Guidelines for Arbovirus Surveillance Programs in the United States
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Enzootic transmission of EEE virus occurs regularly in freshwater swamp habitats along the Atlantic and Gulf Coasts of the U.S. Isolated foci occur in southern Michigan¹⁷⁷, Ohio, and upstate New York²⁰³ (Fig. 3-1). In Canada, EEE virus has been isolated occasionally in Ontario, Alberta, and Quebec.⁶ During periods of intense transmission, the virus is dispersed from these foci by infected mosquitoes or viremic birds. These vectors or bird hosts initiate secondary transmission cycles outside the swamp habitat during the summer or early fall, which can lead to equine or human cases. EEE virus has been recovered in most other U.S. states east of the Mississippi River, although enzootic cycles are not known in those states.²⁰²

Figure 3-1. Distribution of confirmed and presumptive cases of eastern equine encephalomyelitis in the United States, 1964-1992.

Epidemics of EEE are cyclic, with an interval between epidemics of about 9 years (Fig. 3-2). There seems to be no clear-cut relationship between epidemics and any known environmental factors. It is likely that a complex of environmental conditions must simultaneously impact on several parameters, such as vertebrate host population density, brood size and nutritional status, vector population density and longevity, and winter survival of both vectors and vertebrate hosts.

Figure 3-2. Reported cases of confirmed and presumptive human cases of eastern equine encephalomyelitis in the United States, 1964-1992.

Rainfall patterns in Massachusetts and New Jersey have been associated with occurrence of EEE cases. Rainfall more than 20 cm above the average occurring in 2 consecutive years was associated with the beginning of 2-3 year cycles of human EEE outbreaks in Massachusetts.¹⁰⁶ The years 1930-1960 were ranked according to rainfall quantity in Massachusetts. There was an association between EEE outbreaks and years in which heavy rainfall occurred in June through August, preceded by heavy rains in August through October of the previous year. This correlation could not be established for other states. Hayes and Hess¹²⁴ analyzed weather patterns in relation to outbreaks of EEE. They concluded that heavy rainfall during the summer of an outbreak, combined with above average rainfall the preceding fall, produces a favorable environment for an epidemic. An unusually wet fall is probably conducive to successful overwintering of Cs. melanura larvae, and a wet spring facilitates rapid buildup of vector populations.

Letson^d evaluated rainfall patterns in states and locales where human EEE cases occurred between 1983 and 1989. He found a significant association between the occurrence of human cases and excess rainfall in the year when cases occurred. The association was stronger with data from local weather stations than from statewide rainfall averages and the predictive model was best when applied to northern states. The sensitivity and specificity of these measures varied depending on the model used, but the positive predictive value was no more than 50% regardless of the rainfall model applied. Thus, although there appear to be significant associations between excess rainfall and epizootic EEE activity, a useful predictive model has been described only for Massachusetts.

In a retrospective analysis, the sporadic occurrence of human and equine EEE cases in certain northern states was traced by trajectory analysis to the northward movement of cold fronts carrying infected mosquitoes from more southerly locations.²⁵⁷ The validity and possible predictive value of this hypothesis remains to be proven.

A major question in the ecology of EEE is the identity of the bridging vectors that transfer the virus from the enzootic cycle to humans and equines. A variety of species serve as vectors, depending on time of year, environmental conditions, geographic location, and population dynamics.^120,254 These are discussed briefly below.

Aedes albopictus:

(Asian tiger mosquito,²⁴⁹ Forest day mosquito²⁸¹, Common names approved by the Entomological Society of America). Aedes albopictus is a  recently-introduced mosquito native to Asia.^51,273 It has spread rapidly throughout the eastern U.S^199,200. Ae. albopictus probably was introduced into the U.S. in shipments of used tires from Asia.^69,118

In 1991, 14 isolates of EEE virus were obtained from 9,350 Ae. albopictus collected in Polk County, Florida.^53,191 The significance of this observation is unknown at present. Aedes albopictus has the potential to transmit other North American arboviruses, as well.^{103,187,192,262}

The biology and behavior of Ae. albopictus is treated in detail in a recent review by Hawley.¹¹⁷ This species oviposits readily in the CDC ovitrap. Adults respond to the duplex cone trap and to the CDC light trap baited with dry ice. Landing/biting collections, with or without additional dry ice attractant, are effective. Resting females can be collected with the Nasci aspirator or other large suction device (See Appendix II).

