Journal of the American Mosquito Control Association, l9(1):33-38, 2003 Copyright @ 2003 by the American Mosquito Control Association, Inc. ISOLATION OF EEE VIRUS FROM OCH LEROTATU S TAENI ORHYN C H US AND C ULI SETA M EI^AN U RA IN COASTAL SOUTH CAROLINA DIANA I. ORTIZ,I ARTHUR WOZNIAK,' MARSHA W. TOLSON,' PEARL E. TURNER'AND DAVID R. VAUGHAN] ABSTRACT. A l-year arbovirus study was conducted at The Wedge Plantation located in coastal South Carolina to determine the occurrence and level of arbovirus activity in mosquito species inhabiting the site. Mosquito species composition and temporal abundance were also determined. A total of 45,051 mosquitoes representing 27 species in 9 genera was collected and identified during 130 trap-nights between August, 1997, and July, 1998. The most abundant species was Culex salinarius (n:20,954) followed by Ochlerotatus tae- niorhynchus (n : 12,185). Eastern equine encephalomyelitis virus (EEE) was isolated from 2 pools collected in August, 1997; one pool of Oc. taeniorhyncfta.s(minimum infection rate [MIR] - 0.6/1,000) and a second of Culiseta melanura (MIR - 3.8/1,000). This report represents the lirst record of an EEE isolation from Oc. taeniorhynchus and Cs. melanura in South Carolina. KEY WORDS Eastern equine encephalitis, surveillance, epidemiology, mosquitoes, horses Knowledge of the natural history and epidemi- recorded 2 serologically confirmed human cases ology of eastern equine encephalitis in South Car- caused by EEE from South Carolina, one in 1955 olina is limited. However, cases in commercially and another in 1969. From 1970 to 1990,5 human important animals such as horses, donkeys, emus, cases were reported (Tidwell et al. 1984, Wright pheasants, and quail are reported in the state every 1993). No human cases were reported between year (Alexander and Murray 1958, Brody and Mur- 1991 and 1995. However, there was one nonfatal ray 1959, Wozniak et al. 2001). Surveillance for human case in 1996 and 2 cases (one fatal) in 1997. equine encephalitis in the USA in 1971showed that All 3 individuals were residents of counties located 52.27a (35/6'7) of all encephalitis cases caused by in the Atlantic Coastal Plain (Horry County in 1996 eastern equine encephalomyelitis virus (EEE) re- and Charleston County in 199'7) (Wozniak et al. ported in horses were from North and South Car- 2OOl). olina (Maness and Calisher 1981). A review of re- The first statewide mosquito-borne arbovirus sur- ported domestic animal and human encephalitis veillance program in South Carolina was conducted casescaused by EEE in South Carolina showed 458 between 1996 and 1998 by the South Carolina De- horse cases reported in 1955-98, with most occur- partment of Health and Environmental Control ring in the Sandhills and Coastal Plain regions of (SCDHEC). During this study, EEE was detected the state (Table 1). The largest South Carolina epi- in pools of Anopheles crucians Wiedernann, Culex zootic resulting from EEE infection was reported in erraticus (Dyar and Knab), Anopheles punctipennis 1991 and involved at least 71 equine cases, with (Say), Anopheles quadrimaculatus Say, Culiseta about 55Vo occurring in the lower coastal plain re- melanura (Coquillet), and Coquilletidia perturbans gion (Clemson University Veterinary Diagnostic (Walker) collected in Allendale, Bamberg, Beau- Centet Columbia, SC, unpublished data). ford, Charleston, Clarendon, Colleton, Dillon, Human eastern equine encephalomyelitis cases Hampton, Horry, Lee, and Sumter counties (Woz- in South Carolina are infrequent. McGowan et al. niak et al. 2001). The role of these mosquito spe- (1973) listed 3 human cases from South Czrolina cies in the epidemiology of EEE in South Carolina reported to the Centers for Disease Control and Pre- remains unclear. vention (CDC) during 1955-71, although no state The purpose of our study was to determine the records exist substantiating these cases (Tidwell et level of arbovirus activity in mosquitoes collected al. 1984). Unpublished information from the CDC at The Wedge Plantation, Charleston County, SC, between 1997 and 1998. In addition, mosquito spe- cies composition and temporal abundance were also I University of Texas Medical Branch, Center for Trop- determined. ical Diseases, Department of Pathology, Room 4.142. The Wedge Plantation is located 72 km northeast Keiller Building, 301 University Boulevard, Galveston, of Charleston. SC. at the northern end of Charleston TX 775s5-0609. County on the South Santee River, approximately 'South Carolina Department of Health and Environ- mental Conffol, Bureau of Laboratories, 8331 Parklane 8 km inlandfrom the Atlantic coast.This site