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Andreadis Et Al JME 1998 Multiple Isolations of Eastern Equine Encephalitis and Highlands J Viruses from Mosquitoes (Diptera: Culicidae) During a 1996 Epizootic in Southeastern Connecticut THEODORE G. ANDREADIS,1 JOHN F. ANDERSON,1 AND SHIRLEY J. TIRRELL-PECK2 J. Mecl. Entomol. 35(3): 296-302 (1998) ABSTRACT Thirty-six isolations of eastern equine encephalitis virus were obtained from 8 species of mosquitoes collected from 5 September through 18 October 1996 during an epizootic in south- eastern Connecticut. These included Culiseta melamira (Coquillett) (19 isolates), Culex pipiens L. (8), Culiseta morsitans (Theobald) (3), Aedes sollicitans (Walker) (2), Aedes cantator (Coquillett) (1), Aedes trivittatus (Coquillett) (1), Aedes vexans (Meigen) (1), and Coquillettidia perturbans (Walker) (1). Isolations from Ae. cantator and Ae. trivittaus are new to North American records, and those from Ae. cantator and Ae. sollicitans represent the first infections of human-biting, salt-marsh mosquitoes with eastern equine encephalitis virus in Connecticut. With one exception, eastern equine encephalitis-infected Cs. melamira were found at all sites where eastern equine encephalitis virus was isolated. The large number of eastern equine encephalitis isolations from Cs. melamira and the collection of infected mosquitoes in residential vvoodlots and coastal salt marshes away from traditional red maple or white cedar swamp habitats, reaffirm the importance of local populations of this mosquito for viral amplification and dispersal from swamp foci. Highlands J virus was more widespread geographically, but fewer isolations of this vims were made from fewer species of mosquitoes. These included Cs. melamira (8 isolates), Cx. pipiens (5), Ae. vexans (3), Aedes canadensis (Theobald) (1), Ae. cantator (1) and Cs. morsitans (1). No human or horse cases of eastern equine encephalitis were reported, although this represents the largest number of isolations for eastern equine encephalitis ever recovered from field-collected mosquitoes in Connecticut. KEY WORDS eastern equine encephalitis, Highlands J, arbovirus, isolation, mosquito, epizootic EPIZOOTICS OF EASTERN equine encephalitis have oc- lations from Culiseta melamira (Coquillett) from curred irregularly in Connecticut since 1938 (Andrea- 157,556 mosquitoes that were collected in areas with dis 1993). The majority of these outbreaks have been a history of eastern equine encephalitis activity in reported from the eastern portion of the state during domestic animals over a 10-yr period, 1969-1978. late summer and early fall (August-October). Cases of These isolations were coincident with the reporting of eastern equine encephalitis have been limited to multiple human and horse cases in southeastern Mas- horses and domestic pheasants, and most sites where sachusetts during 1970-1975 (Grady et al. 1978). More eastern equine encephalitis have typically been in recently, Andreadis et al. (1994) obtained no isola- close proximity to freshwater swamp habitats (red tions of eastern equine encephalitis from 21,347 mos- maple or white cedar) or in swamp-forest border quitoes (21 species in 7 genera) collected from areas locations. There has been no discernible periodicity, in southeastern Connecticut where confirmed horse but eastern equine encephalitis activity in Connecti- cases had occurred the previous year. cut historically has coincided with major outbreaks in In late August and early September 1996, an unusu- humans and horses in southeastern Massachusetts ally large number of eastern equine encephalitis-in- (Edman et al. 1993) and Rhode Island (Gettman fected mosquitoes was detected in and around a pre- 1993). The most recent epizootic in horses (5 cases) sumed focus at Chapman Swamp in Westerly, Rhode occurred in 1990-91 and was confined largely to Island (Markowski 1996). In response to this finding, southeastern Connecticut (Andreadis et al. 1994). we initiated an emergency mosquito arbovirus sur- Despite this activity, there have been very few iso- veillance program in adjacent regions of southeastern lations of eastern equine encephalitis virus from field- Connecticut. Our objectives were to determine the collected mosquitoes in Connecticut. Wallis et al. identity, distribution, and relative abundance of east- (1960) obtained a single isolation from Aedes vexans em equine encephalitis-infected mosquitoes in this re- (Meigen) among 11,625 mosquitoes trapped between gion. The results of this investigation are reported next. 1955-59. Main et al. (1979) subsequently made 4 iso- 1 The Connecticut Agricultural Experiment Station, 123 Hunting- Materials and Methods ton Street, P.O. Box 1106, New Haven, CT 06504. 