Larval diet and temperature alter mosquito immunity and development: using body size and developmental traits to track carry-over effects on longevity.

BACKGROUND: Estimating arbovirus transmission potential requires a mechanistic understanding of how environmental factors influence the expression of adult mosquito traits. While preimaginal exposure to environmental factors can have profound effects on adult traits, tracking and predicting these effects remains challenging. METHODS: Using Aedes albopictus and a structural equation modeling approach, we explored how larval nutrition and temperature jointly affect development rate and success, female body size, and whether these metrics capture carry-over effects on adult female longevity. Additionally, we investigated how larval diet and temperature affect the baseline expression of 10 immune genes. RESULTS: We found that larval development success was primarily determined by diet, while temperature and diet both affected development rate and female body size. Under a low larval diet, pupal wet weight and wing length both declined with increasing temperature. In contrast, responses of the two morphometric measures to rearing temperature diverged when females were provided higher larval nutrition, with pupal wet weight increasing and wing length decreasing at higher temperatures. Our analyses also revealed opposing relationships between adult female lifespan and the two morphometric measures, with wing length having a positive association with longevity and pupal weight a negative association. Larval diet indirectly affected adult longevity, and the time to pupation was negatively correlated with longevity. The expression of eight immune genes from the toll, JAK-STAT and Imd pathways was enhanced in mosquitoes with higher nutrition. CONCLUSIONS: Our results highlight deficiencies from using a single body size measure to capture carry-over effects on adult traits. Further studies of larval development rate under varying environmental conditions and its potential for tracking carry-over effects on vectorial capacity are warranted.

Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide.

Proliferative Vitreoretinopathy (PVR) is a refractory retinal disease whose primary pathogenesis involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. At present, there is no effective treatment other than surgery for PVR. The purpose of this study was to investigate the effect of αB crystallin peptide (αBC-P) on EMT in PVR. We have previously shown that this peptide is antiapoptotic and regulates RPE redox status. Subconfluent primary human RPE (hRPE) cells were stimulated by TGFß2 (10 ng/mL) with or without αBC-P (50 or 75 µg/mL) for 48 h and expression of EMT/mesenchymal to epithelial transition (MET) markers was determined. Mitochondrial ROS (mtROS) generation in hRPE cells treated with TGFß2 was analyzed. The effect of TGFß2 and αBC-P on oxidative phosphorylation (OXPHOS) and glycolysis in hRPE was studied. RPE cell migration was also assessed. A PVR-like phenotype was induced by intravitreal dispase injection in C57BL/6J mice. PVR progression and potential therapeutic efficiency of αBC-Elastin-like polypeptides (ELP) was studied using fundus photography, OCT imaging, ERG, and histologic analysis of the retina. αSMA, E-cadherin, Vimentin, Fibronectin and, RPE65, and CTGF were analyzed on Day 28. Additionally, the amount of VEGF-A in retinal cell lysates was measured. The EMT-associated αSMA, Vimentin, SNAIL and SLUG showed a significant upregulation with TGFß2, and their expression was significantly suppressed by cotreatment with αBC-P. The MET-associated markers, E-cadherin and Sirt1, were significantly downregulated by TGFß2 and were restored by αBC-P. Incubation of hRPE with TGFß2 for 24 h showed a marked increase in mitochondrial ROS which was noticeably inhibited by αBC-ELP. We also showed that after TGFß2 treatment, SMAD4 translocated to mitochondria which was blocked by αBC-ELP. Mitochondrial oxygen consumption rate increased with TGFß2 treatment for 48 h, and αBC-P co-treatment caused a further increase in OCR. Glycolytic functions of RPE were significantly suppressed with αBC-P (75 µg/mL). In addition, αBC-P significantly inhibited the migration from TGFß2 treatment in hRPE cells. The formation of proliferative membranes was suppressed in the αBC-ELP-treated group, as evidenced by fundus, OCT, and H&E staining in dispase-induced PVR in mice. Furthermore, ERG showed an improvement in c-wave amplitude. In addition, immunostaining showed significant suppression of αSMA and RPE65 expression. It was also observed that αBC-ELP significantly reduced the expression level of vimentin, fibronectin, and CTGF. Our findings suggest that the antioxidant αBC-P may have therapeutic potential in preventing PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells.

