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Am. J. Trop. Med. Hyg., 75(6), 2006, pp. 1140-1147
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES IN CULISETA MELANURA AND CULISETA MORSITANS FROM AN ENDEMIC FOCUS OF EASTERN EQUINE ENCEPHALITIS VIRUS IN NEW YORK

GOUDARZ MOLAEI*, JOANNE OLIVER, THEODORE G. ANDREADIS, PHILIP M. ARMSTRONG, AND JOHN J. HOWARD
The Connecticut Agricultural Experiment Station, New Haven, Connecticut; Arthropod-Borne Disease Program, New York State Department of Health Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, New York; Arthropod-Borne Disease Program, New York State Department of Health, Office of Public Health, Syracuse, New York


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eastern equine encephalitis (EEE) virus perpetuates in an enzootic cycle involving ornithophilic mosquito vectors, principally Culiseta melanura (Coquillett) and avian amplification hosts. To better understand the role of Cs. melanura and Culiseta morsitans (Theobald) in the epizootiology of EEE virus, we collected blood-fed mosquitoes between 31 May and 15 October 2004 at two sites associated with an EEE virus focus in central New York and identified the source of vertebrate blood by nucleotide sequencing of polymerase chain reaction (PCR) products of the cytochrome b gene. Analysis of 484 Cs. melanura and 122 Cs. morsitans revealed that 94.2% and 86.9%, respectively, acquired blood solely from avian hosts. Blood meals derived exclusively from mammals were detected in 0.8% of Cs. melanura and 1.6% of Cs. morsitans. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were also detected in 5.0% of Cs. melanura and 11.5% of Cs. morsitans. Wood thrush constituted the most common vertebrate host for Cs. melanura (23.6%) and Cs. morsitans (30.9%), followed by American robin, song sparrow, ovenbird, red-eyed vireo, and common yellowthroat. Mammalian-derived blood meals were identified as white-tailed deer, horse, domestic cat, and eastern pipistrelle bat. There were three isolations of EEE virus from Cs. melanura and one from Cs. morsitans. These results suggest that wood thrush and a few other passerine birds may play key roles in supporting EEE virus transmission in the northeast and possibly throughout the geographic range of EEE in North America. The frequency of mammalian feedings also suggests that Cs. melanura and Cs. morsitans may play a role in the transmission of EEE virus to equines, in addition to maintaining enzootic transmission among avian hosts. We report the first isolation of arboviruses from mosquito vectors concomitant with the identifications of their blood meal sources.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eastern equine encephalitis (EEE) is a highly pathogenic arthropod-borne virus that perpetuates in an enzootic cycle involving wild birds and ornithophilic mosquito vectors. In the northeastern United States, the two principal enzootic vectors are Culiseta melanura (Coquillett) and, to a lesser degree, Culiseta morsitans (Theobald).1–5 These mosquitoes are particularly efficient vectors because they feed almost exclusively on avian hosts.4,6,7 However, occasional feeding on mammals has been documented,8–11 suggesting they may potentially serve as epizootic vectors as well.

Serologic surveys of wild birds indicate that many species, particularly members of Passeriformes inhabiting fresh water swamp foci, are exposed to EEE virus.12–16 It is not known, however, which avian species are critical for virus amplification. Most studies on the host feeding patterns of Cs. melanura and Cs. morsitans have not identified specific avian hosts because they were based largely on identification of mosquito blood meals using a panel of broadly reactive antisera. Recent technological advances using polymerase chain reaction (PCR)-based methods have permitted the identification of both avian- and mammalian-derived blood meals to the species level for the first time.17,18 These methods allow us to directly estimate vector contact with different bird species, which is essential to evaluating their relative importance as potential amplification hosts of EEE virus.

