1. Mosquitoes Associated with Equine West Nile Virus Cases in Southeast Georgia Calvin W. Hancock & William S. Irby Dept. of Biology, Georgia Southern University, Statesboro, GA INTRODUCTION West Nile Virus is a mosquito-borne flavivirus first encountered in Africa in 1937. Since its introduction into Queens, New York in 1999, the virus has spread exponentially across the United States and viral activity (infected mosquito pools, birds, equines, or humans) has been reported in 48 states. It appeared in Georgia during the summer of 2001 and since then, there have been more than 50 equine infections in the southeastern part of the state. In 2003, we began a two year study monitoring mosquito populations associated with West Nile Virus (WNV) equine infection case sites. Mosquitoes were obtained through use of light traps as well as aspiration. Samples are tested for the presence of the virus and sources of mosquito blood meals will be identified in hopes of establishing the significance of each vector species to the enzootic and epizootic transmission cycles. The main objectives of the research are summarized as follows; to determine which mosquito species are associated with equine WNV infections in Southeastern Georgia, determine which mosquito species are positive for WNV, and ultimately to determine which species may serve as bridge vectors between avian reservoirs and mammal (equine) hosts. RESULTS & DISCUSSION To date, we have collected over 10, 500 mosquitoes throughout the 14 month project. Representative data (Table 1) shows that Culex nigripalpus, Aedes vexans, and Culex erraticus were the most abundant species encountered in our area. However, the majority of the Aedes vexans collected were from light traps during the 2004 season only, and those were limited to a few single traps. Culex quinquefasciatus, the mosquito species most commonly associated with West Nile Virus by surveillance data throughout the United States, was rarely encountered and the majority of the specimens were collected in two samples. Culex nigripalpus and Culex erraticus were collected more evenly and in high abundance, indicating that these species may have a more significant role in transmission than previously assumed. To date, we have tested 7,660 specimens from 2003 and 2004 (Table 2) for the presence of WNV, but all results have been negative. Temporal data for each collection method are shown in Figures 1, 2, and 3. Searching for a commonality between populations encountered at different sites, diversity was analyzed with the Shannon-Weaver Index (Table 3). However, it indicated that diversity was uncommon among trapping sites. We hope that complete testing of samples will reveal which species are most associated with arbovirus transmission in our area. In light of the lack of abundance of Culex quinquefasciatus, we suspect that Culex nigripalpus or other species may have a more significant role in local virus transmission. Blood meal analyses will be conducted in fall 2005 and collection data is summarized in Table 4. ACKNOWLEDGMENTS I would like to thank Dr William Irby for his support and guidance throughout the project. This work was partially funded by a grant from the CDC to Michael Womack and partially through a Georgia Southern University faculty grant. PCR-Heteroduplex Assays: PCR products of cytochrome B amplification are made from each test animal and mixed with either northern cardinal or Carolina chickadee “driver” cytochrome B PCR products. The mixture is then heated to 99 o C to denature DNA, followed by slow cooling to promote heteroduplex formation. Homologous and heterologous duplexes form, where the number of duplexes = 2 n, where n = number of different cytochrome B genes in mix. The resulting products are distinguished by relative mobilities by Polyacrylamide Gel Electrophoresis (PAGE) and any products not matching standards are sequenced. See figure 2 for example. These tests will be concluded in the fall of 2005. West Nile virus West Nile virus Mosquito vector Incidental infections Bird reservoir hosts Incidental infections Temporal Mosquito Abundance for CDC-LT Traps (2003-2004) 1 10 100 1000 Date Average #/sample (log) 2003 2004 Aug Sep Oct Figure 1 Temporal Mosquito Abundance by Aspiration (Feb-Oct 2004) 0 50 100 150 200 Date Average #/sampl Feb Mar Apr May Jun Jul Aug Sep Oct Figure 3 Temporal Mosquito Abundance by Aspiration (Aug-Nov 2003) 0 10 20 30 40 50 Date Average #/sample Aug Sept Oct Nov Figure 2 Table 4: Mosquito Blood meals to be Analyzed Table 3: Shannon-Weaver Index by Site -- Shannon-Weaver index measures