertaken which employed entomological indicators to check for transmission recrudescence., Methodology/principal Findings: In-depth entomologic assessments were performed in 18 communities in the three endemic foci of Mexico. None of the 108,212 Simulium ochraceum s.l. collected from the three foci were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in a maximum upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.035/2,000 flies examined. This is an order of magnitude below the threshold of a 95%-ULCI of less than one infective fly per 2,000 flies tested, the current entomological criterion for interruption of transmission developed by the international community. The point estimate of seasonal transmission potential (STP) was zero, and the upper bound of the 95% confidence interval for the STP ranged from 1.2 to 1.7 L3/person/season in the different foci. This value is below all previous estimates for the minimum transmission potential required to maintain the parasite population., Conclusions/significance: The results from the in-depth entomological post treatment surveillance surveys strongly suggest that transmission has not resumed in the three foci of Mexico during the three years since the last distribution of ivermectin occurred; it was concluded that transmission remains undetectable without intervention, and Onchocerca volvulus has been eliminated from Mexico.

The suite of pheromones that promote communal oviposition by Simulium vittatum, a North American black fly species, was identified and characterized using gas chromatography-mass spectrometry, electrophysiological, and behavioral bioassays. Behavioral assays demonstrated that communal oviposition was induced by egg-derived compounds that were active at short range and whose effect was enhanced through direct contact. Three compounds (cis-9-tetradecen-1-ol, 1-pentadecene, and 1-tridecene) were identified in a non-polar solvent extract of freshly deposited S. vittatum eggs that were capable of inducing the oviposition response. Electroantennography demonstrated that two of these three compounds (1-pentadecene and 1-tridecene) actively stimulated antennal neurons. Identification of the oviposition pheromones of this family may be helpful in developing control measures for nuisance black flies and for medically-important species such as Simulium damnosum sensu lato.

Background: Entomological indicators are considered key metrics to document the interruption of transmission of Onchocerca volvulus, the etiological agent of human onchocerciasis. Human landing collection is the standard employed for collection of the vectors for this parasite. Recent studies reported the development of traps that have the potential for replacing humans for surveillance of O. volvulus in the vector population. However, the key chemical components of human odor that are attractive to vector black flies have not been identified., Methodology/principal Findings: Human sweat compounds were analyzed using GC-MS analysis and compounds common to three individuals identified. These common compounds, with others previously identified as attractive to other hematophagous arthropods were evaluated for their ability to stimulate and attract the major onchocerciasis vectors in Africa (Simulium damnosum sensu lato) and Latin America (Simulium ochraceum s. l.) using electroantennography and a Y tube binary choice assay. Medium chain length carboxylic acids and aldehydes were neurostimulatory for S. damnosum s.l. while S. ochraceum s.l. was stimulated by short chain aliphatic alcohols and aldehydes. Both species were attracted to ammonium bicarbonate and acetophenone. The compounds were shown to be attractive to the relevant vector species in field studies, when incorporated into a formulation that permitted a continuous release of the compound over time and used in concert with previously developed trap platforms., Conclusions/significance: The identification of compounds attractive to the major vectors of O. volvulus will permit the development of optimized traps. Such traps may replace the use of human vector collectors for monitoring the effectiveness of onchocerciasis elimination programs and could find use as a contributing component in an integrated vector control/drug program aimed at eliminating river blindness in Africa.

Background: Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO2 had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated., Methodology/principal Findings: Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission., Conclusions/significance: These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination.