The current research initiative was undertaken to identify the specific avian hosts of Cs. melanura and Cs. morsitans and to clarify the role of these hosts in the epizootiology and ecology of EEE virus. Accordingly, blood-fed mosquitoes were collected between 31 May and 15 October 2004 at two sites associated with the Toad Harbor-Big Bay Swamp (THS), a known EEE virus focus in central New York.19,20 Vertebrate blood meals were identified by sequencing PCR products of the cytochrome b gene of mitochondrial DNA.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mosquito collections. Mosquitoes were collected between 31 May and 15 October 2004 at two study sites, the swamp perimeter and the Village of Central Square, associated with the THS complex, Town of West Monroe, Oswego County, NY (Figure 1Go). These sites have been continuously monitored since 1977 as part of a research and regional EEE surveillance program (J. J. Howard, unpublished data). During the epizootics in 1990 and 1991, EEE virus was isolated from Cs. melanura and Cs. morsitans collected at both sites,20 although the virus has not been detected in either site since 1996 despite continuous surveillance (J. J. Howard, unpublished data). Both sites were used in previous local host preference studies of Culiseta.4,7


Figure 1
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  		    FIGURE 1. Location of resting shelter collection sites and avian study area at Toad Harbor–Big Bay Swamp complex, Oswego County, NY. The web site http://mapserver.maptech.com was used to create the figure.

 
Ten resting shelters (RSs) were set at each site according to the established protocol.21 The swamp perimeter site (43°16'01'' N, 76°05'24'' W) comprises a stand of mature Eastern hemlock (Tsuga canadensis) and beech (Fagus grandifolia) on a peninsula that extends east ~100 m into the swamp (Figure 1Go). The 10 shelters were positioned on the west-facing slope of the peninsula with the openings toward the swamp. The Village site (43°16'43'' N, 76°08'08'' W) is 5 km west of the swamp site in a wooded area ~100 m east of an apartment complex where a young child was exposed to EEE virus in 1971.22 Ten shelters were spaced between trees on the west edge of a plantation of white spruce (Picea glauca).

Mosquito collections and handling. Specimens were collected from the RS by chloroform anesthetization 2 consecutive days per week according to the following protocol. One to 2 mL of chloroform was placed on the box lid, the lid was fitted to the box, and the closed box laid upright (lid facing up). After closing four to five boxes, the units were opened, and the anesthetized mosquitoes were gently shaken onto a paper towel. The anesthetized blood-fed mosquitoes were transferred with forceps to a 120-mL glass bottle fitted with a plastic snap cap (Wheaton, Millville, NJ). The remaining mosquitoes were transferred into a separate 120-mL glass bottle. Each bottle was labeled by site and date. Generally, 90% or more of the specimens revived within 20 minutes of collecting from all 10 boxes. Bottles were placed in paper bags and stored in a cooler on wet ice. The collection bottles from the first day sampling of each week were held on wet ice for 24 hours until the next day’s collection were made, after which specimens were transported to the NYS Department of Health Laboratory (NYSDOH) at the College of Veterinary Medicine, Cornell University, Ithaca, NY. Mosquitoes were cold-killed, identified to species, and examined for evidence of blood using a Nikon SMZ645 dissecting microscope using keys appropriate for the collection sites.23 Non–blood-fed and gravid females and males were identified to species and enumerated. Data were recorded for date by species for the total collected from 10 shelters for the following three categories: non–blood-fed females, blood-fed and gravid females, and males. Yearly collections are expressed as the mean per shelter day (MSD; total collected/number of shelters collected per day). Only the blood-fed mosquitoes reported here were tested for viruses. Blood-fed mosquitoes were individually placed in 0.6-mL snap-capped microcentrifuge tubes and sealed with parafilm. Tubes were labeled with a numerical code representing the year and consecutive specimen number. Tubes containing Culiseta were stored in racks at –80°C until shipped on dry ice to the Connecticut Agricultural Experiment Station, New Haven, CT, for molecular analysis and virus isolation attempts.