Cascade of ecological consequences for West Nile virus transmission when aquatic macrophytes invade stormwater habitats.

Artificial aquatic habitats are ubiquitous in anthropogenic landscapes and highly susceptible to colonization by invasive plant species. Recent research into the ecology of infectious diseases indicates that the establishment of invasive plant species can trigger ecological cascades which alter the transmission dynamics of vector-borne pathogens that imperil human health. Here, we examined whether the presence or management of two invasive, emergent plants, cattails (Typha spp.) and phragmites (Phragmites australis), in stormwater dry detention basins (DDBs) alter the local distribution of vectors, avian hosts, or West Nile virus (WNV) transmission risk in an urban residential setting. Mosquitoes and birds were surveyed at 14 DDBs and paired adjacent residential sites. During the study period, emergent vegetation was mowed by site managers in three DDBs. In the absence of vegetation management, the overall abundance and species composition of both adult vectors and avian hosts differed between residential and DDB habitats; however, WNV entomological risk indices were equivalent. Communal bird roosts composed primarily of three species, European Starlings (Sturnus vulgaris), Red-winged Blackbirds (Agelaius phoeniceus), and Common Grackles (Quiscalus quiscula), representing a broad range of WNV reservoir competence, were observed at half (three out of six) of the DDBs containing unmanaged stands of phragmites; however, their presence was associated with a lower seasonal increase in vector infection rate. Conversely, mowing of emergent vegetation resulted in a significant and sustained increase in the abundance of WNV-infected vectors in DDBs and the increase in risk extended to adjacent residential sites. These findings indicate that management of invasive plants in DDBs during the growing season can increase, while presence of communal bird roosts can decrease, WNV transmission risk.

Evaluation of Household Bleach as an Ovicide for the Control of Aedes aegypti.

Accumulations of dormant eggs in container habitats allow Aedes aegypti populations to survive harsh environmental conditions and may frustrate control interventions directed at larval and adult life stages. While sodium hypochlorite solutions (NaOCl) have long been recognized as ovicides for use against dengue vectors, the susceptibility of eggs to spray applications has not been robustly evaluated on substrate materials representative of the most frequently utilized artificial container habitats. Experiments were performed under controlled and natural conditions by applying dilutions of household bleach (52.5 ppt NaOCl) as a spray to eggs on plastic, rubber, and concrete surfaces, with and without a smectite clay thickener. Laboratory assays identified the minimum NaOCl concentrations required to eliminate eggs on plastic (10 ppt), rubber (20 ppt) and concrete (20 ppt) surfaces. Addition of smectite clay reduced the minimum effective concentration to 10 ppt NaOCl for all 3 substrates. A minimum exposure period of 24 h was required to completely eliminate egg viability on concrete surfaces, even at the highest NaOCl concentration (52.5 ppt). Field experiments verified that spray application of a 1∶3 dilution of household bleach mixed with smectite clay can reduce egg hatching by ≥ 99% in shaded and sun-exposed plastic containers. Similarly, 4∶1 dilution of household bleach (with or without smectite clay) eliminated ≥ 98% of eggs from concrete surfaces in outdoor, water-filled drums. In this study, we propose a practical, effective and safe strategy for using household bleach to eliminate Ae. aegypti eggs in a range of artificial container habitats.

Characteristics of a dengue outbreak in a remote pacific island chain--Republic of The Marshall Islands, 2011-2012.

Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1-4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10-29 years, and ∼95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.

Use of the CDC autocidal gravid ovitrap to control and prevent outbreaks of Aedes aegypti (Diptera: Culicidae).

