EASTERN EQUINE ENCEPHALOMYELITIS VIRUS ACTIVITY IN SOUTH CAROLINA1

M. A. Tidwe1l2, D. M. Forsythe2, Margaret A. Tidwe1l3, Richard L. Parker'. and A. J. Main5

Abstract: A review of South Carolina state records maintained since 1969 revealed onc case of eastern equine encephalitis (EEE) in human beings, four cases in birds, and 198 confinned cases in horses. A positive correlation was observed between the number of EEE cases and the mean arulUa! rainfall The highest incidence of reported cases was in the Sandhill and Coastal Plain Regions of the State.

During 1981, EEE hemagglutination-inhibition antibody titers of 1:20 or greater were detected in 4 of 39 sentinel chickens maintained at a study site near South Santee, Charleston County, se. Seasonal distribution data are given for mosquitoes collected during 1981 and 1982. A total of 28 species including 10 potential vectors of EEE was collected from the area. No virus was isolated.

Key words: Eastern equine encephalomyelitis virus, South Corolino, rainfall, mosquitoes, sentinel chickens, horses, humans.

J. Agric. Enoomol. 1(1): 43-52 (January 1984)

Eastern equine encephalomyelitis (EEE) virus has been reported from aU Atlantic coastal states from Maine to Florida, although little published information is available on the occurrence of this virus in South Carolina. Alexander and Murray (1958) reported 27 cases of encephalitis in South Carolina horses during 1956 with three EEE virus isolations from brain tissue. In a 1957 report summarizing arthropod-borne encephalitis sUIVeillance activities, Brody and Murray (1959) reported EEE antibodies in wild birds and feral swine from South Carolina and Georgia. No virus was isolated from mosquitoes, small mammals or fowl; however, they reported four EEE virus isolations from 59 equine cases of encephalitis in South Carolina. McGowan, et al. (1973) list three human cases of EEE from South Carolina reported to the Centers for Disease Control (CDC) during 1955-1971, although no state records exist substantiating these cases. Unpublished information from CDC recorded two serologically confirmed cases from South Carolina in 1955 and another in 1969 (Leslie D. Beadle, personal communication). Maness and Calisher (1981) reported 12 equine cases in South Carolina during 1971 and 23 additional horses with monotypic EEE antibody (i.e., in the absence of western equine encephalomyelitis antibody).

During 1981, a preliminary surveillance program was conducted to detennine whether EEE virus activity could be detected at a study site in the South Santee area of Charleston County. The results of this surveillance program 8S well as a

1 Too rellearch v..al lupported by The Citadel De\'elopment FoundlltJon grant 21130·V001, The Uni....ersity or South Carolina, Inumlltional Center for Public: Health Research, the South Carolina Depattlntnt or Health and Emironmental CA:>ntrol grant 21130·L003, and the National Inllituto or Health grant lROIAI0984. Received for publication 31 March 1983; ac:cepted 22 June 1983.

2 Vedor Biology Ruearc:h program, Department of BiolotD'. The Citadel Charlelton, SC 29409. 3 lntel'nlltional Center for Public: Health Reloll!arch. Unh·enity of South Carolina, Mc:Clellanville, SC 29458. 4 Burellu of DilJelLM ControL South Carolina Department of Health and Eovimnmenc.1 ControL Columbia. SC 29201. 6 Vale ArbovinJI Reloll!ucb Unit., Department of EpidemiolOJ)' and Public Health, V.le Univenily, 60 CoUIIge Street, P. O.

Bo. 3333. Ne. Haven, CT 06510.

43

44 J. Agric. EnromoL VoL 1, No.1 (1984)

summ8lY of state records of EEE cases in domestic fowl and equines are presented herein

REVIEW OF SOUTH CAROLINA STATE RECORDS

An inspection of the recorda from the Bureeu of Diseese Contro~ South Carolina Depertrnent of Health and Environmental Control and from the Clemson University Livestock-Poultry Health Division was made to detennine the distribution and incidence of EEE in South Carolina. ConfIrmation of EEE in equines was based on histopathology of brain tissue and!or virus isolation.

Records maintained since 1969 revealed 8 total of 198 confIrmed equine cases of EEE with reported cases every year except 1977,1978, and 1980. From 1969 to 1975, the number of cases per year ranged from a high of 47 in 1971 to 11 in 1972. In 1976, the volunteer equine encephalitis reporting system was discontinued; the numher of recorded cases decreased to six for the period 1976-1981 (Tahle 1). An outbreak in 1982 resulted in 18 recorded equine cases of EEE.

Table 1. Annual cases of eastern equine encephalitis reported in South Carolina horses from 1969-1982.