Aedes canadensis:

(Woodland pool mosquito²⁸¹). Aedes canadensis is widely distributed in the U.S. and Canada. A subspecies, Ae. c. mathesoni, is found in the southeastern U.S. EEE virus has been isolated from this species in New York.¹³⁷

Larval habitats consist of woodland pools formed by melting snow or spring rains.⁴⁸ Larvae are most often found in pools with dead and decaying leaves on the bottom. Other larval habitats include roadside puddles, sink holes, wooded freshwater swamps, and isolated oxbows of small woodland streams. Adults of this species are abundant from March until October. There may be more than one generation per year.

Few estimates of daily survival have been attempted, but adults are said to live for several months.⁴⁸ In Newfoundland, where Ae. canadensis is univoltine, ovarian dissections confirmed the long life of this species. The gonotrophic cycle was estimated at 3 weeks, and 2-, 3-, and 4-parous females were estimated to have lived 6, 9, and 12 weeks respectively.¹⁹⁴ From these data the upper limit of daily survival can be estimated at 0.996 per day. The flight range of this species is reported to be short. Females rarely migrate far from larval habitats.⁴⁸ Ae. canadensis feeds primarily on mammals. In Maryland, 47% of bloodfed Ae. canadensis collected in the Pokomoke Cypress Swamp had fed on deer.¹⁶² Interestingly, 16% of the females had fed on reptiles.

This species is readily collected in CDC and New Jersey light traps. Landing-biting collections are also effective.

Aedes sollicitans:

(The salt marsh mosquito²⁸¹*). Ae. sollicitans has been implicated as a bridging vector of EEE in New Jersey.^62,66 It may be an important vector in other parts of its range, as well. This species is common along the Atlantic and Gulf coastal plains, extending into Texas and Oklahoma. However, isolated population foci have been reported from brackish water in states as diverse as Arizona, North Dakota and Michigan.⁷¹

In coastal sites, Ae. sollicitans is associated with salt-marsh grasses.¹³⁵ In Louisiana coastal marshes, saltgrass (Distichlis spicata) was the best predictor of Ae. sollicitans habitat.¹⁰² In North Carolina coastal dredge sites, egg laying was associated with new stands of Aster subulatus.²⁵⁵ Inland larval habitats have been associated with oil fields in various areas,⁴⁶ with sewage and high sulfate content in Michigan,⁵⁸ and with septic tank overflow plus road salt accumulation in western New York.²²

Aedes sollicitans has 5-8 broods per year in New Jersey, and breeding is continuous in more southern areas such as Texas 135. The eggs of some populations are photosensitive and enter diapause under short day conditions.²²⁵

During the day, adults rest on vegetation such as salt hay (Spartina patens) and saltgrass,^48,68 where they can be collected by vacuum aspiration. Adults are strong fliers and, during migratory flights, may fly as far as 64 km (40 mi) with wind assistance. A "large swarm" was once encountered by a ship 166 km (100 mi) east of coastal North Carolina.¹³⁵ They commonly disperse in large swarms from larval habitats in search of hosts, leaving about dusk, and may fly 5 to 10 miles in a single night. They are attracted to lights and thus to urban areas where they are a significant pest problem as well as potential vectors of EEE. Females return to marsh habitats to oviposit following the initial migratory flight. In New Jersey, parous females do not engage in repeated dispersal. They remain close to the marsh during later gonotrophic cycles, thereby concentrating potential human exposure in the marsh area.⁶⁷

Aedes sollicitans females feed almost exclusively on mammals. In Florida, 97% of Ae. sollicitans females had fed on mammals, and 3% had fed on ciconiiform birds. Of the mammal feedings, 79% were on rabbits.⁸⁹ In New Jersey, 98% of blood meals came from mammals, with slightly more than 1% of meals from birds.⁶⁸ Deer were the most frequent mammalian host. In upland areas, avian hosts were most often passerine and gallinaceous birds, while in salt marsh areas virtually all meals came from ciconiiform birds. The low rate of feeding on birds may still be sufficient to account for the importance of Ae. sollicitans as an epizootic EEE vector given the high population density of this species.⁶⁸