Populations ofAedes aegypti (L.) can be managed through reductions in adult mosquito survival, number of offspring produced, or both. Direct adult mortality can be caused by the use of space sprays or residual insecticides to mosquito resting sites, and with a variety of residual insecticide-impregnated surfaces that are being tested, such as curtains, covers for water-storage vessels, bednets, and ovitraps. The fertility ofAe. aegypti populations can be reduced by the use of autocidal oviposition cups that prevent the development of mosquitoes inside the trap by mechanical means or larvicides, as well as by releasing sterile, transgenic, and para-transgenic mosquitoes. Survival and fertility can be simultaneously reduced by capturing gravid female Ae. aegypti with sticky gravid traps. We tested the effectiveness of the novel Centers for Disease Control and Prevention autocidal gravid ovitrap (CDC-AGO trap) to control natural populations ofAe. aegypti under field conditions in two isolated urban areas (reference vs. intervention areas) in southern Puerto Rico for 1 yr. There were significant reductions in the captures of female Ae. aegypti (53-70%) in the intervention area The presence of three to four AGO control traps per home in 81% of the houses prevented outbreaks of Ae. aegypti, which would be expected after rains. Mosquito captures in BG-Sentinel and AGO traps were significantly and positively correlated, showing that AGO traps are useful and inexpensive mosquito surveillance devices. The use of AGO traps to manage Ae. aegypti populations is compatible with other control means such as source reduction, larviciding, adulticiding, sterile insect techniques, induced cytoplasmic incompatibility, and dominant lethal gene systems.

A novel autocidal ovitrap for the surveillance and control of Aedes aegypti.

We describe an inexpensive autocidal ovitrap for Aedes aegypti that uses cross-linked polyacrylamide (PAM) gel as the oviposition substrate. Aedes aegypti females readily laid eggs on PAM gel that had been hydrated with either hay infusion or water. Aedes aegypti larvae that hatched from their eggs desiccated on the surface of the PAM gel. We tested the effects of gel hydration, texture, and type of attractant on trap performance, and compared the capture rates of standard ovitraps with those of PAM gel ovitraps in the field. The results showed that the number of eggs did not vary over a range of gel hydration levels (40-100%) and that more eggs were recovered from ovitraps containing coarse gel than from those containing homogenized gel. The PAM gel hydrated with hay infusion was more attractive to gravid female mosquitoes than gel hydrated with water. In the field, the number of eggs recovered from autocidal ovitraps with PAM gel was similar to that recovered from standard ovitraps with hay infusion.

An improved autocidal gravid ovitrap for the control and surveillance of Aedes aegypti.

BACKGROUND: Limited success has been achieved using traditional vector control methods to prevent the transmission of dengue viruses. Integrated control programs incorporating alternative tools, such as gravid ovitraps (lethal ovitraps and sticky ovitraps) may provide greater potential for monitoring and reducing vector populations and dengue virus transmission. We had developed an autocidal gravid ovitrap (AGO) as a simple, low-cost device for surveillance and control of Ae. aegypti without the use of pesticides that does not require servicing for an extended period of time. The purpose of our study was to improve the efficacy and efficiency of this device. METHODS: Competitive assays were performed in the laboratory and an outdoor cage to evaluate whether modifications to the structure and appearance of our original trap design (AGO-A), and the addition of an olfactory bait (hay infusion), improve trap function. The performance of a modified trap design (AGO-B) was then assessed and compared with conventional ovitraps in a series of field tests in San Juan City, Puerto Rico. Generalized linear mixed models were used to analyze adult Ae. aegypti capture data from the laboratory, outdoor cage and field experiments. RESULTS: Increasing the size of the trap entrance, altering the color of trap components, and increasing the volume/surface area of the aqueous bait significantly improved the performance of the AGO in the outdoor cage. In a subsequent field comparison, captures of Ae. aegypti females were 3.7 fold greater in the improved trap (AGO-B), compared with the original design (AGO-A). An infusion bait produced "in situ" significantly improved capture rates of the improved trap under both semi-natural and field conditions. Semi-weekly collections of Ae. aegypti females in the AGO-B were significantly correlated with cumulative rainfall 8 to 28 days prior to sampling, whereas egg collections in paired conventional ovitraps were not. When vector abundance was low, the AGO-B provided greater sensitivity and precision as a surveillance device, compared with paired conventional ovitraps. CONCLUSIONS: The AGO-B can be used to efficiently attract and capture gravid Ae. aegypti females for more than 8 weeks without the need for trap maintenance.