Bird population. Bird population estimates were based on the analysis of mist-netting data from a 5-year (1986–1990) study on the avian hosts of EEE virus.16 From May through September annually, birds were captured in mist nets set in the swamp, dry woods, an overgrown orchard, hedgerows, and field-edge ecotones, all within walking distance of the NYSDOH encephalitis field station19 on the northern edge of THS (Figure 1Go). The field station (43°16'05'' N, 76°04'01'' W) is 2 km east of the perimeter resting box site. All birds were banded with US Fish and Wildlife Services bands. Data on species, age, sex, date, and location of capture were recorded by band number. Avian nomenclature followed the sixth edition of the American Ornithologists’ Union24 (AOU) and grouping of birds into orders and families followed the AOU classification.24

DNA isolation from blood-fed mosquitoes. With the aid of a dissecting microscope, mosquito abdomens were removed and reserved for blood-meal analysis. Each mosquito was dissected individually on a new microscope slide by using flame-sterilized forceps to avoid cross-contamination. DNA was isolated from the abdominal contents of blood-fed mosquitoes individually by using DNA-zol BD, (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s recommendation with some modifications.18 Briefly, individual mosquito abdomens were homogenized with heat-sealed pipette tips in 1.5-mL tubes containing DNA-zol BD solution. The homogenates were incubated at room temperature for 5–10 minutes, mixed, and centrifuged at 10,000g for 10 minutes. DNA was precipitated by adding isopropanol and 3–4 µL Poly Acryl Carrier (Molecular Research Center). The DNA pellet was washed twice with 75% ethanol, air dried briefly, reconstituted in TE buffer (10 mmol/L Tris-HCl [pH 8.0], 1 mmol/L EDTA), and stored at –20°C for further analysis.

Blood-meal analysis. Isolated DNA from the mosquito blood meals served as DNA templates in subsequent PCR reactions. PCR primers were either based on a multiple alignment of cytochrome b sequences of avian and mammalian species obtained from GenBank or previously published primer sequences.18 All DNA templates were initially screened with avian- and mammalian-specific primer pairs, and the sequences were analyzed. Avian-specific PCR primers were 5'-GAC TGT GAC AAA ATC CCN TTC CA-3' (forward) and 5'-GGT CTT CAT CTY HGG YTT ACA AGA C-3' (reverse) with amplified product size of 508 bp. PCR cycling condition included an initial reaction activation step at 95°C for 5 minutes, followed by 36 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 50 seconds, and extension at 72°C for 40 seconds. The final cycle was completed with 7 minutes of extension at 72°C. Mammalian-specific PCR primers were 5'-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3' (forward) and 5'-TGT AGT TRT CWG GGT CHC CTA-3' (reverse) with amplified product size of 772 bp. Initial PCR reaction activation step was performed at 95°C for 10 minutes followed by 36 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1.5 minutes. The final cycle was completed with 7 minutes of extension at 72°C. In a few cases, other primer pairs were additionally used to resolve ambiguous sequences.18 A Taq PCR Core Kit (Qiagen, Valencia, CA) was used for all PCR reactions according to the manufacturer’s recommendation. A 50-µL reaction volume was prepared with 3 µL template DNA, 4 µL each primer (0.1–0.5 µmol/L), 5 µL 10x QIAGEN PCR Buffer (containing 15 mmol/L MgCl2), 1 µL dNTP mix (10 mmol/L each), 0.25 µL Taq DNA Polymerase (1.25 U/reaction), and 32.75 µL water. PCR reactions were performed with the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) using previously described thermal-cycling conditions.18 PCR-amplified products were purified by using QIAquick PCR Purification Kit (Qiagen) and sequenced directly in cycle sequencing reactions by using the sequencer, 3730xl DNA Analyzer (Applied Biosystems) at the Keck Sequencing Facility, Yale University, New Haven, CT. Sequences were annotated by using ChromasPro version 1.22 (Technelysium Pty Ltd., Tewantin, Australia) and identified by comparison to the GenBank DNA sequence database (NCBI available online). The performance of the molecular-based assay was previously validated by isolating DNA from the blood of a number of known vertebrate species, subjecting them to PCR amplification and sequencing.18

