INTRODUCTION
In 1997, a new Mosquito Management and Virus Testing Program for
eastern equine encephalitis (EEE) was established in the State
of Connecticut. The impetus for this program stemmed from the
potentially serious crisis in September of 1996, when an unprecedented
number of EEE isolations were made from human-biting mosquitoes
in southeastern Connecticut (Andreadis et al., 1998). The new
program involves The Connecticut Agricultural Experiment Station
which is responsible for trapping, identifying and testing mosquitoes
for arboviruses, the Department of Environmental Protection which
is responsible for monitoring mosquito breeding sites and providing
technical assistance to municipalities regarding mosquito control,
and the Department of Public Health which handles all public health
issues. The program is health based and focuses on preventive
efforts and mosquito monitoring for early detection of EEE. The
results of the surveillance aspect of this program for mosquito-borne
viruses are reported herein.
MATERIALS AND METHODS
Mosquito Collection and Identification Techniques. Thirty-seven locations throughout the state of Connecticut (Fig. 1) were selected to trap mosquitoes for virus testing. These sites included: (1) locations where EEE-infected mosquitoes were found in 1996, (2) locations where recent EEE-related horse deaths had occurred (Andreadis 1993, Andreadis et al. 1994) (3) red maple/white cedar swamps that support populations of Culiseta melanura. Mosquito trapping was conducted from June 9 through October 16 with CO2-baited CDC miniature light traps. Traps were set once every 10 days at each location. Mosquitoes were transported live to the laboratory where they were immediately frozen on dry ice and then identified microscopically on a chill table using the keys of Carpenter and LaCasse (1955), Darsie and Ward (1981) and Means (1979, 1987). Specimens were pooled by species, site, and collection date. The number of mosquitoes per pool was 50.
Virus Assays. The virus testing was conducted at the Yale Arbovirus Research Laboratory. Each frozen mosquito pool was homogenized in phosphate buffered saline containing 0.5% gelatin, 30% rabbit serum, antibiotic, and antimycotic. The homogenate was centrifuged for 10 min at 520 g to clear the mixture of mosquito debris. A 0.1-ml aliquot of each supernatant then was inoculated into a 25-cm2 flask containing a monolayer of Vero cells and incubated at 37oC in 5% CO2 for up to 7 d (Tesh et al. 1992). One uninoculated flask was kept as a negative control. The remainder of the supernatant was stored at -70oC.
Flasks were examined daily for cytopathic effect. If cytopathic
effect was noted, the cells were scraped from the flask and a
cell lysate antigen was prepared (Ansari et al. 1993). Isolates
were identified by enzyme immunoassy using reference antibodies
that were prepared in mice. These included: Cache Valley, EEE,
Highlands J (HJ), Jamestown Canyon (JC), La Crosse, and St. Louis
encephalitis virus antibodies. Positive control cell lysates were
run at least daily. Highlands J and EEE antibodies crossreact
in the enzyme immunoassy, but were distinguishable on the basis
of titer and speed of color development. Identity of selected
HJ and EEE isolates was confirmed by plaque reduction neutralization
test in Vero cells.
RESULTS AND DISCUSSION
The mosquito collection and virus isolation data are shown in Tables 1 and 2. A total of 44,556 female mosquitoes representing 25 species in 7 genera were collected from the field and assayed for arboviruses. The most abundant species were Coquillettidia perturbans and Cs. melanura. Thirteen species of Aedes were collected, among which, Aedes canadensis, Aedes cinereus, Aedes abserratus and Aedes vexans were the most common. Culex restuans was the most abundant Culex species. Uranotaenia sapphirina also was relatively abundant and comparatively few Culiseta morsitans were trapped.