No direct estimates of survival appear to have been made for Ae. sollicitans. In New Jersey, 36.3%, 53.5%, 8.8% and 1.4% of females had completed 0, 1, 2 and 3 gonotrophic cycles, respectively.⁸⁷ This yields survival estimates of between 16.2% and 31.4% per gonotrophic cycle. Another study in the same area over a two-year period gave estimates of 30.4% and 50.6% survival per generation.⁸⁸ In Connecticut, a similar study found 53.9%, 37.1%, 9,0% and 0% of females had completed 0, 1, 2, and 3 cycles, leading to an estimate of 40.8% survival per gonotrophic cycle.¹⁶⁸

Aedes sollicitans is readily collected in light traps, with and without CO₂. Resting females can be collected by vacuum aspiration or with a sweep net.⁶⁸ Large numbers of host-seeking females can be collected in landing-biting collections.⁸⁷

Aedes vexans:

(The inland floodwater mosquito,¹³⁶ vexans mosquito²⁸¹*). EEE virus has been recovered from Ae. vexans in several states.²⁵⁴ It is thought to be involved in the transmission of EEE to horses and humans in Massachusetts.

Aedes vexans is found throughout the Holarctic, Oriental and Pacific regions. In the New World, it is found throughout Canada and the U.S., extending southward through Mexico to Belize and Guatemala.^154,71 Adults appear in much of the U.S. in May, and are active through September.¹³⁶ Seasonal abundance is strongly affected by rainfall and flooding. Adults may disappear during long summer droughts.¹³⁶ (For an extensive review of the biology and behavior of this mosquito, see Horsfall et al.¹³⁶).

Larvae are found in newly-flooded depressions created by river flooding, irrigation runoff, or rainfall. Specific sites include river flood plains, upland woods, wet prairies, ditches, canals and irrigated pasture.¹³⁶ Larvae usually can be found around the periphery of these habitats, particularly in the early instars.¹³⁶

Newly-emerged adults rest in shrubs and grasses at the margins of the larval habitat. Later, they can be found in vegetation (grasses, flower beds, shrubs, etc.) in and near urban centers and farm buildings, or in livestock pastures and other areas where hosts may be found.¹³⁶ Aedes vexans engages in dispersal flights from larval habitats. Depending on wind conditions, adults may fly or be carried as much as 48 km (30 mi) from emergence sites.¹³⁶ Flight activity is almost entirely crepuscular.

Aedes vexans readily bites humans, and is a major pest species in the U.S. Although primarily a mammal feeder, this species also will feed on birds.^136,260 In host preference studies in several areas of California, 60-66% of female Ae. vexans fed on mammals, with 10-13% feeding on humans.²⁴³ In a Florida study, 99.5% of blood meals were from mammals. The primary hosts were ruminants, armadillos and rabbits.⁸⁹ In a study at rural and playa lake habitats in Hale County, Texas, 95% of blood meals were from mammals, with less than one percent of meals from humans. Host abundance varied between habitats. Forage ratios for domestic mammals were 12.1 and 10.0 at rural and playa lake habitats, respectively.¹²⁶

Despite the importance and widespread abundance of Ae. vexans, daily survival has rarely been estimated for this species. Horsfall and associates estimated adult life at three weeks in summer and six weeks in spring.¹³⁶ In northern Colorado, daily survival between June and August was estimated at 0.665 by the apodeme banding method, and 0.688 by parity measurement.¹⁹³

This species is readily collected by light traps, with or without CO₂. Power aspirators can be used to collect resting adults, and host-seeking adults can be collected in landing/biting collections.