Virus isolation and identification. The head and thorax of each blood-fed mosquito was processed for virus isolation by homogenizing them in 1 mL of phosphate-buffered saline containing 30% heat-inactivated rabbit serum, 0.5% gelatin, and 1x antibiotic/antimycotic by using a copper BB and vibration mill as previously described.25 Mosquito homogenates were centrifuged at 4°C for 10 minutes at 520g, and 100 µL of the supernatant was inoculated onto a monolayer of confluent Vero cells growing in minimal essential media, 5% fetal bovine serum, and 1x antibiotic/antimycotic. Cells were maintained at 37°C in 5% CO2 and examined daily for cytopathic effect from Day 3 through Day 7 after inoculation. RNA from infected cell supernatants was extracted by using a viral RNA kit (Qiagen) and screened for West Nile virus and EEE viruses by real-time RT-PCR assays.26,27

Statistical analysis. Mosquito and bird data were analyzed using SAS, Version 9.1 for windows (SAS Institute, Cary, NC). Within- and between-year captures of individual birds were tracked through the creation of two variables as described.16 Briefly, the two variables were the number of captures this year (CTY) and number of total captures (CTOT). The first time a bird was captured and banded, the value of CTY and CTOT were each 1. At each recapture during the same year, the values of each were increased by one. Thus, a bird captured four times in 1 year had CTY and CTOT values of 4. If a bird was recaptured in a year subsequent to the banding year, the CTY value was reset to 1 but the CTOT was increased by 1. The number of individuals on site for the 5-year study was based on the number of birds that were first banded during the study (i.e., where CTY and CTOT = 1) plus the first between-year capture of any bird banded in a previous year (i.e., where CTY = 1 and CTOT > 1). {chi}2 analysis for blood feeding pattern by site and species was performed online at http://www.georgetown.edu/faculty/ballc/webtools/web_chi.html. Analysis of frequencies for blood-meal source and individual bird captures for passerines also used {chi}2 with {alpha} = 0.05 (SAS version 9.1 for Windows).


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mosquito collections. Between 31 May and 15 October 2004, mosquitoes were collected twice per week for a total of 26 days from the swamp perimeter and 25 days from the Village site. Collections contained Cs. melanura, Cs. morsitans, Anopheles punctipennis (Say), Anopheles quadrimaculatus Say, and Culex territans Walker. At the swamp perimeter, the MSDs for Cs. melanura were 6.01, 1.48, and 13.46 for non–blood-fed females, blood-fed and gravid females, and males, respectively. For Cs. morsitans, the MSDs were 1.98, 0.51, and 0.96, respectively. At the Village site, the means for Cs. melanura were 1.91, 0.87, and 0.87, and for Cs. morsitans, the means were 2.34, 0.65, and 1.69, respectively. In the previous 27 years, the number of days collections were made at these sites each season has ranged between 20 and 106 for the swamp perimeter and 18 and 56 days for the Village site. The MSDs for 2004 are consistent with means calculated for previous years (J.J.H., unpublished data). There were a total of 513 blood-fed Cs. melanura and 125 Cs. morsitans in the collections. Of these, there were 307 Cs. melanura collected from the swamp site and 206 from the Village site, and for Cs. morsitans, there were 35 and 90 blood-fed specimens collected, respectively, from the two sites.

DNA analysis. Blood-meal sources were successfully identified by DNA sequencing from 484 of 513 Cs. melanura and 122 of 125 Cs. morsitans (Table 1Go). Of the 484 Cs. melanura analyzed, 456 (94.2%) contained solely avian blood, 4 (0.8%) contained mammalian blood, and 24 (5%) had both avian and mammalian blood. Of the 122 Cs. morsitans analyzed, 106 (86.9%) contained avian blood, 2 (1.6%) contained mammalian blood, and 14 (11.5%) contained both avian and mammalian blood.