A simple inexpensive trap (Esperanza window trap) was shown recently to collect significant numbers of Simulium ochraceum sensu lato, a major vector of Onchocerca volvulus in Mesoamerica. Here, we report studies optimizing this trap for the collection of Simulium damnosum s.l., the major vector of O. volvulus in Africa. A shortened, blue and black striped version of the Esperanza window trap, when baited with a combination of CO2 and worn trousers, rivalled human landing collections in the number of S. damnosum s.l. females collected. Traps baited with a commercially available human skin lure and CO2 resulted in collections that were not significantly different than those obtained from traps baited with worn trousers and CO2. This suggests that the Esperanza window trap may offer a replacement for human landing collections for monitoring onchocerciasis transmission in Africa., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Human landing collections are currently the standard method for collecting onchocerciasis vectors in Africa and Latin America. As part of the efforts to develop a trap to replace human landing collections for the monitoring and surveillance of onchocerciasis transmission, comprehensive evaluations of several trap types were conducted to assess their ability to collect Simulium ochraceum sensu lato, one of the principal vectors of Onchocerca volvulus in Latin America., Methodology/principal Findings: Diverse trap designs with numerous modifications and bait variations were evaluated for their abilities to collect S. Ochraceum s.l. females. These traps targeted mostly host seeking flies. A novel trap dubbed the "Esperanza window trap" showed particular promise over other designs. When baited with CO2 and BG-lure (a synthetic blend of human odor components) a pair of Esperanza window traps collected numbers of S. Ochraceum s.l. females similar to those collected by a team of vector collectors., Conclusions/significance: The Esperanza window trap, when baited with chemical lures and CO2 can be used to collect epidemiologically significant numbers of Simulium ochraceum s.l., potentially serving as a replacement for human landing collections for evaluation of the transmission of O. volvulus.

In temperate regions, seasonal epidemics of many mosquito-borne viruses are triggered when mosquito populations shift from feeding on avian to mammalian hosts. We investigated effects of temperature on the timing of bird-to-mammal shifts using an 8 year dataset of blood-meals from a mosquito (Culex erraticus) in Alabama, USA. As expected, Cx. erraticus shifted from avian to mammalian hosts each year. The timing of the shift, however, varied considerably among years. Harshness of the preceding winter (chill accumulation) explained 93 per cent of the variation in the timing of bird-to-mammal shifts, with shifts occurring later in years following harsher winters. We hypothesize that winter temperatures drive the timing of bird-to-mammal shifts through effects on host reproductive phenology. Because mosquitoes target birds during the nesting season, and bird nesting occurs later in years following colder winters, later nesting dates result in a concomitant delay in the timing of bird-to-mammal host shifts. Global increases in winter temperatures could cause significant changes in the timing of seasonal host shifts by mosquitoes, with prolonged periods of epidemic transmission of mosquito-borne diseases.

We review and analyze approaches over a 65 year period that have proven successful for onchocerciasis control in several different epidemiological settings. These include vector control with the goal of transmission interruption versus the use of mass drug administration using ivermectin (Mectizan(®)) monotherapy. Ivermectin has proven exceedingly effective because it is highly efficacious against Onchocerca volvulus microfilariae, the etiological agent of onchocercal skin and ocular disease and the infective stage for the vector. For these reasons, the drug was donated by the Merck Company for regional control programs in Africa and the Americas. Recurrent treatment with ivermectin at semi-annual intervals also impacts adult worms and result in loss of fecundity and increased mortality. Using a strategy of 6-monthly treatments with high coverage rates, the Onchocerciasis Elimination Program for the Americas has interrupted transmission in seven of the thirteen foci in the Americas and is on track to eliminate onchocerciasis in the region by 2015. Treatments given annually or semi-annually for 15-17 years in three hyperendemic onchocerciasis foci in Mali and Senegal also have resulted in a few infections in the human population with transmission levels below thresholds postulated for elimination. Follow-up evaluations did not detect any recrudescence of infection or transmission, suggesting that onchocerciasis elimination could be feasible with Mectizan(®) treatment in some endemic foci in Africa., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Thrombostasin (TS) is an anticlotting protein found in saliva of Haematobia irritans (horn flies). The polymorphic nature of the ts gene was first associated with success of horn flies blood feeding on a laboratory host, New Zealand White rabbits. In this study, we report results of similar studies testing blood uptake of horn flies feeding on a natural host, cattle. These studies confirmed the association of ts genotype with blood uptake of horn flies and showed that it was host species specific. In contrast to rabbits, blood uptake volumes of homozygous ts10 horn flies were lower than those of other ts genotypes when fed on control (ovalbumin-vaccinated) cattle. Cattle vaccinated with recombinant protein isoforms, rTS9 or rTB8, resisted horn fly feeding by yielding lower blood volumes compared with flies feeding on control cattle. The specific impact of vaccination, however, varied by ts genotype of flies. Cattle vaccinated with isoform rTS9 resisted flies of ts2, ts9, and tb8 genotype. Vaccination with isoform rTB8 produced resistance to ts8, ts9, and tb8 genotype flies. Horn flies of genotype ts10 were not affected by vaccination with either TS isoform and fed as well on rTS9- and rTB8-vaccinated as on control-vaccinated cattle. These experimental results confirm the efficacy of vaccines targeting horn fly salivary proteins and provide new insight into the dynamics of horn fly-cattle interactions in nature.