An improved trap to capture adult container-inhabiting mosquitoes.

Although dengue viruses are thought to be transmitted by Aedes aegypti in Puerto Rico, Aedes mediovittatus, the Caribbean tree hole mosquito, is also a potential vector. This species is native to the Greater Antilles and has been shown to be a competent vector of dengue viruses in the laboratory. Consequently, it has been suggested that Ae. mediovittatus could be acting as a secondary vector or virus reservoir. This study was part of an ongoing investigation into this, and it aimed to determine whether BG-Sentinel traps (BGS traps) could be used to collect adults of this mosquito and could be modified to increase the number of captures of this species in the field. We conducted experiments to test the relative attractiveness of BGS traps to Ae. mediovittatus and Ae. aegypti and explored the effects of chemical lures (BG-Lure, CO2, octenol) and optical properties (color, size) on the capture rates of BGS traps in a large, outdoor cage in San Juan city, Puerto Rico. We also conducted field tests to compare modified BGS traps with the original traps in a rural community in Patillas municipality, Puerto Rico. Results obtained from the large, outdoor cage experiments indicated that trap captures of both mosquito species could be significantly enhanced by using black instead of white BGS traps combined with BG-Lure. Field experiments revealed that the modified traps captured a significantly greater number of Ae. aegypti, Ae. mediovittatus, and Culex quinquefasciatus, with greater sensitivity for the latter 2 species, and also captured a larger number of mosquito species and a smaller ratio of Ae. aegypti to Ae. mediovittatus, with greater than expected species co-occurrences.

Vertebrate hosts of Aedes aegypti and Aedes mediovittatus (Diptera: Culicidae) in rural Puerto Rico.

The distribution of Aedes (Stegomyia) aegypti (L.), the main vector of dengue viruses (DENV) worldwide, overlaps with Aedes (Gymnometopa) mediovittatus (Coquillett), the Caribbean treehole mosquito, in urban, suburban, and rural areas. Ae. mediovittatus is a competent vector of DENV with high rates of vertical DENV transmission in the laboratory. This study determined whether Ae. mediovittatus feeds on humans and compared its feeding patterns with co-occurring Ae. aegypti in two rural communities of Puerto Rico. Adult mosquitoes were captured for three consecutive days every week from July 2009 to May 2010 using BG-Sentinel traps with skin lures that were placed in the front yard of houses in both communities. Three methods were used to identify the 756 bloodmeals obtained in this study: a multiplex polymerase chain reaction (PCR) for humans and dogs targeting cytochrome b; a PCR targeting the 16S rRNA; and a nested PCR targeting cytochrome b. Ae. mediovittatus fed mostly on humans (45-52%) and dogs (28-32%) but also on cats, cows, horses, rats, pigs, goats, sheep, and chickens. Ae. aegypti fed mostly on humans (76-79%) and dogs (18-21%) but also on cats, horses, and chickens. Our results indicate that Ae. mediovittatus may have a relatively high rate of vector-human contact, which might facilitate virus transmission or harborage in rural areas of Puerto Rico.

Population dynamics of Aedes aegypti and dengue as influenced by weather and human behavior in San Juan, Puerto Rico.