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TABLE 1
Number and percentage of avian, mammalian, and mixed blood meals identified from Cs. melanura and Cs. morsitans collected at two sites associated with the Toad Harbor–Big Bay swamp in central New York
 
An analysis of 508 avian and mammalian blood-meal sources for Cs. melanura is shown in Table 2Go. We identified 52 species as avian hosts for Cs. melanura. A majority of the bloods (N = 120, 23.6%) were from wood thrush, Hylocichla mustelina, followed by American robin, Turdus migratorius (N = 46, 9.1%), song sparrow, Melospiza melodia (N = 42, 8.3%), ovenbird, Seiurus aurocapilla (N = 30, 5.9%), and red-eyed vireo, Vireo olivaceus (N = 29, 5.7%). The 52 species were members of eight avian orders, but the majority were species from the order Passeriformes, 80.8% (N = 42), followed by Anseriformes (5.8%; N = 3), Ciconiiformes (3.8%; N = 2), and 1.9% (N = 1) each of the Apodiformes, Charadriiformes, Columbiformes, Cuculiformes, and Strigiformes. Mammalian hosts of Cs. melanura were identified as white-tailed deer, Odocoileus virginianus (N = 18, 3.5%), domestic horse, Equus caballus (N = 9, 1.8%), and domestic cat, Felis catus (N = 1, 0.2%). There were no significant differences between the two collection sites in overall composition of blood meals acquired by Cs. melanura from avian, mammalian, or mixed sources ({chi}2, 2 df, P ≤ 1.0).


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TABLE 2
Number and percentage of avian and mammalian blood meals identified from Cs. melanura collected in 2004 from two sites associated with Toad Harbor–Big Bay Swamp
 
The composition of 136 avian- and mammalian-derived blood meals for Cs. morsitans is shown in Table 3Go. We identified 21 species of avian hosts for Cs. morsitans. A majority of the bloods were from wood thrushes, (N = 42, 30.9%), followed by common yellowthroats, Geothlypis trichas (N = 10, 7.4%), red-eyed vireos, (N = 9, 6.6%), and 8 each (5.9%) from song sparrows and American robins. Mammalian hosts for Cs. morsitans were white-tailed deer (N = 10, 7.4%), horse (N = 5, 3.7%), and an eastern pipistrelle bat, Pipistrellus subflavus (0.7%). There were no significant differences between the two collection sites in overall composition of blood meals acquired by Cs. morsitans from avian, mammalian, or mixed sources ({chi}2, 2 df, P ≤ 1.0).


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TABLE 3
Number and percentage of avian and mammalian blood meals identified from Cs. morsitans collected in 2004 from two sites associated with Toad Harbor Swamp
 
Avian populations. Culiseta fed on 56 species representing eight avian orders, but 60% of blood meals were derived from only six avian hosts: wood thrush (N = 162, 25.2%), American robin (N = 54, 8.4%), song sparrow (N = 50, 7.8%), red-eyed vireo (N = 38, 5.9%), ovenbird (N = 34, 5.3%), and common yellowthroat (N = 28, 4.3%).

From 1986 to 1990, there were a total of 6,906 captures of 5,296 individuals of 96 species. A majority of the species (N = 81, 84.4%) and captures (N = 5,121, 96.7%) were members of the Passeriformes. The remaining captures were from the Piciformes (N = 123, 2.3%), Apodiformes (N = 21, 0.4%), Cuculiformes (N = 13, 0.2%), Charadriiformes (N = 10, 0.2%), 4 (<0.1%) each of the Falconiformes and Galliformes, and 1 Columbiformes. The 83 species of Passeriformes were members of 20 families, but > 75% of all captures were species from five Passerine families: Parulidae (warblers; N = 1,138, 22.2%), Emberizidae (sparrows; N = 949, 18.5%), Turidae (thrushes; N = 771, 15.1%), Mimidae (mockingbirds; N = 756, 14.8%), and Tyrannidae (flycatchers; N = 324, 6.3%). There were 4,956 birds captured only once and 340 between-year recaptures. Of the 5,296 individuals captured, 4,956 were only captured once (CTY and CTOT = 1), and 340 were captured in a year after their initial capture (CTY = 1 and CTOT > 1). The remaining captures were within-year recaptures of previous banded birds. Of these 5,296 individuals, 4,846 were (91.2%) were passerines in 18 families.