Background: A site near Tuskegee, Alabama was examined for vector-host activities of eastern equine encephalomyelitis virus (EEEV). Land cover maps of the study site were created in ArcInfo 9.2 from QuickBird data encompassing visible and near-infrared (NIR) band information (0.45 to 0.72 microm) acquired July 15, 2008. Georeferenced mosquito and bird sampling sites, and their associated land cover attributes from the study site, were overlaid onto the satellite data. SAS 9.1.4 was used to explore univariate statistics and to generate regression models using the field and remote-sampled mosquito and bird data. Regression models indicated that Culex erracticus and Northern Cardinals were the most abundant mosquito and bird species, respectively. Spatial linear prediction models were then generated in Geostatistical Analyst Extension of ArcGIS 9.2. Additionally, a model of the study site was generated, based on a Digital Elevation Model (DEM), using ArcScene extension of ArcGIS 9.2., Results: For total mosquito count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 5.041 km, nugget of 6.325 km, lag size of 7.076 km, and range of 31.43 km, using 12 lags. For total adult Cx. erracticus count, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 5.764 km, nugget of 6.114 km, lag size of 7.472 km, and range of 32.62 km, using 12 lags. For the total bird count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 4.998 km, nugget of 5.413 km, lag size of 7.549 km and range of 35.27 km, using 12 lags. For the Northern Cardinal count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 6.387 km, nugget of 5.935 km, lag size of 8.549 km and a range of 41.38 km, using 12 lags. Results of the DEM analyses indicated a statistically significant inverse linear relationship between total sampled mosquito data and elevation (R2 = -.4262; p < .0001), with a standard deviation (SD) of 10.46, and total sampled bird data and elevation (R2 = -.5111; p < .0001), with a SD of 22.97. DEM statistics also indicated a significant inverse linear relationship between total sampled Cx. erracticus data and elevation (R2 = -.4711; p < .0001), with a SD of 11.16, and the total sampled Northern Cardinal data and elevation (R2 = -.5831; p < .0001), SD of 11.42., Conclusion: These data demonstrate that GIS/remote sensing models and spatial statistics can capture space-varying functional relationships between field-sampled mosquito and bird parameters for determining risk for EEEV transmission.

Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report, the effect of TS isoforms on blood feeding was assessed using individual flies that carried corresponding ts allelles. Laboratory studies of horn fly blood feeding were conducted using colony-reared flies fed on New Zealand White (NZW) rabbits. After timed 20-min feeding periods, each fly was characterized for gender, blood volume uptake, and ts genotype. The results showed that mean blood volumes obtained by individual flies were not related to fly gender but were correlated to the ts genotype(s) carried by each fly. A fly having one or both ts alleles coding for the TS9 isoform took less blood than those possessing one or both ts alleles coding for the TS10 isoform. These results confirm the significant role that TS plays in horn fly feeding and highlight the differential impact of TS protein isoforms that vary by as few as three amino acids.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was first detected in the Tennessee Valley and in Alabama in August 2001. In summer 2002, intensive viral activity was seen, but in subsequent years, viral activity settled into an enzootic pattern. Here, we report an analysis of viral activity in the mosquito fauna in the Mid-South from 2002 (the first year viral activity was detected in mosquitoes) through 2005. Eight mosquito species were infected with WNV during 2002. However, viral activity was only detected in four species--Culex salinarius Coquillett, Culex erraticus Dyar & Knab, Coquillettidia perturbans Walker, and Aedes vexans Meigen--in multiple years. The greatest number of positive pools was in Cx. erraticus and Cx. salinarius. Despite being specifically targeted for collection, Aedes albopictus Skuse was only found to be infected during the epiornitic year (2002), suggesting that under enzootic transmission conditions its role as a bridge vector in the region may not be significant. Virus-positive pools of Cx. erraticus were identified from winter-resting and early season dry ice-baited trap collections in 2005, implicating this species in WNV overwintering in Alabama. Molecular analysis of individuals initially identified as members of the Culex pipiens L. complex suggested that alleles characteristic of Cx. pipiens predominated in mosquitoes collected in Huntsville, AL, whereas alleles in the Auburn, AL, population were predominately characteristic of Culex quinquefaciatus Say. The southern boundary of the overlap zone of the two species seems to be located primarily between Huntsville and Auburn, a distance of 350 km.

Eastern equine encephalitis virus (EEEV) is one of several arthropod-borne viruses (arboviruses) endemic to the United States. Interactions between arthropod (mosquito) vectors and avian amplification host populations play a significant role in the dynamics of arboviral transmission. Recent data have suggested the hypothesis that an increased rate of successful feeding on young-of-the-year (YOY) birds might play a role in the dynamics of EEEV transmission. To test this hypothesis, we developed a model to explore the effect of the interactions of the vectors and avian host populations on EEEV transmission. Sensitivity analyses conducted using this model revealed eleven parameters that were capable of disproportionately affecting the predicted level of EEEV infection in the vertebrate reservoir and vector populations. Of these, four parameters were related to the interaction of the vector with young-of-the-year birds. Furthermore, adult birds could not substitute for young-of-the-year in initiating and maintaining a predicted enzootic outbreak of EEEV. Taken together, the model predicted that young-of-the-year birds play a key role in establishing and maintaining enzootic outbreaks of EEEV.

A reanalysis of several published reports indicates that when community-wide biannual treatment disrupts transmission of new infection, a profound macrofilaricidal effect of ivermectin (Mectizan) occurs that is accelerated for individuals treated 4 times a year (4x/yr). The effect is particularly obvious on adult male worms and suggests that this gender is susceptible to repetitive treatment after transmission has been blocked or greatly reduced as a result of community treatment.

The chromosomal locations of two genes encoding the salivary protein products Simulidin (SVAT) and Simulium vittatum erythyma protein (SVEP) were identified using high-resolution cytogenetic mapping. Chromosomal subsection levels were determined from larval and adult salivary gland polytene chromosomes and adult Malphigian tubule chromosomes. Syntenic relationships occurred for both loci from a wild population of Simulium vittatum IIIL-1 collected in central Alabama and the colonized IS-7 S. vittatum sibling. cSVAT mapped to the short arm of chromosomes III, IIIS-72a4.5 and cSVEP mapped to the long arm of chromosome III, IIIL-96b1. cSVAT sits proximal to a common IIIS-2 paracentric inversion, which occurs predominately as the standard sequence in the IIIL-1 sibling and as the inverted sequence in the IS-7 sibling. cSVEP sits close to the differentiated X chromosomes in the IIIL-1 sibling.

Thrombostasin (TS) is a thrombin inhibitor found in the salivary glands of horn flies (Haematobia irritans). It is produced as an inactive form with a 76-amino acid propeptide in the N-terminus preceding the mature TS. A minimal recognition sequence by subtilisin-like proprotein convertases, Arg-Xaa-Xaa-Arg, is localized C-terminal to the propeptide. This study demonstrated that a gene cloned from the salivary glands of the horn fly encodes a new convertase, subsequently named horn fly proprotein convertase (HFPC), and that the recombinant HFPC expressed in insect HighFive cell culture specifically cleaves recombinant pro-thrombostasin, produced in E. coli, at the expected site. The relative cleavage efficiency of rHFPC was compared with that of recombinant human furin, a commercially available proprotein convertase. The result indicated that this newly identified proprotein convertase is of importance for the proteolytic maturation of thrombostasin, a protein secreted in horn fly saliva and used by the insect to counteract its host's haemostatic response.