Vear Number of Cases

1969 28 1970 12 1971 47 1972 11 1973 26 1974 17 1975 33 1976' 1 1977 o 1978 o 1979 3 1980 o 1981 2 1982 18

Total 198 ·Volunteer equine enoophalitis reporting sys1cm discontinued

Seventy percent of South Carolina counties reported one or more equine cases of EEE during the 14-year period (1969-1982) with more than 50% of the counties recording three or more cases. The highest incidence of EEE was in the Sandhill and Coastal Regions of the State. In general, these areas were located east and south of a line running from Aiken County to Chestemeld County (Fig. 1). Horry and Kershaw counties reported the highest number of equine cases, 26 and 22, respectively. Sumter County ranked third with 21 cases. Recorded dates were not available for all cases; however, most occurred from June through September. During seasonal outbreaks, coincident cases could occur at widely scattered loca­tions. For example, at the beginning of the 1975 season, cases were recorded on 21, 22 and 24 June from Clarendon, Hampton and Horry counties, respectively.

45 TIDWELL et at: EEE in South Carolina

N

1

.::--"""':::---::".m

Fig. 1. Distribution of eastern equine encephalitis caees in South Carolina horses (1969-1982). "W' indicates The Wedge study site. Arrow indicates approx­imate upper margin of Sandhill Region.

It should be noted that the EEE reporting system waa based on voluntary participation and probably does not reflect tbe actual number or distribution of horse cases. For example, the year with the highest recorded prevalence was 1971, when interest in equine disease was undoubtedly increased throughout the southern U.S. 88 8 result of the Venezuelan equine encephalomyelitis outbreak in Mexico and southern Texas. In addition, the use of EEE vaccines may reduce the number of susceptible equines and render them unreliable 8S a sentinel to indicate virus activity in a region.

From 1977-1982, four cases of EEE in pen-raised quail and pheasant were confirmed by virus isolation. The birds were submitted from diseased flocks in Berkeley, Dorchester and Sumter Counties.

One non-fatal human case of EEE from Beaufort County in 1980, was comumed during the period 1970-1982. This case waa diagnosed as EEE baaed on a four­fold or greater rise in antibody titer by complement fuation and hemagglutination­inhibition (HI) tests.

46 J, Agric, Entomol. Vol. 1. No, 1 (1984)

METHODS

The arbovirus surveillance was conducted at The Wedge Plantation, Interna­tional Center for Public Health Research, University of South Carolina, at McClellanville and a nearby plantation, The Hampton, Both plantations lie in the Altantic coastal plain cs. 72 Ian NE of Charleston, SC, The Wedge with cs. 547 ha lies adjacent to the Santee River and contains a variety of habitats including salt and freshwater marshes, freshwater swamps and pine and hardwood forests. The Hampton site, 6.4 km further inland on the Santee River, contains a large freshwater swamp.

A sentinel flock of domestic chickens (Gallus gallus) located at The Wedge and live-trapped mosquitoes collected at The Wedge and The Hampton were utilized for the surveillance study. Venous blood samples were collected on 16 June, 23 July and 23 November 1981, from color-marked sentinel chickens using standard arbovirus techniques (Sudia et al. 1970), A 0,2 ml blood sample was mixed with 1.8 ml of saline solution, centrifuged at 1,000 X g for 10 min and the clot discarded The serum was stored at _12°C until serological tests could be conducted Hemagglutination-inhibition (HI) tests were conducted in microtiter plates using serial two-fold dilutions of acetone-treated sera and four to eight hemagglutination units of sucrose acetone extracted antigen (Clarke and Casals 1958). Plates con­taining the extracted sera and hemagglutinin were incubated overnight at 4°C and goose cells diluted 1:24 with appropriate pH (6,2) adjusting diluents were added,

Mosquitoes were collected on 12, 14 September and 23 October from 10 stations using New Jersey (AC and DC) light traps supplemented with dry ice and equipped with net bags to facilitate live capture, The CDC catching bags were removed prior to sunrise and held over wet ice in a cooler. Bloodfed mosquitoes were aspirated from the sentinel chicken house on 12 September and 23 November. All specimens were transported to the lab, immobilized in a freezer or with CO2,

placed on cold plates, and identified to species. The identified specimens were placed in poola of 6 to 75 females in labelled Nunc'" tubes and maintained at -70°C until tested. Each pool was triturated in a sterile mortar with alundum as an abrasive, Two ml of 20% newborn calf serum in a phosphate-buffered saline (pH 7.2) with penicillin and streptomycin was added. The suspensions were centrifuged at 1500 X g for 10-20 min, and 0,02 ml of supernatant from each pool was inoculated intracerebrally into each of eight suckling Webster Swiss mice, Tech­niques for mosquito collection and processing for virus detection were modified from those of Sudia and Chamberlain (1967). All serological testing and virus isolation studies were conducted at the Yale Arbovirus Research Unit