We compared the proportion of host blood meals by family for the 558 Cs. melanura and Cs. morsitans that had fed on passerines with the proportion of individuals during 1986 to 1990 (Table 4Go). The proportion of mosquitoes that had fed on wood thrushes and other members of the family Turidae (42.1%; American robin, veery, hermit thrush, and Eastern bluebird) was significantly higher than would be expected based on their proportion of the netted population (cell {chi}2 = 165.52, deviation = 131.12; Table 4Go). This was also true for the number of host blood meals from the Icteridae (blackbirds), Thrupidae (tanagers), and Vireonidae (vireos), although the proportions for these families were not as great for the Turidae (Table 4Go). Families where the number of blood meals was lower than expected based on species abundance were the Mimidae (mockingbirds), Emberizidae (sparrows), Parulidae (warblers), Paridae (chickadees), and Bombycillidae (waxwings; Table 4Go). The most under-represented family was the Mimidae (cell {chi}2 = 53.97, deviation = –65.51; Table 4Go), although the gray catbird was the most frequently netted bird in both multi-year bird studies conducted on site.13,16 The second highest netted species during these studies was the song sparrow, but the Emberizidae was also significantly under-represented as a host blood-meal source (cell {chi}2 = 22.85, deviation = –48.67; Table 4Go).


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TABLE 4
Comparison by passerine families of host blood meals for 558 blood-fed Culiseta and 4,846 individual birds netted at Toad Harbor Swamp 1986–1990
 
Virus isolations. There were four isolations of EEE virus from 513 Cs. melanura and 125 Cs. morsitans blood-fed mosquitoes. Two were isolated from Cs. melanura and one from a Cs. morsitans collected at the Village site, and one was from a Cs. melanura collected at the swamp perimeter site. Infected Cs. melanura were collected on 25 August (one at each site) and 8 September (from the Village site). The infected Cs. morsitans was collected at the Village site on 14 September. Sources of blood meals from EEE virus–infected Culiseta were identified as wood thrush, song sparrow, and ovenbird. West Nile virus was also isolated from a Cs. melanura collected at the Village site on 10 August, and it had fed on a red-eyed vireo.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study provides insight into the host associations of Cs. melanura and Cs. morsitans in this region of the northeastern United States. We found that these mosquitoes feed primarily on passerine birds and focus their feeding activity on several species including wood thrush, American robin, song sparrow, ovenbird, and red-eyed vireo that could support EEE virus transmission. A moderate proportion of Cs. melanura (5.0%) and Cs. morsitans (11.5%) acquired mixed blood meals from both avian and mammalian sources, suggesting that they could also facilitate transmission of EEE virus to incidental hosts such as horses and possibly humans. For the first time, we report the isolation of arboviruses from vector mosquitoes concomitant with the identification of the wild vertebrate hosts on which they had fed.

Our findings that > 90% of blood-fed Cs. melanura acquired blood meals from avian hosts and that 80% of blood meals were derived from passerines are consistent with the results of other studies that have used precipitin testing,6–10 polyclonal antibodies,28 or PCR and PCR–heteroduplex reactions.17,29,30 Our PCR-based method took advantage of the conservation and diversity of the cytochrome b gene of the mitochondrial sequences as a useful marker in identifying the source of vertebrate blood from mosquitoes to the species level. While this method offers clear advantages over serologic identification of blood meals, a small percentage of the avian samples did not exactly match any of the DNA sequences currently available in the GenBank database. Evidence also suggests that a single mosquito could have acquired blood meals from two avian or two mammalian species; however, further modifications of the technique may be required to determine the source of blood meals to the species level in the aforementioned categories. The amount of blood acquired by mosquitoes, the time between capturing mosquitoes and processing for blood meal analysis, quality of isolated DNA, availability of the species-specific cytochrome b gene sequences in the database, the degrees of sequence homology among the vertebrate hosts, particularly bird species present in the study area, and the possibility of mixed blood meals either from two avian or mammalian species are among the factors that contribute to successful identification of the blood-meal source.