The objective of this study was to examine nodules from Mexico, Guatemala, and Ecuador collected over a one-year period (2001) to determine the effects of semi-annual ivermectin treatments on Onchocerca volvulus macrofilarial populations. Nodules were sectioned, stained with hematoxylin and eosin, and histologic findings were compared between countries and with historical data prior to the introduction of ivermectin into the region. Nodules from Ecuador had 10 times more dead or moribund worms than the historical control (66.6% versus 6.5%); nodules from patients from Mexico and Guatemala did not differ from the control. More than 80% of the female worms in each country were uninseminated and producing unfertilized oocytes. Nodules containing males differed in each country from the historical control (P < 0.0001), with presence of males ranging from 19.7% in Mexico to 13.6% in Ecuador versus 73% in the control. Nodules with females producing active microfilariae ranged from 7.8% (Mexico) to 2.7% (Ecuador) versus 60% in the historical control (P < 0.0001). Nodules from Ecuador and Mexico were significantly smaller in size than those from Guatemala or historical controls (P < 0.0005). These results depict a deteriorating condition of adult O. volvulus populations in Mexico, Guatemala and Ecuador, indicating that semi-annual ivermectin treatment of >/=6 years has had a profound effect on survival and reproduction of this species.

The susceptibility of four strains of Aedes albopictus (Skuse) to permethrin, deltamethrin, resmethrin, chlorpyrifos, malathion, propoxur, fipronil, imidacloprid, spinosad, and Bacillus thuringiensis variety israelensis (Bti) was determined. The HAmAal and MAmAal strains were collected in 2002 and 2003, respectively, from Huntsville and Mobile, AL, and the VBFmAal and SFmAal strains were collected in 1998 from Vero Beach and southern Florida, respectively. The HAmAal strain showed a 22-fold elevated level of resistance to deltamethrin compared with the susceptible Ikaken laboratory strain, whereas the VBFmAal strain showed a six-fold lower sensitivity to deltamethrin compared with Ikaken. However, comparison of resistance ratios for deltamethrin at LC50 and LC90 (21-fold) and the gradual slopes of dose-response curves indicated that the field population of this mosquito strain was heterogenous in response to deltamethrin. All four mosquito strains showed elevated levels of resistance to chlorpyrifos, with resistance ratios from 10 to 33. Nevertheless, except for the relatively low resistance to deltamethrin and chlorpyrifos, all mosquito strains showed a similar susceptibility or lower tolerance to the remaining insecticides tested compared with the susceptible Ikaken strain, even though some, such as permethrin, resmethrin, malathion, and Bti, have been used in the field for a long time, especially in Alabama. These results indicate that the development of resistance to insecticides in Ae. albopictus is slow and conventional insecticides, such as permethrin, resmethrin, malathion, and Bti, and relatively new insecticides, such as fipronil, imidacloprid, and spinosad, may all be valuable for the management of this important mosquito.

Uranotaenia sapphirina, Culex erraticus, and Cx. peccator were collected in an enzootic eastern equine encephalomyelitis (EEE) virus focus in central Alabama (Tuskegee National Forest) from 2001 to 2003 and analyzed for virus as well as host selection. EEE virus was detected in each species every year except 2003, when pools of Cx. peccator were negative. Most (97%) of the 130 Cx. peccator blood meals identified were from ectothermic hosts; 3% were from birds. Among blood meals from reptiles (approximately 75% of the total), 81% were from Agkistrodon piscivorus (cottonmouth); all amphibian blood meals (approximately 25%) were from Rana spp. with > 50% taken from the bullfrog R. catesbeiana. Host identifications were made from 131 of 197 Cx. erraticus, but only 3 (2%) were derived from ectothermic species. Identification of Ur. sapphirina blood meals proved difficult and only 2 of 35 hosts were determined. Both were from R. catesbeiana. Ectothermic species are possible EEE virus reservoirs in the southeastern United States where species such as Cx. peccator and Ur. sapphirina occur with large, diverse reptilian, amphibian, and avian populations such as those at the Tuskegee site.

