Journal of the American 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 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 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 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 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 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 (), 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 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; al- fication keys of Darsie and Ward (1981). During bopictus (Skuse) (63Vo, n : 6ll), Cx. restuans identification, mosquitoes were placed in petri dish- Theobald (29Vo, n : 259), Cx. territans Walker es held on dry ice in a minicooler and viewed with (8Vo, n = 76), and Cx. salinarius (27o, n = 18). a dissecting microscope. Specimens were pooled by None of these larval pools were positive for arbo- species, trap location, and date of collection and viruses. A total of 2 adult mosquito pools, collected stored in sterile cryovials at -70'C until tested. Ap- in August 1997 using CDC miniature light traps, proximately S-lOVo of each mosquito pool was ex- were positive for the EEE virus, I pool frorn Oc. amined for parity status using the tracheation meth- taeniorhynchas (Wiedemann) (MIR : 0.6/1,000) od (Meadows 1968). and another from Culiseta melanura (Coquillet) Virus isolations and EEE and Saint Louis equine (MII{ : 3.8/1,000). Additional pools coffesponding encephalitis (SLE) antigen-capture immunoassays to these 2 species tested by AC-EIA were all neg- (AC-EIA) were performed with the species-specific ative for EEE virus. Parity determination performed mosquito pools following previously published in August 1997 for Oc. taeniorhyncy'ras specimens methods (Tsai et al. 1987, Day and Stark 1996, showed 66Vo (n = 37/56) parous and gravid fe- Wozniak et al. 2001). Mosquito pools for virus iso- males, whereas insufficient numbers of Cs. mela- lation consisted of 1-100 specimens. If 10-15 nura specimens were obtained to determine parity pools for virus isolation were obtained, additional in August samples. pools of 40-50 mosquitoes were used for AC-EIA; Mosquito temporal abundance and species com- one half of the homogenized volume of each pool position found at The Wedge Plantation are typical was used for the EEE AC-EIA and the other half for the southeastern U.S, coastal saltmarsh,/swamp for the SLE AC-EIA. habitats. Coastal areas of South Carolina generally Serological assays, in addition to Vero cell tissue support large population densities of Oc. sollicitans cultures, were perfofined primarily to save resourc- and Oc. taeniorhynchus, Cx. salinarius, and An. es and time in processing and testing the large crucians complex (Mekuria et al. 1994). In fact, amount of mosquito specimens collected during our these 4 species represented 89Vo of the total collec- study. Vero cell cultures demonstrating cythopathic tion in our study. Our data show a peak of abun- effect were confirmed by virus neutralization test- dance between April and May, mostly due to mas- ing using a panel of mouse hyperimmune ascitic sive Cx. salinarius and An. crucians complex fluids (MHIAFs) containing antibodies against emergence, and another peak between July and Au- EEE, SLE virus, encephalitis virus (CE), gust, mostly from emergent populations of Oc. tae- westem equine encephalomyelitis (WEE) virus, and niorhynchus and Oc. sollicitans. A 22-month sur- Highlands J virus (HJ). Mosquito pools testing pos- vey to determine mosquito species composition and itive by viral AC-EIA were confinned by antigen- abundance at The Wedge Plantation and adjacent capture inhibition testing using MHIAFS containing South Santee area reported 9OVo of their total col- antibodies against EEE and SLE (Tsai et al. 1987, lection contained all 4 species (Tidwell et al. 1984). Day and Stark 1996, Wozniak et al. 2O0l). Adults Tissue culture isolates of EEE virus from Oc. reared from immature stages collected in the field taeniorhynchas and Cs. melanura in August 1997 were also tested using Vero cell cultures and AC- at The Wedge Plantation represent the first isolation EIA using t}re same criteria as with the main col- of EEE from these 2 mosquito species in South lection. Finally, minimum infection rates (MIR) of Carolina. These isolations coincided with a number arbovirus-positive mosquito species were calculat- of human, horse, and bird cases reported through- ed by mosquito species and month of collection. out the state during the same year. Few studies have A total of 45,051 adult mosquitoes representing reported EEE in birds and mosquitoes in coastal 27 species in9 genera were collected and identified South Carolina. Tidwell et al. (1984) conducted between August 12, 1997, and July 2L, 1998, dur- EEE surveillance with domestic chickens and mos- ing 130 trap-nights at The Wedge Plantation. Mos- quito collections between June and November 1981 quito collections by species are shown in Table 2. at The Wedge Plantation and at a second nearby Culex salinarius Coquillett was the most common plantation. Eastern equine encephalomyelitis hem- species collected and tested, followed by Ochero- agglutination inhibition (HI) antibody titers of tatus taeniorhynchus (Wiedemann). Oc. sollicitans l:2O-1:16O were detected in 4 of 39 (lOVo) sentinel JouRNAr-oF THE AMERTCANMoseurro CoNrnol AssocrRrtoN Vor-. 19. No. I

Table 2. Adult mosquito species collected at The Wedge Plantation and tested for arboviruses, 1997-98.