An additional survey was begun in March 1981 at The Wedge Plantation and adjacent South Santee area to determine species composition and relative abun­dance of mosquitoes. Three New Jersey light traps were set one night each week, and weekly samples were obtained from two Malaise traps. None of these mos­quitoes was tested for the presence of arboviruses. In addition, two tree holes, two artificial containers and three Aedes aegypti oviposition traps were monitored to detect eggs and/or larvae of Ae, aegypti (Linnaeus) and Ae, triseriatus (Say) in the area.

47 TIDWELL et aL: EEE in South ClllOlina

RESULTS

Eastern equine encephalomyelitis III antibody titers of 1:20 or greater were detected in 4 of 39 chickens sampled at The Wedge Plantation during 1981. HI titers in these birds ranged from 1:20 to 1:160 confirming infection with EEE virus. These sera also were tested for antibody to Highlands J virus and none was positive.

EEE virus was not detected in 40 pools of mosquitoes which included 53 Ae. soUicitans (Walker), 127 Ae. taeniorhynchus (Wiedemann), 26 Ae. vexam; (Meigen), 297 Anopheles bradleyilcrucians, 5 An. punctipennis (Say), 18 An. quadrimaculatus Say, 23 Coquillettidia perturbam; (Walker), 1,079 Culex salinarius CoquiUett and 195 Culiseta melanura (Coquillett). Ninety bloodfed mosquitoes including 16 An. bradleyilcrucians, 53 An. quadrimaculatus, 2 Cx. e"aticus (Dyar and Kuab), 15 Cx. Quinquefasciatus Say and 4 eX. salinarius collected from the sentinel chicken house were also negative for EEE virus.

Mosquito light and Malaise trap surveys in The Wedge/South Santee area yielded 27 species (Table 2). One additional species, Ae. triseriatus, was collected in the egg and larval stages during 1981 and 1982. Adult mosquitoes were collected every month of the year from this region. Most frequently collected were repre­sentatives of the An. bradleyVcrucians complex, ex. salinan'us, and the two salt marsh mosquitoes, Ae. taeniorhynchus and Ae. sollicitans.

DISCUSSION

The enzootic cycle of EEE virus principally involves the freshwater swamp breeding mosquito, Culiseta melanura; and wild birds. This cycle usually goes undetected until disease occurs in horses or human beings. Most host preference studies of Cs. melanura indicate that approximately 80·99% of adult females obtain bloodmeals from birds particularly passennes and only rarely from inci­dental hosts such as man, horse or chicken (Joseph and Bickley 1969, Edman et al. 1972). Therefore, it is likely that EEE virus is primarily transmitted to these hosts by other mosquito vectors (Chamberlain 1980).

Mosquito surveys at The Wedge and nearby South Santee area revealed 28 species of which at least 10 IAe. sollicuam;, Ae. triseriatus, Ae. vexam;, Cq. perturbans, ex. erraticus, ex. restuans Theobald, Cs. melanura, Orthopodomyia signifera (Coquillett), Psorophora ciliata (Fabricius), and Ps. columbiae (Dyar and Kuab)1 have been reported as potential vectors of EEE virus (Davis 1940, Chamberlain et al. 1954, Chamberlain 1958). Most of these species occur in both the Coastal Plain and Sandhill Regions of South Carolina where reported periodic outbreaks of EEE in equines most frequently have occurred. Coastal areas of South Carolina generally support high population densities of Ae. sollicitans and Ae. taeniorhynclws. These populations can be attributed, in p~ to extensive acreages of man-made impound­ments located throughout the area Crans (1977) reported that sufficient data have been accumulated to incriminate Ae. sollicitans as an epidemic vector of EEE in New Jersey.

Various studies have indicated that an abundance of rainfall apparently can be a contributing factor in epidemics and epizootics of EEE (Beadle 1959, Dalrymple et al. 1972). Hayes and Hess (1964) demonstrated that outbreaks in human beings and equines in Massachusetts and New Jersey were associated with excessive

------------- ------ ----

---------

Table 2. Species composition and relative abundance of mosquitoes from weekly light and Malaise trap collections from three sites in The Wedge/South Santee area of Charleston County, South Carolina, March 1981-Decemher 1982.