The potential for controlling blood-feeding by the cattle pest, Haematobia irritans irritans (horn fly), was tested by vaccination against thrombostasin (TS), an inhibitor of mammalian thrombin that is released into skin during horn fly blood-feeding. The increase in blood meal size that occurred for flies feeding on sensitized non-vaccinated hosts was blocked and egg development in female flies was delayed when horn flies fed on rabbits and cattle immunized with recombinant TS. This demonstration of the impact of disrupting TS action by vaccination provides a novel approach toward control of this veterinary pest and offers a paradigm for limiting blood-feeding in other medically-important insect species.

The species composition and population dynamics of adult mosquitoes in a wetland near Iuka, MS, were analyzed over a 6-yr period (1997-2002) and reverse transcription-polymerase chain reaction (PCR) detection rates of arboviruses determined during five of those years. Blood meals of three likely vector species were identified using a PCR-based method that allows identification of the host to species. Culex erraticus (Dyar & Knab) composed 51.9% of the population during the 6-yr period with 295 females collected per trap night. Eastern equine encephalomyelitis (EEE) virus was detected in six genera of mosquitoes [Coquillettidia perturbans (Walker), Culex restuans Theobald, Culex salinarius Coquillett, Culex erraticus (Dyar & Knab), Anopheles crucians Wiedemann, Anopheles quadrimaculatus Say, Aedes vexans (Meigen), Ochlerotatus triseriatus Say, and Psorophora ferox Humboldt) with positive pools occurring in 1998, 1999, and 2002. Culiseta melanura Coquillett occurred at a low level (< 1%) and was not infected. Saint Louis encephalitis virus was detected once in a single pool of Cx. erraticus in 1998. Neither West Nile virus nor LaCrosse virus was found. Minimum infection rates per 1000 females tested of competent vectors of EEE virus were variable and ranged from 0.14 for Cx. erraticus to 40.0 for Oc. triseriatus. Thirty-nine species of birds were identified in the focus with blood-engorged mosquitoes found to contain meals (n = 29) from eight avian species. The majority of meals was from the great blue heron, Ardea herodias L. (n = 55%), but when bird abundance data were adjusted for avian mass, the brown-headed cowbird, Molothrus ater (Boddaert); blue jay, Cyanocitta cristata (L.); and northern mockingbird, Mimus polyglottos (L.), were overrepresented as hosts.

Insecticide resistance and cross-resistance was determined for three strains of Culex quinquefasciatus Say in the southeastern United States. HAmCq and MAmCq strains were collected in 2002 from Huntsville and Mobile, AL. The VBFmCq strain was collected in 1998 from Vero Beach, FL. VBFmCq, HAmCq, and MAmCq larvae showed resistance to permethrin with resistance ratios of 13, 100, and 940, respectively, compared with the susceptible S-Lab strain. Levels of resistance in HAmCq and MAmCq larvae were 200- and 830-fold to resmethrin and 4- and 70-fold to malathion, respectively. VBFmCq, HAmCq, and MAmCq strains all demonstrated a great ability to develop tolerance and/or cross-resistance to different insecticides, including deltamethrin (50-, 100-, and 300-fold), chlorpyrifos (150-, 33-, and 720-fold), fipronil (10-, 5-, and 15-fold), and imidacloprid (7.5-, 5- and 10-fold, respectively). Comparison of resistance ratios for pyrethroids, organophosphates, and imidacloprid at LC50 and LC90 and gradual slopes of dose-response curves indicated that VBFmCq, HAmCq, and MAmCq were heterozygous in response to these insecticides. All three strains showed high levels of susceptibility to Bacillus thuringiensis variety israelensis (Bti) and spinosad, although these mosquitoes had been extensively exposed to Bti. Thus, we conclude that Bti and spinosad may be valuable for the management of Cx. quinquefasciatus, especially in situations where local strains are highly resistant to other insecticides.