con- Road. Cofumbia. SC 29223. sists of 2O2 ha of upland freshwater swamps and r Richland County Health Department, Mosquito Con- pine and hardwood forests and 4O4 ha of marsh trol Program, 2000 Hampton Street, Columbia, SC 292O4. habitat dominated by marsh elder (lva frutescens), JJ JoURNALop'rug ArurBnrcANMoseulTo Coxrnol AssocrnrroN Vol. 19. No. I Table 1. Number of reported domestic animal and human casesof easternequine encephalitis(EEE) virus in South Carolina(per county) from 1955to 1998(estimate).' County Horses Birds Emus Donkeys Dogs Human Aiken l2 2 0 0 0 I 15 Anderson I 0 0 0 0 0 1 Bamberg z 0 0 0 0 1 J Barnwell 16 0 0 0 0 0 l6 Beauford 18 0 0 0 0 2 20 Berkeley l8 J 0 0 0 0 21 Calhoun z 0 0 0 0 0 2 Charleston2 6 2 0 0 0 2 l0 Chesterfield 1 0 0 0 0 0 I Clarendon 15 I 0 0 0 0 l6 Colleton 18 0 1 0 o 0 to Darlington 5 z z 0 0 I 10 Dillon l0 0 I 0 0 0 n Dorchester 8 2 I 0 0 0 11 Florence t7 0 0 0 0 0 t7 Ceorgetown J 0 0 0 0 0 J Greenville 2 0 0 0 0 0 2 Hampton o 0 0 0 0 0 9 Horry 40 0 I I 0 2 44 Jasper 0 0 0 0 0 4 Kershaw 28 0 I 0 0 0 29 Laurens 3 0 0 0 0 0 3 Lee 6 0 I 0 0 0 7 Lexington 8 I 0 0 0 0 9 Marion l1 0 0 0 0 0 1l Marlboro 0 0 I 0 0 0 1 McCormick 1 0 0 0 0 0 I Orangeburg lo 0 I 0 0 0 11 Pickens I 0 0 0 0 0 I Richland l3 I 0 0 1 0 l5 Spartanburg I 0 0 0 0 0 I Sumter 30 I 0 0 0 0 31 Williamsburg 7 0 0 0 0 0 7 York J 0 0 o 0 0 J Unknown Co. 129 2 0 0 0 2 133 Total 458 tt 10 I I II 498 rNumbers based on Tidwell et al. (1984), wright (1993), South Crolina Department of Health and Environmental Control, and Clemson University records. 2 County where study area was located, black rush (luncus roemeriatus), broomsedge (An- study to increase sample size and to attract other dropogon virginicus), foxtail grass (Setaria spp.), mosquito species less frequently collected in the panic grass (Panicum spp.), glasswort (Lilacopsis CDC light traps. All CDC light traps were activated carolinensis), saltgrass (Distichlis spicata), salt- at dusk and mosquitoes retrieved the next morning. marsh bulrush (Scirpus robustus), sea myrtle (Bdc- Fay-Prince and gravid traps were operated both day caris halimifulia), sea oxeye (Borrichia frutescens), and night and captured mosquitoes retrieved every and smooth cordgrass (Spartina alternffiora). The 8-10 h. Trap location was based on presence or abundance of diverse mosquito habitats, site acces- absence of mosquito breeding sites, amount of veg- sibility, and previous reports of arbovirus activity etation, and accessibility to the area. Mesh bags in the area were the main criteria for selection of containing collected mosquitoes were placed in a The Wedge Plantation as our study site. styrofoam cooler containing dry ice to kill the mos- Adult female mosquitoes were collected for 2 quitoes and preserve them during transport to the consecutive days once a month from August 12, laboratory for virus testing. 1997, to JuLy 21, 1998, using 6-8 COr-baited CDC In addition, mosquito larvae and pupae were sur- miniature light traps (Sudia and Chamberlain 1962) veyed and collected using a standard l-pint mos- (Hausherr's Machine Works, Toms River, NJ). Ad- quito dipper for 2 consecutive days once a month ditionally, I Fay-Prince trap (Fay and Prince 1970) during August, October, and November, 1997, and (John W. Hock Co., Gainesville, FL) and 2 gravid March and May, 1998. Collection sites included traps (Reiter 1983) (Hausherr's Machine Works and woodland pools, ditches, and artificial containers BioQuip, Gardenia, CA) were also used during our located throughout the property. Immatures were MARCH2003 EEE Vrnus ISoLATToNSrN SourH CnnoLrNa 35 placed in mosquito breeding chambers (BioQuip), (Walker), and An. crucians complex, respectively. transported to the laboratory where they were trans- Mosquitoes were most abundant in the months of ferred to pans inside 46-cm3 mosquito cages August 1997, and April, May, and July 1998 (Fig. (BioQuip), and reared to adulthood. Adults were 1). collected 24-48 h postemergence using an insect Forty-six percent (20,847145,051) of the mosqui- aspirator (Hausherr's Machine Works), frozen, toes were placed into 399 species-specific pools placed in petri dishes, and stored at -70oC until and tested for arboviruses. Additionally, a total of identified and tested for arboviruses. 964 larvae were reared to the adult stage in the Collected mosquitoes were stored in 15- x lfi)- laboratory and 9OVoof both male and female mos- mm Petri dishes at -70"C until processed. Female quitoes tested for arboviruses. Four species were mosquitoes were identified to species using identi- collected as immatures during our study; Aedes al- fication keys of Darsie and Ward (1981).