Coquillettidia perturbans:

(Irritating mosquito, salt and pepper mosquito²⁸¹). EEE virus has been isolated frequently from Cq. perturbans. This species is believed to be an important bridging vector involved in transmission of the virus to equines.²⁵⁴ In Florida, the minimum field infection rate (MFIR) for this species over a 20-year period was 1:34,980 (0.03 per 1,000).³⁰

Coquillettidia perturbans occurs throughout most of the U.S. and southern Canada. It is absent or rare in the plains and southwestern states, but extends southward into Mexico along the Gulf coast.⁷¹ This species normally has only one generation per year except in Florida, where there are two and occasionally even three generations.^48,167 In south Florida, adults of the first generation emerge in mid-March through mid-July. Those of the second generation emerge from mid-July to mid-October. In more northerly parts of the range, a single peak occurs between June and August.²

Coquillettidia perturbans larval habitats are freshwater marsh areas. The larvae attach to the submerged roots of aquatic plants by a specially adapted siphon. They are typically associated with cattails (Typha spp.), sedges (Carex spp.) and floating plants such as water hyacinth (Pistia spp.). In Florida, Cq. perturbans were found in significantly greater numbers where the bottom had a thick layer of detritus and in sites adjacent to wooded shorelines.⁴³

Adults rest on leaves of grass and other low vegetation in cool, shaded locations during the day. Males may be especially abundant in grasses and rushes near the water.¹³⁵ The adults of Cq. perturbans are strong fliers, and will move several miles from larval habitats to surrounding populated areas to seek hosts.¹³⁵ They are readily attracted to CDC and New Jersey light traps, with or without CO₂. Swarming has been observed in Florida.²²² This species readily enters houses and bites humans.¹³⁵ Biting occurs mostly at dusk, with a second peak after midnight.¹³⁵ In shaded situations, females also will bite during the day.³¹ In a Florida study, more than 90% of blooded Cq. perturbans females had fed on mammals. Most feeds were on ruminants (the most abundant hosts in the study area), while armadillos and rabbits were also well represented.⁸⁹

Culex nigripalpus:

(No common name²⁸¹). EEE virus has been isolated from Cx. nigripalpus on a number of occasions. The significance of this species in the ecology of EEE has not been clearly established.²¹⁶ In Florida, the minimum field infection rate (MFIR) for this species over a 20-year period was 1:21,150 (0.05 per 1,000).³⁰ For a discussion of the biology of Cx. nigripalpus, see Chapter 5 - Western Equine Encephalomyelitis, SLE.

Culex salinarius:

(Unbanded saltmarsh mosquito²⁸¹). EEE virus has been isolated from Cx. salinarius in Florida, Alabama, South Carolina, Maryland and New Jersey.²⁵⁴ The role of this species as an epizootic or epidemic vector is uncertain. This and several other species probably serve as vectors depending on time of year, environmental conditions, geographic location and dynamics of the vector populations.²⁵⁴

Culex salinarius occurs throughout most of the eastern United States, and is especially common along the Atlantic and Gulf Coasts. Despite its name, Cx. salinarius is not found predominantly in salt-or brackish-water habitats.¹³⁵ However, in coastal Louisiana, oviposition sites were associated with saltgrass stands.¹⁰² Larval habitats consist of semi- permanent ponds, ditches, springs, seeps, and artificial containers.¹³⁵ Freshwater impoundments in coastal areas may generate large populations of this species.²⁶⁸

Adults can be found during the day in buildings, culverts, and similar cool, shaded sites. Overwintering adults have been collected in dwellings,¹³⁵ but not in animal burrows.²⁶⁸ In New Jersey, adults begin to appear in light trap collections in May, with peak abundance in July.²⁶⁶ Activity continues late into the fall, well after other species have entered diapause. Although fall collections are virtually all nulliparous, the first collections of adult females in the spring were more than 90% parous.²⁶⁶ This could be a result of winter or early spring feeding, or a negative response to light traps before the first blood meal in overwintering females.

This species apparently engages in migratory flight, and unobstructed flights over water of 12.8 km (7.7 mi) have been reported in Delaware.¹³⁵ In Louisiana, marked females were recaptured 2 km (1.2 mi) from a release site within 26 hr after release.¹⁶⁰ The latter specimens were presumed to be engaging in host-seeking dispersal, since they were collected in CO₂ - baited light traps.