Our results also confirm the ornithophilic blood feeding pattern of Cs. morsitans.4,10 We found that Cs. melanura or Cs. morsitans fed on 56 avian species representing eight orders, and whereas Passeriformes comprised > 80% of all avian blood meals, the wood thrush represented 24% of the total blood meals and 27% of the blood meals from passerines. The predominance of wood thrushes as a host suggests a specific host preference rather than opportunistic feeding based on avian species abundance.9,29 Virtually all avian host species identified as blood-meal sources have been reported from THS based on mist netting records13,16 or direct observation.19 However, the proportion of mosquitoes that fed on wood thrushes and other members of the family Turidae (American robin, veery, hermit thrush, and Eastern bluebird) is significantly higher than would be expected based on their proportion of the netted population (Table 4Go). The under-representation of the Mimidae and Emberizidae was unexpected because gray catbirds and song sparrows are the two most frequently netted species at THS.16 Although mist-netting results are biased estimates of the avian population because they underestimate canopy species,31 they provide reasonable estimates of avian abundance at 2- to 3-m heights. Both Cs. melanura and Cs. morsitans prefer to host-seek at ground level in an open field.32 Our avian population data were from a study completed 15 years ago, but the ecology of the THS complex has remained relatively unchanged since our original description,19 because over two thirds of the area is a NYS game wildlife area. Furthermore, two long-term studies on the role of birds in the epizootiology of EEE virus have been conducted at THS.13,16 In the earlier study, over 3 years (1978–1980), there were 4,272 individuals of 93 species banded and a total of 6,292 captures.13 The species abundance for the two studies was similar, and some of the individuals banded during the first study were recaptured during the latter.16 An interesting observation between the two studies is that no scarlet tanagers (Thrupidae) were banded during 1978–1980, whereas 19 individuals were banded between 1986 and 1990, and we herein report that 3.8% of Culiseta blood meals (N = 604) were from this species.

Many species of birds support enzootic transmission of EEE virus in North America, although the relative contribution of each species is unclear. We recently reported the current total is 66 species from which EEE virus has been isolated.16 Studies have reported that certain species seem to be more important in the enzootic cycling of EEE virus and wood thrushes and other dominant species reported here are among that group. In Alabama, there were 42 isolations of EEE virus from > 3,000 birds bled, but there were more from wood thrush than any other species,12 and one half the isolations were from four species: wood thrush (N = 7), gray catbird and veery (N = 5 each), and red-eyed vireo (N = 4). In New Jersey, early season virus isolations from a gray catbird (among others) and high EEE virus prevalence rates for summer residents, including the wood thrush (59.9%) American robin (30.4%), ovenbird (37.1%), red-eyed vireo (21.0%), and scarlet tanager (11.1%), were reported as evidence of a cryptic EEE virus cycle.15 In Massachusetts, isolation of EEE virus from gray catbird and American robin has been reported,14 but the highest EEE antibody prevalence rates were found in wood thrush (26.7%) followed by swamp sparrow (24.5%), American robin (20.9%), and ovenbird (18.2%).

Overall, the blood-feeding patterns reported here for Cs. melanura and Cs. morsitans are consistent with the results of other studies in indicating the importance of these species as enzootic hosts of EEE virus. Although Cs. melanura is largely ornithophilic, infrequent feeding on mammals is indicated by nearly 6% of Culiseta tested in this study. The only study reporting exclusive mammalian feeding by Cs. melanura was based on precipitin testing of specimens collected from the Pocomoke Swamp of Maryland.33 The validity of this study, however, was later challenged because only 37% (N = 130) of the blood meals were identified, and none of the reactors were of avian origin.34 A study conducted a few years later at the same swamp reported that 12.7% (N = 1,556) of Cs. melanura blood meals were of mammalian origin.8 Recently, feeding on deer (N = 3), humans (N = 2), and a raccoon was reported for six Cs. melanura identified with non-avian hosts collected in New Jersey,17 but most reports in the northeast indicate minimal mammalian blood feeding by Cs. melanura or Cs. morsitans.4,7,10 Only three mammalian blood meals were identified from > 2,000 blood-fed Cs. melanura collected at sites in Oswego County including THS,7 and there were only 12 blood meals identified as mammalian from > 3,000 Cs. melanura collected from sites associated with two swamps in southeastern Massachusetts.10 Ours is the first report of Cs. morsitans feeding on a bat, although it was reported that both species were attracted to but did not feed on little brown bats (Myotis lucifugus) confined in lard can traps.35