species “evenness” -- community structure at different sites is weakly correlated with diversity -- range of sites reflects a diversity of mosquito communities -- no commonality among sites, except Cx. nigripalpus present at all MATERIALS AND METHODS Collection of mosquitoes: Adult mosquitoes were collected, starting in August, 2003 as follows: By regular sampling using modified-CDC light traps: mosquitoes were collected from 22 sites, in a 5 county area, where avian or equine cases of West Nile Virus had occurred in 2002 or 2003 (above right). If notification of a case was received within ~3 weeks, trapping was initiated as soon as possible. For some cases, trapping was conducted at positive sites one year later, in order to capture mosquitoes that were representative of populations that would have been present during the time of infection. For avian cases, modified CDC light traps (right) baited with dry ice were placed at the site were the infected bird was located. For equine cases, light traps were similarly placed. Trapping in 2003 was conducted from 29-VIII-03 to 16-X-03, and in 2004 from 3-IX-04 to 23-X-04. Trapping seasons were chosen based on encompassing all onset dates. By regular sampling using modified-CDC backpack aspirator: resting mosquitoes were collected by aspiration of light trapping sites. Vertical surfaces of structures which may serve as potential mosquito resting sites (barns, feed sheds, etc.) were sampled by aspiration using a battery-powered modified CDC backpack aspirator (right). Collection by aspirator was conducted from 30-VIII-03 to 24-X-04. Processing of mosquitoes: Testing for the presence of West Nile Virus: upon collection, mosquitoes were placed on ice until they could be returned to the laboratory and frozen. A chill plate was used to keep the mosquitoes frozen while they were separated by gonotrophic level and keyed to species. Unfed females were pooled by species, site, date, and collection method in pools of ≤50 individuals. These pools are analyzed using the antigen assay test, VecTest®. Specimens of Anopheles spp. and males were not tested due to their low likelihood of involvement in the transmission cycle. Blood meal analyses: blood meals are first identified to vertebrate class (mammal, avian, reptile/amphibian) using screening antisera produced in New Zealand White Rabbits through the use of Enzyme-Linked Immunosorbent Assays (ELISAs). This antisera is produced by intranodal and interdermal injection of a mix of sera from representatives of major groups within each class and Freud’s adjuvant to induce an immune response. Blood meals determined to be of mammalian or reptile/amphibian sources will be analyzed by use of ELISAs and species-specific antisera produced in the same manner as the screening antisera described above. Blood meals determined to be of avian source will be analyzed using PCR- based Heteroduplex Assays (HDAs) described by Lee and others (2002) and further outlined in the following section. Equine WNV in Georgia, 2003 Collection Sites Figure 2: Heteroduplex assay of cytochrome B PCR products from avian and human samples. All patterns are unique. northern cardinal heteroduplex driver Carolina chickadee heteroduplex driver 1: Carolina chickadee 2: eastern towhee 3: white-throated sparrow 4: field sparrow 5: blue jay 6: house finch 7: house sparrow 8: swamp sparrow 9: orchard oriole 10: tufted titmouse 11: blue grosbeak 12: brown-headed cowbird 13: red-winged blackbird 14: American goldfinch 15: northern cardinal 16: northern bobwhite 17: human (no HDA pattern) 1-15: Order Passeriformes; 16: Order Galliformes * * * = homologous driver Lee, Joon H; Hassan, Hassan; Hill, Geoff; Cupp, Eddie; Higazi, Tariq; Mitchell, Carl; Godsey, Marvin; Unnasch, Thomas. 2002. Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis. American Journal of Tropical Medicine and Hygiene, 66: 599-604. Table 1: Mosquito abundance at equine WNV case sites in southeastern GA (August 2003-October 2004) *() relative percentages of total individuals collected Mosquito Species 20032004 PoolsTotal SpecimensPoolsTotal Specimens Ae. albopictus7141878 Ae. vexans1532722184 Cx. melanura11 Cq. perturbans6712359 Cx. erraticus24211521535 Cx. quinquefasciatus16 Cx. nigripalpus27503652288 Cx. restuans5716 Cx. tarsalis33 Cx. territans47 Cx. salinarius202551253 Oc. atlanticus510228740 Oc. canadensis523 Oc. fulvus pallens38 Oc. triseriatus11612 Ps. ciliata832 Ps. columbiae41015254 Ps. ferox111081 Ur. sapphirina37 Totals12411603107660 Table 2: Mosquito pools tested for WNV, EEEV and SLEV from equine WNV sites, 2003-4