Culex salinarius is a general feeder that feeds primarily on mammals in some habitats. In a study of two Florida localities, the ratio of bird to mammal feeding was 1.3:1 at one site and 1:19 at a second site.⁹⁰ In another study, populations from Minnesota were found to have fed primarily on passerine birds, while populations from Texas fed entirely on mammals.²⁸⁴ This species feeds readily on humans mostly out-of-doors but occasionally inside buildings. Feeding is heaviest at dusk. In New Jersey, most host-seeking females were collected in the first two hours after sunset, but host-seeking activity continued through the night.²⁶⁷ Adults may be collected from diurnal resting shelters or by use of light traps. Pigeon traps have also been used to collect this species.²⁶⁷

Culiseta melanura:

(Blacktailed mosquito²⁸¹). Cs. melanura is the primary enzootic vector of EEE in the U.S. In Florida, the MFIR for this species over a 20-year period was 1:1,825 (0.55 per 1,000).³⁰ Transovarial transmission of EEE in Cs. melanura has been suspected since several workers have reported virus in males⁵⁴ or in larvae.¹²² However, later laboratory and field studies in New York,²⁰⁵ Massachusetts,¹²² and Maryland,^254,272 did not detect evidence of transovarial transmission.

This species occurs in the eastern United States from Canada to the Gulf of Mexico. It has been collected in all states east of the Mississippi River except Vermont and West Virginia. However, it is uncommon or rare throughout much of its range due to the lack of suitable larval habitats. Adult emergence begins in late May or early June in New York,²⁰⁷ and in late April in Maryland.¹⁴⁷ Emergence is somewhat earlier in more southerly states. Oviposition occurs from mid- to late June through October. There may be 2, 3, or more adult emergence peaks during the season, depending on temperature and rainfall conditions. There are two summer generations and one overwintering generation in Maryland.¹⁴⁷ Adults are most numerous during late summer and early fall and persist until October. This species overwinters in the larval stage.¹⁴⁷

Culiseta melanura larvae are most often found in heavily shaded sites associated with uprooted or decaying trees in permanent freshwater hardwood swamps.¹⁴⁷ These sites are frequently characterized by the presence of an interwoven root mat with a matrix of peaty soil.²¹⁰ Indicator tree species are red maple (Acer rubrum), swamp white oak (Quercus bicolor) and white cedar (Thuja occidentalis) in northern states;²⁰³ and with baldcypress (Taxodium distichum), sweetgum (Liquidambar styraciflua) and tupelo (Nyssa aquatica) in the southeastern U.S.^152,276 Although artificial containers do not constitute a primary habitat for this species, larvae have been found on several occasions in discarded tires.²⁵¹ Larvae also have been found in water in a concrete-lined pit in a utility tunnel ²⁷¹and in water collecting at the bottom of a resting box.²⁰⁷

Adult Cs. melanura can readily be found during the day in natural resting sites such as tree holes or fallen logs.²⁰⁷ Adults seek daytime shelter both at the swamp edge and at upland "congregating sites" where they probably gather following host-seeking flights.¹³⁸

Adult females are most active during the evening twilight period, but some activity continues throughout the night. Very little adult activity occurs during the daylight hours.²⁰⁷ Mark-release-recapture studies in New York showed that Cs. melanura females moved a mean distance of 9 km (5.6 mi) from the release site. Thus, Cs. melanura may play an active role in transporting EEE virus to upland areas.¹³⁸ This may be particularly important when parous females make up a large percentage of the dispersing population.²¹⁵

Host-seeking activity begins shortly after sunset, peaks within the first 2 hours after dark, and then continues at a relatively constant level throughout the night.²¹⁴ Culiseta melanura feeds primarily on passeriform birds, feeding uniformly at heights between 1.5 and 7.6 m.^93,206,213 Other birds, mammals and reptiles are less frequent hosts.^147,206 Humans are rarely bitten.¹²³

Little is known about survival rates of Cs. melanura. A single study in Massachusetts estimated daily survival at 0.749 to 0.814.²¹⁵ There is no apparent relationship between body size and either parity or infection with EEE virus,¹⁶⁶ as might be expected for a species with stable, nutrient-rich larval habitats.¹⁰¹

Adult Cs. melanura can be collected in both CDC and New Jersey light traps.^147,171 Adult females are also attracted to bird-baited traps, and can be collected from artificial resting shelters.¹³⁸ In one study, significantly more parous females were collected in CO₂-baited CDC light traps than in resting boxes.²⁰⁷ As with most mosquito species, blooded females are rarely collected in either regular or CO₂-baited CDC light traps.^138,210 Resting boxes collect the largest numbers of blooded females.¹⁴⁷