Month traps were utilized to collect mosquitoes- Total Species Jan. Feb. Mar. Apr. May Jun. JuL Aug. Sep. Oct. Nov. Dec. mosquitoes

Aedes atlanticus c. canadensis fulum paUens infirmaJus milchellae sollicitans tceniorhynchus uerans

Anopheles atropos bradleyi/ crucians punctipennis quadrimaculatus

Coquillettidia perturbans

Guier erraticus nigripalpus quinquelasciatus resluans salinariu.s terruans

443 208

5 27 "" >50 ';.

"29,068 25,126

~1,421 '" S 3 !2­

30 0 <: ~84,356 !" Z8 P

2,104 ~

~

1,093 <0

e'"149

8 14 82

26,256 29

Culiseta inomata melanura

Psorophora ciliata columbiae {eroz howardii

Uranotc.enia sapphirina

Orthopodomyia signi{era

177 1,909

27 169 177

17

10,069

10 Solid linn indicate monthl in whim 100 or more specimelUl ...-ere coUeCUd.

... '"

50 J. Agric. EntomoL Vol 1, No.1 (1984)

rainfall during the summer of the outbreaks (June-August) and the preceding autumn (August-October). While we were unable to observe an association of EEE cases and rainfall during these two 3-month periods, we did obtain a positive correlation between the mean annual rainfall (recorded from nine sites in the central and coastal portions of the State) and the number of EEE cases reported from South Carolina from 1969-1981 (Fig. 2). Generally, periods of heavy precipi­tation enhance conditions for the production of Cs. melanura, the enzootic vector, and may provide favorable conditions for other species involved in the transmission of EEE virus to man, borses and domestic birds. Although Wallis et al (1974) did not include rainfall data, they reportad that a substantial increase in the es. melanura population coincided with the onset of an epizootic of EEE in Con­necticut.. EEE virus is frequently recovered from Cs. melanura collected from freshwater swamp foci in other states. EEE virus and/or serological evidence of the virus is often encountered in wild birds from these same aress (Schaeffer et a1 1958, Dalrymple et al. 1972). Efforts to isolate EEE virus from other species of mosquitoes usually are not as successful. Generally, large numbers of mosquitoes are utilized to obtain virus isolations (Wallis et al 1958, Cbamberlain 1958). Evidence for the occurrence of EEE virus was provided by the seroconversion of birds in the sentinel flock at The Wedge. The relatively sman number of mosqui­toes (2,003) tested from tlUs area apparently precluded the isolation of virus.

50

40

IQ, 1400 i( 1\

I I \I 30I \I I I \

I0 1300 I

~

" E I

I 20 U"'

~

f w ~ 1200 I W

W

~ ec: rY' 10 2;;;

'" 1100

1.,-~~-::r--::,:"~~~:::::==~4==:""'-~c:;.J.0 1969 70 71 72 73 74 75 76 77 78 79 80 81

Fig. 2. Number of cases of eastern equine encephalitis in South Carolina horses in relation to the mean annual rainfall from nine stations in the central and coastal portions of the State (1969-198L).

TIDWELL et at: EEE in South Carolina 51

Wallis and Main (1974) speculated on the source of virus involved in recurrent outbreaks of EEE. They noted several possible methods for the overwintering of the virus in natural enzootic areas including transovanan transmission in mosqui­toes and the overwintering of virus in mosquitoes, other arthropods, and hibernating vertebrates. While any of these factors could be responsible, the maintenance of the virus in areas with wanner climates like South Carolina may he due to 8

different combination of factors and might actually be more readily accomplished in these areas. The high populations of wintering birds and the presence of potential vector mosquitoes during much of the year could perhaps contribute to the enzootic cycle of the virus in warmer climates. The coincident reappearance of EEE cases in scattered South Carolina foci provides some support that enzootic cycles are being maintained in these areas.

ACKNOWLEDGMENTS

The authors wish to express appreciation to J. Dale Hutto and Sue C. Ferguson for providing mosquito-collection data from the Richland County Health Department, to Mark Perry, Research and Statistical Services, South Carolina State Climatologist, Columbia, SC, for providing climatological data, to Ms. Ellen Peterson, Ohio Department of Health, Columbus, OH, and William P. Kelly, Memphis and Shelby County Health Department, Memphis, TN, for providing helpful information and suggestions on processing avian blood and mosquitoes for arbovirus surveillance. We would like to thank Tommy Strange, Supervisor of the Santee Coastal Reserve and Donald Fergerson, Superintendent, Hampton Plantation State Park for providing assistance and unlimited access to these parks for collecting purposes. We are especially greatful to Drs, Carl E. Boyd, Director, William T. Derieux, and Gaffney H. Blalock, Clemson University Livestock-Poultry Health Division, Elgin. SC, for providing infonnation on avian and equine cases of EEE. We would also like to thank Jack Finger and Mark Ottelin for their help in gathering and analyzing data used in this project.