Two isolations of EEE from 2 mosquito species (Cs. melanura and Cs. morsitans), 22 isolations of HJ from 6 mosquito species (Ae. triseriatus, Ae. vexans (2), , Cs. melanura (15), Cx. pipiens (2); and Ur. sapphirina) and 7 isolations of JC virus from 5 mosquito species (Ae. abserratus (2), Ae. canadensis (2), Ae. triseriatus, An. punctipennis and Cq. perturbans) were made. There were no isolations of Cache Valley, La Crosse, or St. Louis encephalitis. The two EEE isolations were obtained from the same location in Stonington. The isolations of HJ were more extensive but with one exception (Cromwell), they were generally confined to mosquitoes collected from the southeastern corner of the state. In contrast, the JC isolates were widely distributed, occurring in mosquitoes trapped from 5 of 8 counties (Fairfield, Litchfield, Middlesex, New London and Tolland).
The first EEE isolation was made on September 9 from a pool of Cs. melanura mosquitoes that were collected behind the Stonington High School. In response to this, trapping frequency was increased in the affected region and additional traps were placed in nearby locations within the town of Stonington (West Vine Street School, Osbrook Point, Industrial Park). A precautionary warning was issued for citizens in the affected area to avoid mosquitoes with personal protective measures (Phase One of Contingency Plan). One additional EEE isolation was made at the same site from another pool of Cs. morsitans mosquitoes trapped on September 29, but no further EEE isolations were made from either bird or human-biting mosquitoes that were collected in traps set in other nearby locations.
On October 20, we were notified of the death of 3 emu birds due
to infection with EEE. These highly susceptible birds were penned
at the Waterford Country School in Quaker Hill and were afflicted
from September 27 to October 1. This location was approximately
6.3 miles north of our trap site in Great Neck, Waterford. Upon
visiting this area on November 4, we found a large swamp that
was adjacent to the school where we have arranged to trap mosquitoes
next season. We also visited Deep Hollow Emu Farm in Oakdale,
which was only 3 miles from the school and were informed that
none of the birds had shown any symptoms of EEE. These observations
reinforce the highly focal nature of EEE which can be limited
to mosquitoes and birds in a single swamp, and demonstrate our
need for continued trapping and testing of mosquitoes in high
risk areas.
ACKNOWLEDGMENTS
I wish to acknowledge the assistance of J. Shepard, C. Moser,
S. Cantu, J. R. Dubicki, D. Dunaj, R. Ferrucci, J. Taneja-de Bruyne
(Connecticut Agricultural Experiment Station); S. Tirrell-Peck
(Yale Arbovirus Research Unit); David Florin (U.S. Navy); P. Capotosto,
W. Bradshaw, D. Shaw, F. Shaw (Connecticut Department of Environmental
Protection) who participated in various aspects of the surveillance
program.
REFERENCES
Andreadis, T. G. 1993. Epidemiology of eastern equine encephalitis in Connecticut. J. Fla. Mosq. Control Assoc. 64: 97-103.
Andreadis, T. G., J. F. Anderson & S. J. Tirrell-Peck. 1998. Multiple isolations of eastern equine encephalitis and Highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut. J. Med. Entomol. 35:296-302.
Andreadis, T. G., P. M. Capotosto, R. E. Shope, and S. J. Tirrell. 1994. Mosquito and arbovirus surveillance in Connecticut, 1991-1992. J. Am. Mosq. Control Assoc. 10: 556-564.
Ansari, M. Z., R. E. Shope, and S. Malik. 1993. Evaluation of Vero cell lysate antigen for ELISA of flaviviruses. J. Clin. Lab. Anal. 7: 230-237.
Carpenter, S. J., and W. J. LaCasse. 1955. Mosquitoes of North America (North of Mexico). University of California Press, Berkeley.
Darsie, R. F., Jr., and R. A. Ward. 1981. Identification and geographic distribution of mosquitoes of North America, north of Mexico. Mosq. Syst. Suppl. 1: 1-313.
Means, R. G. 1979. Mosquitoes of New York. Part I. The genus Aedes Meigen with identification keys to genera of Culicidae. N. Y. State Mus. Bull. 430a.
Means, R. G. 1987. Mosquitoes of New York. Part II. Genera of Culicidae other than Aedes occurring in New York. New York State Mus. Bull. 430b.