Previous studies on the influence of weather on Aedes aegypti dynamics in Puerto Rico suggested that rainfall was a significant driver of immature mosquito populations and dengue incidence, but mostly in the drier areas of the island. We conducted a longitudinal study of Ae. aegypti in two neighborhoods of the metropolitan area of San Juan city, Puerto Rico where rainfall is more uniformly distributed throughout the year. We assessed the impacts of rainfall, temperature, and human activities on the temporal dynamics of adult Ae. aegypti and oviposition. Changes in adult mosquitoes were monitored with BG-Sentinel traps and oviposition activity with CDC enhanced ovitraps. Pupal surveys were conducted during the drier and wetter parts of the year in both neighborhoods to determine the contribution of humans and rains to mosquito production. Mosquito dynamics in each neighborhood was compared with dengue incidence in their respective municipalities during the study. Our results showed that: 1. Most pupae were produced in containers managed by people, which explains the prevalence of adult mosquitoes at times when rainfall was scant; 2. Water meters were documented for the first time as productive habitats for Ae. aegypti; 3. Even though Puerto Rico has a reliable supply of tap water and an active tire recycling program, water storage containers and discarded tires were important mosquito producers; 4. Peaks in mosquito density preceded maximum dengue incidence; and 5. Ae. aegypti dynamics were driven by weather and human activity and oviposition was significantly correlated with dengue incidence.

Evidence of vertical transmission of West Nile virus in field-collected mosquitoes.

Male and nulliparous female mosquitoes were surveyed for evidence of vertical WNV infection in East Baton Rouge Parish, Louisiana. Adult male mosquitoes collected by trapping and aspiration, and adult male and nulliparous female mosquitoes reared from field-collected larvae were tested. Adult male Culex spp., female Aedes albopictus (Skuse), and female Culex quinquifasciatus Say mosquitoes that were collected as larvae were test-positive for WNV RNA. Infectious WNV was detected using virus isolation in field-collected male Aedes triseriatus Say and Culex salinarius Coquillett; these data represent the first field evidence of vertical transmission of WNV in Ae. triseriatus and Cx. salinarius.

Host feeding patterns of Culex mosquitoes (Diptera: Culicidae) in East Baton Rouge Parish, Louisiana.

Host feeding patterns were examined for four species of Culex mosquitoes collected from 18 sites in or adjacent to East Baton Rouge Parish, LA, from November 2002 to October 2004. Host DNA from 37 bloodfed Culex coronator Dyar and Knab, 67 bloodfed Cx. salinarius Coquillett, 112 bloodfed Cx. nigripalpus Theobald, and 684 bloodfed Cx. quinquefasciatus Say were identified. The percentages of bloodmeals containing mammalian DNA were 94.6% for Cx. coronator, 82.1% for Cx. salinarius, 66.1% for Cx. nigripalpus, and 40.1% for Cx. quinquefasciatus. Human DNA was detected in 7% of the bloodmeals from Cx. quinquefasciatus and 2.7% of the bloodmeals from Cx. nigripalpus. The northern cardinal was the most frequent avian host of Cx. quinquefasciatus and Cx. nigripalpus. In 2003 and 2004, there was no significant relationship from May through October between the proportion of Cx. quinquefasciatus feeding on mammalian hosts and the date of collection. Of the six avian species most frequently fed on by Cx. quinquefasciatus, the northern cardinal, northern mockingbird, common grackle, and brown thrasher were fed on more frequently than expected based on their abundance. House sparrows were fed on less frequently than expected based on their abundance. These data support the conclusions of previous studies that Cx. quinquefasciatus is the most important vector for both the enzootic amplification and transmission of West Nile virus to humans in southern Louisiana.

Dynamics of Aedes aegypti and Culex quinquefasciatus in septic tanks.