Dual feeding on birds by Cs. melanura was shown in field experiments that used caged chickens. Eight percent (N = 189) of blood-fed Cs. melanura were found to have fed on blood containing both rubidium- and cesium-labeled blood.36 Mixed blood meals from mammals and birds are more frequently reported than sole mammal feedings. In the Massachusetts study, the percent of mixed meals ranged from 0.04% (N = 1,116) to 11.0% (N = 471) depending on year and site. The highest percentage was from blood-fed Cs. melanura collected from the swamp perimeter where 21 bloods were a mix of passerine and rabbit and 32 were a mix of passerine and unspecified mammal.10 However, our finding of mixed blood meals from avian and large mammalian hosts, horses and white-tailed deer, is unique and suggests that Cs. melanura or Cs. morsitans in the northeast may play a role in the transmission of EEE virus to horses. Results from a mark–recapture study37 defined the area at risk to EEE based on the dispersal of Cs. melanura and Cs. morsitans and equine cases have occurred within definable limits of Culiseta breeding swamps in central New York.20 It is more logical to infer that the reappearance of disease at the same site can be attributed to the dispersion of an infective vector species rather than the complex concept of primary and secondary or bridge vectors interacting with avian hosts establishing cycling of EEE virus that leads to equine disease.1–3 Our finding that Culiseta fed on white-tailed deer is also consistent with reports of EEE in free-living white-tailed deer in Houston County, GA,38 and Kent and Montcalm counties, MI (Michigan Department of Health, unpublished data). In both epizootics, equine EEE was also recorded from these counties.

Isolation of viruses from vector mosquitoes concurrent with host identification of mosquito blood meals has not been reported in the literature. Virus was isolated from the head and thorax of blood-fed Culiseta, indicating that they were infected, but it cannot be determined if the mosquito acquired the virus from the current host or the previous gonotrophic cycle. While laboratory studies have shown the dissemination of EEE virus in Cs. melanura within 17 hours of feeding and the presence of virus within 48–72 hours of feeding on an infectious host,3 most of the infected mosquitoes were collected from the Village during August and September when populations are dominated by older, previously fed females.4,7 However, it is noteworthy that the host blood meals for the infected mosquitoes were from bird species that may be important avian hosts of EEE virus in central New York and possibly throughout the range of EEE virus in North America.

Received March 27, 2006. Accepted for publication August 17, 2006.

Acknowledgment: We are grateful for the technical assistance of our support staff: John Sheppard, Michael Thomas, Shannon Finan, and summer research assistant, Alberto Distefano.

Financial support: Funding for this research was provided by Laboratory Capacity for Infectious Disease Cooperative Agreement U50/CCU6806-01-1 from the Centers for Disease Control and Prevention, United States Department of Agriculture (USDA) Specific Cooperative Agreement 58-6615-1-218, and USDA-administered Hatch funds CONH00768.

* Address correspondence to Goudarz Molaei, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06504. E-mail: Goudarz.Molaei{at}po.state.ct.us Back

Authors’ addresses: Goudarz Molaei, The Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, CT 06504, Telephone: 203-974-8487, Fax: 203-974-8502, E-mail: Goudarz.Molaei{at}po.state.ct.us. JoAnne Oliver, Arthropod-Borne Disease Program, New York State Department of Health, Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, Telephone: 607-253-4297, Fax: 607-253-3907, E-mail: jo59{at}cornell.edu. Theodore G. Andreadis, The Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, CT 06504, Telephone: 203-974-8510, Fax: 203-974-8502, E-mail: theodore.andreadis{at}po.state.ct.us. Philip M. Armstrong, The Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, CT 06504, Telephone: 203-974-8461, Fax: 203-974-8502, E-mail: philip.armstrong{at}po.state.ct.us. John J. Howard, Arthropod-Borne Disease Program, New York State Department of Health, Office of Public Health, 217 South Salina St., Syracuse, NY 13202, Telephone: 315-477-8157, Fax: 315-477-8581, E-mail: jjh08{at}health.state.ny.us.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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