Number Percent Number Number Virus isolation results Species collected of total tested of pools (MIR) 240 0.5 214 4 vexans 235 0.5 t2r 5 Anopheles cructans 2,8r2 6.2 t,913 36 quadrimaculatus 225 0.5 t62 lt Coquilletidia

perturbans 1,958 +.J r,227 Culex erraticus 442 1.0 346 8 nigripalpus 9 >0.1 5 I restuans l8 >0.1 l5 2 salinarius )o q\4 46.5 7,369 ll3 Culiseta tnornata 3 >o.l 0 0 melanura 870 1.9 719 19 r EEE(3.8/1,000) Ochlerotatus atlanticus 112 0.2 103 6 canadensis c- 36 >0.1 t7 2 fulvus pallens I >0.1 0 0 hendersoni 8 >0.1 0 0 infirmatus l16 0.3 98 4 mitchellae I >0.1 I 1 sollicitans 4 l4\ 9.2 I otn JO taeniorhynchus 12,185 27 6,120 t02 l EEE (0.6/l,000) triseriatus 6 >0.1 o 0 Orthopodomyia signifera I >0.1 0 7 >0.1 2 2 columbiae 597 I.-l 467 12 ferox 7 >0.1 5 3 howardii a >0.1 1 I Uranotaenia sapphirina 55 >0.1 22 3 Total 45,O51 100+ 20,847 399 2 EEE

chickens, indicating local EEE transmission. None The occurrence of EEE infection in domestic an- of the mosquito pools collected during their study, imals and humans along with the isolation of the including those collected inside chicken cages, test- virus fiom Cs. melanura and, Oc. taeniorhynchus ed positive for EEE in suckling mice or serological suggest that an active transmission cycle was oc- assays. The small number of mosquito samples (n curring in this region of coastal South Carolina be- : 2,003) and time of collection (mid-September to tween July and August 1997. During 1997, 15 cases late November) may have reduced the chance of of EEE infection in domestic animals (12 horses, 2 isolating the virus. Durden et al. (1997) studied the emus, and I donkey) were reported in the state. prevalence of wild bird blood parasites and EEE Seven cases occurred between July and August in and SLE in 2 southeastern coastal areas, including Colleton, Dorchester, and Charleston counties, all our study site. Although no EEE was isolated from located in the coastal region of South Carolina 218 bird sera, serum neutralizing antibodies to EEE (Clemson University Veterinary Diagnostic Center, were found rn3Vo (4/121) of the samples collected. Columbia, SC, surveillance data). Two human cas- The positive sera were from Carolina wrens (Thry- es (one fatal) were reported from Charleston Coun- othorus ludoviciantus) and northern cardinals (Car- ty during the same period. The fatal case, a 76-year- dinalis cardinalis), which are considered nonmi- old woman, occurred 5 km south of The Wedge grating permanent residents in the area. Plantation with an Ausust 7 date of onset. The non- Mrncs 2003 EEE Vnus ISoLATToNSrN Souru CenolrNe

35000 30000 fi zsooo E zoooo

:1s000 t E 10000 z 5000 HH!24 3 e H Month

Fis. 1. Seasonal distribution of adult mosouitoes collected at The Wedge Plantation, 1997-98. fatal case, an 8l-year-old man, occurred about 65 monly used pesticide in the region (Mekuria et al. km southwest of our study site with a September 1994). l7 date of onset (SCDHEC, Reportable Diseases Although our study included only 12 months of Case Reports, 1997). During our study, EEE was mosquito activity at The Wedge Plantation, it gives detected in 2 mosquito pools, I in Cs. melanura additional information about temporal distribution and another in Oc. taeniorhynchus, both collected of mosquito vector populations and arbovirus activ- on August 12-13, 1997. Arbovirus surveillance in ity in coastal South Carolina. The fact that EEE was mosquito populations at The Wedge Plantation has never detected before in populations of Cs. melan- been seldom reported. Wozniak et al. (2001) re- ura and Oc. taeniorhynchus from EEE endemic ar- ported EEE, in An. quadrimaculatus in June 1996 eas demonstrates the need for surveillance in South and in Cs. melanura in April 1998, both tested by Carolina because little data exists about vector in- virus AC-EIA. No virus isolation was attempted on volvement in the local transmission of EEE. these samples. This project was financially supported by the Culiseta melanura has been widely studied and South Carolina Bureau of Laboratories with the col- is considered the primary enzootic vector of EEE laboration of the University of South Carolina in the USA (Moore et al. 1993). Peak monthly col- School of Public Health. We thank Dr. Harold lections of Cs. melanura at The Wedge Plantation Dowda, Dr. Edward O. Oswald, and Dr. Yilma (37Vo) in 1997 and 1998 were in August 1997 and Mekuria for their support and advice during the (53Vc).In in April 1998 temperate regions, Cs. me- course of this project and Dr. Nick Karabatsos for populations 2 peaks of adult abun- lanura have or 3 his technical assistance. We are grateful to Mr. Wil- dance, typically one each during May-June, July- liam Wills, Dr. Jerome Goddard (Mississippi State August, and August-September (Scott and Weaver Department of Health), and Dr. Abelardo C. Mon- 1989). cayo (WHO Center for Tropical Diseases) for kind- Ochlerotatus taeniorhynchas is probably the ly reviewingthis anicle. most important mosquito in coastal South Car- olina. In Charleston County, 53.8-"79.3Vo of all mosquitoes collected from 1989 to l99l were Oc. REFERENCES CITED sollicitans and Oc. taeniorhynchzs, with the latter being the most numerous (Mekuria er al. 1994). Alexander ER, Munay WA Jr. 1958.-borne en- Health Rep 73:329-339. Oclerotatus taeniorhynchzs females are persistent, cephalitisin 1956.Public Brody JA, Murray WA Jr. 1959.Arthropod-borne enceph- opportunistic feeders that often migrate in large alitis in the United States,1957. Public Health Rep 74: numbers to areas where they become serious pests 461-468. (Carpenter 1974, Edman 1985). This and LaCasse Carpenter SJ, LaCasse WJ. 1974. Mosquitoes of North species is a good experimental vector of EEE and America. Los Angeles: University of California Press. it has been implicated as a bridging vector in sev- DarsieRR Ward RA. 1981.Identification and geographi- eral localities (Karabatzos 1985, Turell et al. 1994). cal distributionof the mosquitoesof , In addition, Oc. taeniorhynchus pop'tlations in North of Mexico.Mosq Systl(Suppl)1:l-313. coastal areas of Charleston and Georgetown coun- Day JR Stark LM. 1996-Eastern equine encephalitis trans- ties have shown resistance to malathion, a com- mission to emus (Dromaius novaeholandiae)in Volusia 38 JounNar- oE rse AvemcAN Moseurro CoNrnol AssocrlrroN VoL. 19, No. I