REFERENCES CITED

Alexander, E. R, and W. A Murray. Jr. 1958. Arthropod·borne encephalitis in 1956. Pub. Hllh. Rep. 73: 329-339.

Beadle, L. D. 1959. Status of mosqui~bome encephalitis in the United States. PubL Hlth Rep. 74:84-90.

Brody, J. A., and W. A. Murray, Jr. 1959. Arthropod·bome encephalitis in the United States, 1957. Pub. Hlth. Rep. 74: 461-468.

Chamberlain, R. W. 1958. Vector relationships of arthropod· borne encephalitides in North Amenca. Pub. Hlth. Rep. 73: 377-379.

Chamberlain, R. W. 1980. Epidemiology of arthropod· borne togavrruses: The role of arthro­pods as hosts and vectors and of vertebrate hosts in natural transmissions cycles. Chapter 6, pp. 175-227. In R. W. Schlesinger ledl, The Togaviruses: Biology, Structure, Replication. Academic Press, New York.

Chamberlain, R. W., R K Sikes, D. B. Nelson, and W. D. Sudin. 1954. Studies on the North American arthropod· borne encephalitides. VI. Quantitative determination of virus·vector relationships. Am. J. Hyg. 60: 278-285.

Clarke, D. H, and J. Casals. 1958. Technique for hemagglutination and hemagglutination­inhibition with arthropod-borne viruses, Am. J. Trop. Med. Hyg. 7: 561-573.

Crans, W. J. 1977. The status of Aedes sollicitans as an epidemic vector of eastern equine encephalitis in New Jersey. Mosq, News 37: 85-89.

Dalrymple, J. M., O. P. Young, B. F. Eldridge, end P. K. Russell 1972. Ecology of arboviroses in a Maryland freshwater swamp m Vertebrate hosts. Am. J. EpidemioL 96: 129-140.

52 J. Agric. EntomoL VoL I, No.1 (1984)

Davis, W. A. 1940. A study of birds and mosquitoes 88 hosts for the vinle or eastern equine encephalomyelitis. Am. J. Hyg. 32: 45-59.

Edman. J. D., L. A. Webber, and H. W. Kale, Jr. 1972. Host-feeding patterns of Florida mosquitoes. n. Culiseta. J. Med Entomol. 9: 429-434.

Hayes, R 0., and A D. Hess. 1964. Climatological conditions associated with outbreaks of eastern encephalitis. Am. J. Trap. Med Hyg. 13: 851-858.

Joseph. S. a. and W. E. Bickley. 1969. Culiseta melcuwra (Coquillett) on the eastern shore of Maryland (Dipiero: Culicidae). Univ. Maryland Agr. Expt Stn. BuU. A-161, 84 pp.

McGowan. J. E., Jr., J. A Bryan, and M B. Gregg. 1973. Surveillance of arboviral encephalitis in the United Statea, 1955-1971. Am. J. Epidemiol. 97: 199-207.

Maness, K S. C., and C. H. Calisher. 1981. Eastern equine encephalitis in the United States, 1971: Past and prologue. Current Microbial 5: 311-316.

Schaeffer, M., R E. KiBsling, and R W. Chamberlain. 1958. Current views on the North American arthropod·borne virus problem. Am. J. Pub. Hlth. 48: 336-343.

Sudia, W. D., and R W. Chamberlain. 1967. Collection and processing of medically important arthropods for arbovirus isolations. USDHEW, PHS, CDC, Atlanta 29 pp.

Sudie, W. D., R. D. Lord, and R. O. Haye•. 1970. Collection and proce88ing of vert.brat.. .pecimena for arbovirus .tudiea. USDHEW, PHS. CDC, Atlanta 65 pp.

WaUl., R. C., E. 1. Jungherr, R. E. Luginbuhl, C. F. Hebnbold4 S. F. Samano, L. A. Williamson, and A. L. Lamson. 1958. Investigation of eastern equine encephalomyelitis. V. Entomologic and ecologic field studies. Am. J. Hyg. 67: 35-45.

Wallis, R. C., J. J. Howard, A. J. Main, C. Frazier, and C. G. Hayes. 1974. An increase of Culiseta melanu.ra coinciding with an epizootic of eastern equine encephalitis in Connecti­cut Mosq. News. 34: 63-65.

Wallis, R. C., and A. J. Main, Jr. 1974. Eastern equine encephalitis in Connecticut Progress and problems. Mem. Conn. Entomol Soc. pp. 117-144.