Tesh, R. B., J. Lubroth, and H. Guzman. 1992. Simulation of arbovirus overwintering: survival of Toscana virus (Bunyaviridae: Phlebovirus) in its natural sand fly vector Phlebotomus perniciosus. Am. J. Trop. Med. Hyg. 47: 574-581.
Table 1. Total number
mosquitoes trapped and tested for viruses in Connecticut, 1997
| Mosquito species | # Mosquitoes | # Pools |
| Aedes abserratus | 2,677 | 141 |
| Aedes aurifer | 631 | 59 |
| Aedes canadensis | 5,586 | 353 |
| Aedes cantator | 583 | 63 |
| Aedes cinereus | 3,163 | 289 |
| Aedes excrucians | 1,002 | 137 |
| Aedes sollicitans | 97 | 23 |
| Aedes stimulans | 114 | 20 |
| Aedes sticticus | 2 | 1 |
| Aedes taeniorhynchus | 10 | 4 |
| Aedes triseriatus | 836 | 176 |
| Aedes trivittatus | 1,368 | 62 |
| Aedes vexans | 2,306 | 172 |
| Anopheles punctipennis | 573 | 179 |
| Anopheles quadrimaculatus | 310 | 77 |
| Anopheles walkeri | 77 | 11 |
| Coquillettidia perturbans | 16,767 | 500 |
| Culex pipiens | 849 | 144 |
| Culex restuans | 1,253 | 180 |
| Culex salinarius | 66 | 9 |
| Culex territans | 12 | 9 |
| Culiseta melanura | 5,704 | 407 |
| Culiseta morsitans | 363 | 89 |
| Psorophora ferox | 174 | 22 |
| Uranotaenia sapphirina | 1,033 | 141 |
| TOTALS | 45,556 | 3,268 |
Table 2. Summary of virus isolation data, 1997
Eastern Equine Encephalitis
| Date | Species | Town | Location |
| September 9 | Culiseta melanura | Stonington | High School | |
| September 29 | Culiseta morsitans | Stonington | High School | |
Highlands J
| Date | Species | Town | Location |
| August 11 | Coquillettidia perturbans | Waterford | Great Neck | |
| September 2 | Aedes vexans | Stonington | Barn Island | |
| Culiseta melanura | Stonington | Barn Island | ||
| Culiseta melanura | Stonington | High School | ||
| September 3 | Uranotaenia sapphirina | Cromwell | Cromwell Meadows | |
| September 8 | Culiseta melanura | North Stonington | Wyassup Lake | |
| September 9 | Aedes triseriatus | North Stonington | Exit 93 | |
| Culiseta melanura | North Stonington | Exit 93 | ||
| Culiseta melanura | Stonington | High School | ||
| September 11 | Culiseta melanura | Stonington | High School | |
| Culiseta melanura | Stonington | High School | ||
| September 13 | Culiseta melanura | Stonington | Industrial Site | |
| September 15 | Culiseta melanura | Stonington | Osbrook Point | |
| September 18 | Aedes vexans | Stonington | High School | |
| Culiseta melanura | Stonington | High School | ||
| Culiseta melanura | Stonington | High School | ||
| September 22 | Culex pipiens | North Stonington | Assekonk Swamp | |
| Culiseta melanura | North Stonington | Assekonk Swamp | ||
| October 2 | Culex pipiens | Stonington | Barn Island | |
| Culiseta melanura | North Stonington | Bell Cedar Swamp | ||
| October 6 | Culiseta melanura | North Stonington | Pawcatuck River | |
| Culiseta melanura | North Stonington | Assekonk Swamp | ||
Jamestown Canyon
| Date | Species | Town | Location | Pool size |
| June 30 | Coquillettidia perturbans | Stafford | Cedar Swamp | |
| July 1 | Aedes canadensis | Ridgefield | Great Swamp | |
| Aedes canadensis | Ridgefield | Great Swamp | ||
| Aedes triseriatus | Ridgefield | Great Swamp | ||
| July 7 | Aedes abserratus | Stonington | High School | |
| July 9 | Anopheles punctipennis | Chester | Cockaponset St. Forest | |
| July 14 | Aedes abserratus | Litchfield | White Memorial | |