Aedes aegypti and Culex quinquefasciatus were found in large numbers emerging from septic tanks in southern Puerto Rico during the dry season. Previous studies suggested that Ae. aegypti uses subterranean aquatic habitats only during dry periods when surface containers do not have water. This research investigated whether septic tanks are alternative aquatic habitats that this mosquito uses during unfavorable times of the year, or whether Ae. aegypti uses this aquatic habitat throughout the year. To assess temporal change, exit traps were used to collect mosquitoes emerging from septic tanks in Playa/Playita, southern Puerto Rico, from November 2006 to October 2007. We also investigated the hypotheses that (1) the production of Ae. aegypti in septic tanks was larger than in surface containers and (2) adult mosquitoes emerging from septic tanks were larger than those emerging from surface containers. This study demonstrated that unsealed septic tanks produced large numbers of Ae. aegypti and Cx. quinquefasciatus throughout the year, without any significant relationship with rainfall. The number of adult Ae. aegypti emerging per day from septic tanks in each community was 3 to 9 times larger than those produced in surface containers. It was also demonstrated that Ae. aegypti emerging from septic tanks were significantly larger than those emerging from surface container habitats. It is recommended that dengue prevention programs include regular inspection and maintenance of septic tanks in communities lacking sewerage.

West Nile virus detection in mosquitoes in East Baton Rouge Parish, Louisiana, from November 2002 to October 2004.

The prevalence of West Nile virus (WNV) was determined in mosquitoes between November 2002 and October 2004 in East Baton Rouge Parish, LA. A total of 244,374 female mosquitoes were collected and tested by viral isolation. Additionally, 131,896 female mosquitoes were collected in 2003 and tested by VecTest and 167,175 female mosquitoes were collected in 2004 and tested by reverse transcriptase-polymerase chain reaction (RT-PCR). West Nile virus was isolated by cell culture from 17 (47.2%) out of 36 mosquito species collected over the study period. In 2003, WNV was detected in 9 (33.3%) out of 27 species tested by VecTest. In 2004, 14 (50%) out of the 28 mosquito species tested by RT-PCR were positive for WNV. The species with the greatest number of WNV-positive pools detected by all 3 testing methods was Culex quinquefasciatus. A significantly greater proportion of Cx. salinarius pools collected in light traps placed at a 3-m height were positive for WNV by viral isolation than in pools collected in light traps placed at a 1.5-m height.

West Nile virus surveillance in East Baton Rouge Parish, Louisiana.

West Nile virus (WNV) was detected for the first time in Louisiana in the fall of 2001. Surveillance data collected from East Baton Rouge Parish in 2002 were examined to establish baseline data on WNV activity, to support the current design of disease surveillance programs, and to target vector control efforts in the parish. The first indications of WNV activity were from a dead Northern Cardinal collected in February and from a live male cardinal sampled on 14 March. In mosquito pools, WNV was first detected on June 11. The onset of the first human case and the first detection of WNV in sentinel chickens occurred concurrently on June 24. The number of reported human cases and minimum infection rates in mosquitoes peaked in July. WNV prevalence in wild birds increased in late August and was highest in December. WNV-positive wild birds and mosquito pools were detected an average of 31 and 59 days in advance of the onset date of reported human cases, respectively, within 5 km of the residence of a human case. Antibodies to WNV were detected in sera from 7 (Northern Cardinal, House Sparrow, Northern Mockingbird, Blue Jay, Hermit Thrush, Yellow-rumped Warbler, and White-throated Sparrow) of the 42 wild bird species tested. Wild bird serology indicated WNV activity during the winter. Out of 18 mosquito species tested, the only species found positive for WNV was Culex quinquefasciatus, a result suggesting that this species was the primary epizootic/epidemic vector.

Detection of West Nile virus RNA in pools of three species of ceratopogonids (Diptera: Ceratopogonidae) collected in Louisiana.

Light traps were used to collect ceratopogonids in East Baton Rouge parish, Louisiana. In total, 46,496 ceratopogonids were sorted from 4,968 light trap collections from 20 November 2002 through 25 November 2004. Two hundred and nine pools containing specimens of 18 species of Culicoides Latreille, seven pools containing specimens of Atrichopogon Kieffer, and five pools containing specimens of Forcipomyia Meigen were tested for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) RNA using real-time reverse transcriptase polymerase chain reaction. Five out of the 209 pools of Culicoides specimens were positive for WNV RNA.