2 Mosquito Collection and Identification Tech- Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, niques. Mosquito trapping was conducted at 80 dif- P.O. Box 208034, New Haven, CT 06520. ferent locations in 20 townships (Fig. 1) from 5 Sep- May 1998 ANDREADIS ET AL.: EEE AND HJ VIRUSES FROM CONNECTICUT MOSQUITOES 297 Health Organization Center for Arbovirus Research and Reference, Yale Arbovirus Research Unit, De- Virus Isolations partment of Epidemiology and Public Health, Yale • EEE A HJ University School of Medicine. These included: Cache o None Valley, eastern equine encephalitis, Highlands J, Jamestown Canyon, La Crosse, and St. Louis enceph- alitis virus antibodies. Positive control cell lysates were run at least daily. Highlands J and eastern equine encephalitis antibodies crossreact in the enzyme im- munoassy, but were distinguishable on the basis of titer and speed of color development. Identity of se- lected Highlands J and eastern equine encephalitis isolates was confirmed by plaque reduction neutral- ization test in Vero cells. Fig. 1. Connecticut state map showing towns where mos- Results quitoes were collected for arbovirus testing and those sites The mosquito collection and virus isolation data are where EEE and Highland J (HJ) viruses were isolated. shown in Table 1 and the specific locations of the collection sites are detailed in Figs. 1 and 2. In total, 6,440 female mosquitoes representing 16 species in 7 tember through 18 October 1996. The majority of genera were collected from the field and assayed for these sites were in the southeastern portion of the arboviruses. The most abundant mosquito species state and included mixed hardwood swamps, isolated were Cs. melamira and Culex pipiens L. Eight species woodlots in residential areas, and coastal salt marshes. of Aedes were also collected, among which, 2 saltmarsh Adult female mosquitoes were collected with dry species, Aedes cantator (Coquillett) and Aedes sollici- ice-baited CDC miniature light traps. One trap per site tans (Walker) and 2 freshwater species, Ae. vexam and per night was used, and the number of trap nights per Aedes canadensis (Theobald), were the most common. site ranged from 1 to 10 (ave. = 2), with the most Uranotaenia sapphirina (Osten Sac ken) also was rel- repetitive sampling generally occurring at those loca- atively abundant. Comparatively few Culiseta morsi- tions where eastern equine encephalitis virus isola- tans (Theobald) and Coquillettidia perturbans (Walk- tions were made. Mosquitoes were transported live to er) were trapped. the laboratory where they were immediately frozen Thirty-six isolations of eastern equine encephalitis on dry ice and then identified on a chill table using the (from 8 mosquito species) and 19 isolations of High- keys of Carpenter and LaCasse (1955), Darsie and lands J (from 6 mosquito species) were made. There Ward (1981) and Means (1979, 1987). Specimens were no isolations of Cache Valley, Jamestown Can- were pooled by species, site, and collection date. The yon, La Crosse, or St. Louis encephalitis. With the number of mosquitoes per pool was ^50 and minimum exception of a single isolation from Ae. sollicitans at a field infection rates (number of positive pools per total salt marsh on Great Island in Old Lyme, all of the number of mosquitoes tested) were calculated for eastern equine encephalitis isolations were made from each species at specific locations. mosquitoes collected in the towns of N. Stonington Rainfall and temperature data were obtained from and Stonington. Highlands J was more widespread 2 National Weather Service Climatological Stations geographically, occurring in 5 different townships. (Groton and Norwich Public Utilities Plant) that were The 1st isolations of eastern equine encephalitis located close to the collection sites (Fig. 1). were made on 8 September from Cs. melanura and Cx. Virus Assays. Each frozen mosquito pool was ho- pipiens. Virus isolations from these 2 species were mogenized in phosphate buffered saline containing made repeatedly thereafter through 30 September 0.5% gelatin, 30% rabbit serum, antibiotic, and anti- (Cs. melanura = 19, Cx. pipiens = 8). Eastern equine mycotic. The homogenate was centrifuged for 10 min encephalitis isolations from Cq. perturbans and salt- at 520 g to clear the mixture of mosquito debris. A marsh Ae. cantator and Ae. sollicitans were made in 0.1-ml aliquot of each supernatant then was inoculated mid-September (12-16th), whereas isolations from into a 25-cm2 flask containing a monolayer of Vero flood water Aedes trivittatus (Coquillett) and Ae. vex- cells and incubated at 37°C in 5% CO2 for up to 7 d ans were made 2 wks later (28 September). (Tesh et al. 1992). One uninoculated flask was kept as Site-specific MFIRs ranged from 1:1 to 1:204 and
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