This species is usually very abundant in years in which EEE epizootics occur. Surveillance of Cs. melanura over a 5-year period in Connecticut, for example, noted a twelve-fold increase in the population during an EEE outbreak year.²⁹⁴

EEE virus activity is most intense in bird populations associated with fresh-water swamp forest habitats. These habitats are the foci for enzootic EEE virus transmission between bird hosts and Cs. melanura during the summer months in the northern states ^{70,98,121,177} and throughout the year in southern states.²⁷⁵

Virus or antibody have been detected in enzootic foci in many bird species, particularly passerines, although some species are more intensely involved than others. Some primary host species are the thrushes (wood, gray-cheeked, Swainson's, Hermit and Veery), catbird, cardinal, rufous-sided towhee, sparrows (song, swamp, white-throated), blue jay, vireos (red-eyed and white-eyed), Carolina wren, tufted titmouse, chickadees (Carolina and black-capped), warblers (Kentucky, black and white, yellowthroat and ovenbird), woodpeckers (downy and hairy), and flycatchers.

Once EEE virus leaves the swamp habitat via an infected mosquito or viremic bird, other bird species and equines may become involved. Some birds that regularly occur in both habitats and that could carry the virus between these habitats are the cardinal, common grackle, red-winged blackbird, American robin, song sparrow and blue jay. The post-reproductive flocking and random movement behavior of some of these species, particularly the more susceptible juvenile birds, may contribute to the dissemination of virus out of the swamp habitats. Recent studies in New Jersey indicate that the glossy ibis may function to move EEE virus out of swamp habitats. Post-reproductive ibises roost at night in the swamp forest and feed outside the swamp during the day. (Crans, W.J., Personal communication).

The wild birds that can function as amplifying hosts in mixed and agricultural habitats outside the swamps are the American robin, American goldfinch, barn swallow, house sparrow, cardinal, common grackle, starling, and red-winged blackbird.

Antibody prevalence in wild birds associated with well-established enzootic EEE foci in fresh-water swamps ranged from 6-85% in Alabama ²⁷⁵ and from 5- 80% in Maryland.⁷⁰ For most of the primary species mentioned above, antibody prevalence averaged between 30-50%. During epizootics outside these "permanent foci", similar antibody prevalence rates in local wild bird populations were observed in Massachusetts , New York,¹²² New Jersey,⁹⁸ and Michigan.¹⁷⁷ In Massachusetts and New York,²⁷⁴ the antibody prevalence in these same wild bird populations fell to <10% after 3 consecutive non-epizootic years.

Mortality from EEE virus infection occurs in wild birds in addition to the well-known mortality in ring-necked pheasants and other exotic game bird species.²⁰² The effect of this mortality on local bird populations must be considered when conducting surveillance using these species. However, some surveillance programs use captive ring-necked pheasants as sentinels and monitor the morbidity and mortality in this species as an indicator of EEE virus activity. Some examples of vertebrate species that have been used for surveillance of EEE virus activity are presented in Appendix III.

Answers to the following questions could greatly improve our understanding of and ability to predict, prevent, or control epidemic transmission of EEE. We suggest that, where possible, programs should collect data that could help to provide those answers. For additional information or assistance in designing studies of this type, consult your state health department, state vector control specialist, or contact the Division of Vector-Borne Infectious Diseases, Centers for Disease Control, Fort Collins, Colorado 80522.

• What is the overwintering mechanism of EEE virus?
• What is the relationship between weather patterns, Cs. melanura population density and EEE virus amplification patterns?
• Is there a usable relationship between degree-day accumulation and EEE virus amplification rates in the field?
• Which mosquito species are involved in epizootic transmission of EEE virus in different regions of the country?
• Which bird species are most important in EEE virus amplification?
• What is the relationship between EEE virus infection rates in the bird population and transmission of virus to mammals by bridge vectors?
• What are the most reliable predictors for human risk of EEE infection?
• Are domestic animals other than horses (e.g., goats, pigs, cattle) useful as sentinels for monitoring epizootic EEE activity?
• What impact, if any, does EEE virus have on the dynamics of endangered or protected bird species other than the whooping crane?
• What is the role of Ae. albopictus in the ecology of EEE in the southeastern U.S.?

e Common names approved by the Entomological Society of America are indicated by '*'.