County, : 1992 through 1994. J Am Mosq Con- Moore CG, Mclean RG, Mitchell CJ, Nasci RS, Tsai TE trol Assoc 12:429-436. Calisher CH, Marfin AA, Moore CG. Gubler DJ. 1993. Durden LA, McClean RG, Oliver JH Jr, Ubico SR. James Guidelines for arbovirus surveillance prrsgrams in the AM. 1997. Ticks, Lyme disease spirochaetes, trypano- United States. Washington, DC: U.S. Department of somes, and antibodies to encephalitis viruses in wild Health and Human Services. birds from coastal Georgia and South Carolina. J Par- Reiter P 1983. A portable, battery-powered trap for col- asitol 83:-1178-1182. lecting gravid Culex mosquitoes. Mosq News 43:496- Edman JD. 1985. Blood feeding behavior. In: JK Nayar, 498. ed. Bionomics and physiology o/ Scott TW, Weaver SC. 1989. Eastern equine encephalo- and Ae. sollicitans, the saltmarsh mosquitoes of Flori- myelitis virus: epidemiology and evolution of mosquito da. Agriculture Experiment Station Technical Bulletin transmission. Adv Virus Res 37'.277-328. 852. Gainesville, FL: IFAS-University of Florida. p 95- Sudia WD, Chamberlain RW. 1962. Battery-operated light 103. trap, an improved model. Mosq News 22:126-129. Fay RW' Prince WH. 1970. A modified visual trap for Tidwell MA, Forsythe DM, Tidwell MA, Parker RL, Main . Mosq News 3O:20-23. AJ. 1984. Eastern equine encephalitis virus activity in Karabatzos, N. 1985. International catalogue of arbovi- South Carolina. J Agric Enbmol l:43-52. ruses including certain other viruses of vertebrates, 3td Tsai TE Bolin RA, Montoya M, Bailey RE, Francy DB, ed. San Antonio, TX: American Society of Tiopical Josan M, Roehrig JT, 1987. Detection of Saint Louis Medicine and Hygiene. encephalitis virus antigen in mosquitoes by capture en- Maness KS, Calisher CH. 1981. Eastem equine encepha- zyme immunoassay. J Clin Microb 25:37O-376. litis in the United States, 1971: past and prologue. Carr Turell MJ, Beaman JR, Neely JW. 1994. Experimental Microb 5:31 1-3 I 6. transmission of eastern equine encephalitis virus by McGowan JE Jr, Bryan JA, Greg MB. 1973. Surveillance strains of Aedes albopictus and A. taeniorhynchus (Dip- of arboviral encephalitis in the United States: 1955- tera: Cufcidae). J Med Entomol 3l:287-290. 1971. Am J Epidem 97:199-2O7. Wozniak A, Dowda HE, Tolson M[ Karabatsos N, Meadows KE. 1968. A simple method of mosquito ovary Vaughan DR, Turner PE, Ortiz DI, Wills W. 2001. Ar- dissection. Fla Entomol 51:31-35. bovirus surveillance in South Carolina. 1996-98. J Am Mekuria Y, Williams DC, Hyatt MG, Zack RE, Gwinn Mosq Control Assoc 17:73-78. TA. 1994. Malathion resistance in mosouitoes from Wright PA. 1993. Epidemiology of eastem equine en- Charleston and Georgetown counties of cbastal South cephalitis in South Carolina. J Fla Mosc Control Assoc Carolina. J Am Mosq Control Assoc 10:56-63. 64:110*111.