An important variable in the amplification and escape from the enzootic cycle of the arboviral encephalitides is the degree of contact between avian hosts and mosquito vectors. To analyze this interaction in detail, blood-fed mosquitoes that were confirmed vectors of eastern equine encephalomyelitis (EEE) virus were collected in 2002 from an enzootic site in central Alabama during the time this virus was actively transmitted. Avian-derived blood meals were identified to the species level of the host, and the proportion derived from each species was compared with the overall composition of the avifauna at the study site. The EEE vector mosquito species fed significantly more on some bird species and less on other species than expected given the overall abundance, biomass, or surface area of the local avifauna. When viewed collectively, these data suggest that these mosquitoes are selectively targeting particular avian species.

A site near Tuskegee, Alabama was examined for vector activity of eastern equine encephalomyelitis (EEE) virus in 2001. More than 23,000 mosquitoes representing 8 genera and 34 species were collected during a 21-week period, and five species, Culiseta melanura, Aedes vexans, Coquillettidia perturbans, Culex erraticus, and Uranotaenia sapphirina, were examined for the presence of virus using a nested reverse transcriptase-polymerase chain reaction for EEE virus. Each species was infected at various times of the mosquito season (May-September) with different minimum infection rates (MIRs). Culiseta melanura had the highest MIR (20.2) and positive pools were detected from late May to mid-September. Aedes vexans had an MIR of 2.2 and was infected early in the season (June), while Cq. perturbans exhibited a much higher field infection rate (9.9) with all positive pools collected in August. Culiseta melanura is a likely endemic vector in central Alabama, while Ae. vexans and Cq. perturbans probably function as bridge vectors. Culex erraticus, the most common mosquito in the habitat (54% of total collections), had an MIR of 3.2, and was persistently infected from mid-June to mid-September. This is the first report of high rates of EEE virus infection in this species, a member of the tropical subgenus Melanoconion. Uranotaenia sapphirina, considered to feed on amphibians and possibly reptiles, had an MIR of 5.6, with positive pools spanning a four-month period. This suggests that species other than birds may serve as a reservoir for EEE in hardwood swamps in the Southeastern United States and elsewhere. The lengthy period of mosquito infection with EEE virus, coupled with the diverse habits of the vectors and their proximity to a population center, indicate the importance of monitoring EEE virus activity in the Mid-South.

Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host-skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor [s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host's skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (pIs) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with pI 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5' and sequential polymerase chain reaction (PCR) amplification. It contained a 5'-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.

Thrombostasin (TS) is a newly described thrombin-inhibiting protein isolated from the saliva of the horn fly (Haematobia irritans), a blood-sucking ectoparasite of cattle. This report provides a detailed characterization of the TS gene and the first analysis of the allelic complexity of a gene for an anti-hemostatic protein from a blood-feeding insect. Multiple point mutations at fixed positions in the TS gene were identified in a cDNA library prepared from mRNA isolated from horn fly salivary glands. When translated, the variant mRNAs would specify five biochemically active peptides that differ in molecular weight, isoelectric point and predicted secondary structure. Allelic variation with the same mutation pattern was revealed in the genomes of individual flies collected in the field and sampled from a long-standing laboratory colony. Approximately 60% of flies examined carried heterozygous alleles, including five additional alleles not found in the cDNA library. Comparative analysis of the allelic mutations and the predicted effects on secondary structures of the active proteins produced suggest that the TS gene may be undergoing evolutionary selection.

Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced significantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous pool-feeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission.

The horn fly, Hematobia irritans (L.), is an important pest of livestock because the adult stage of both sexes are aggressive blood-feeders. Remarkably, even though horn fly adults feed recurrently on their hosts as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilatory antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process as demonstrated by the recalcification time assay. Using this as a baseline, the effects of saliva on recalcification time, activated partial thromboplastin time, prothrombin time, and thrombin time were measured to determine which arm(s) of the coagulation cascade might be impacted. Factor-deficient plasma assays also were used to measure possible perturbations in clotting. Gland-free saliva delayed the recalcification time as well as the activated partial thromboplastin time, prothrombin time, and thrombin time. Saliva also further delayed clotting times of plasmas deficient in factor V, factor VIII, and factor XIII, indicating that other factors in the coagulation cascade were inhibited. Although horn fly saliva did not alter the ability of deficient plasma reconstituted with factor X to clot, it did inhibit deficient plasma reconstituted with factor II (thrombin). Antithrombin activity in saliva was confirmed by its ability to interfere with thrombin hydrolysis of fibrinogen, its normal substrate, and by its inhibition of thrombin action on a chromagenic substrate that mimics the hydrolytic site of fibrinogen. Thus, horn fly saliva contains a factor that specifically targets thrombin, a key component in the coagulation cascade. While the biochemical mechanisms of inhibition may vary, this antihemostatic characteristic is shared with other zoophilic Diptera such as black flies, Simulium spp., and tsetse, Glossina morsitans morsitans Westwood, that feed on ungulates.

The economic impact on livestock production by Haematobia irritans (L.) is estimated to approach $1 billion per year in North America. However, there is little information regarding the blood-feeding strategy used by these insects. Information presented here shows that horn fly saliva interferes with the normal coagulation response as measured by the recalcification time assay. The relative anticoagulant activity on a per-gland basis was more than or equal to that reported for Simulium vittatum Zetterstedt, a common hematophagous black fly that also feeds on cattle. However, unlike S. vittatum, H. irritans salivary factors do not inhibit platelet aggregation using apyrase and have no detectable vasodilative activity. In this regard, the horn fly is strikingly different from blood-feeding species in the lower Diptera and shows a much more limited repertoire of antihemostatic factors.

A cDNA was cloned from the salivary glands of a blood-feeding black fly Simulium vittatum. The encoded protein has been given the name Simulium vittatum erythema protein or SVEP, because of its ability to increase blood perfusion in skin capillaries, resulting in the well-characterized erythema of black fly bites. The full-length cDNA contains 548 base pairs which encode 152 amino acid residues of the nascent protein. Post-translational processing produces a mature, secreted protein of 133 residues with a molecular mass of 15.4 kDa. Recombinant SVEP (rSVEP) was produced in a baculovirus expression system and purified by a one-step reversed-phase HPLC procedure. Analyses of physical properties and biological potency demonstrated fidelity of rSVEP to the native protein. Recombinant SVEP relaxed rabbit aorta preparations when preconstricted with 2 micromol l-1 phenylephrine or 25 mmol l-1 K+ but not with 60 mmol l-1 K+. Further, the rSVEP-induced relaxation response of phenylephrine-constricted aorta was inhibited by glibenclamide (10 micromol l-1), suggesting that at least part of its action to relax smooth muscle may result from the opening of ATP-dependent K+ channels. SVEP is a novel salivary-gland-derived vasoactive protein that may be essential for blood feeding by black flies and could potentially enhance transmission of filarial parasites.

Fogo selvagem is an autoimmune blistering skin disease that principally occurs among rural Brazilians living in geographically clumped disease foci. Exposure to hematophagous black flies possibly is related to the cause of the disease. We compared the occurrence, proportions, and richness of simuliid species immatures and the biting activity of adult females within a recently discovered, high prevalence focus of fogo selvagem, the Limão Verde Terena Reservation, to that of neighboring regions with no reported cases of fogo selvagem. Nine black fly species were collected from 12 stream sites during 5 trips to the fogo selvagem focus. The species showed longitudinal (upstream-downstream) trends in occurrence, proportions, and richness, and the abundance of simuliid immatures was greater at downstream sites. The most prevalent species at the focus, Simulium nigrimanum (Macquart), dominated the stream sites with highly abundant simuliid assemblages, and was the most common black fly in human bait collections. This species was absent or in very low numbers in neighboring valleys and villages that did not have cases of fogo selvagem.