APPENDIX II-BQ:  White, et al, “West Nile Virus in Mosquitoes of Northern Ohio, 2003,” Am. J. Trop. Med. Hyg. 75(2), 2006, pp. 346-349.


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Copyright@2006 The American Society of Tropical Medicine and Hygiene


Am. J. Trop. Med. Hyg. 75(2), 2006, pp. 346-349.




Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana; Department of Biology, Oberlin College,

Oberlin, Ohio; Department of Entomology, University of California, Davis, California; Vector-Borne Disease Program,

Ohio Department of Health, Columbus, Ohio

Abstract. From June 19, 2003 to August 18, 2003, we surveyed the mosquitoes of Oberlin, OH, for West Nile Virus

(WNV) infection using reverse transcriptase-polymerase chain reaction. A total of 12,055 mosquitoes, representing 17

species or species groups and 4 genera, were collected in gravid traps at seven sites throughout the city, with Culex

pipiens/restuans being the most abundant and showing the highest minimum infection rate (MIR) of 0.78. This represents

a decrease in WNV enzootic activity from the previous year. Both Cx. pipiens/restuans abundance and MIR increased

significantly with date. However, we found no correlation between Cx. pipiens/restuans abundance and MIR.


West Nile Virus (WNV; family Flaviviridae, genus Flavivirus)

was first detected in Ohio during the summer of 2001 in

a blue jay (Cyanocitta cristata) collected in the northeast corner

of the state. By the end of 2001, the virus was detected in

24 Ohio counties, and by the end of 2002, it could be found in

all 88 counties (Ohio Department of Health, unpublished

data). A previous study monitored the WNV activity within

mosquito populations in the northern Ohio county of Lorain

during the summers of 2001 and 2002.1 Although they reported

no WNV activity in 2001, during the summer of

2002, > 23% of the mosquito pools tested were WNV positive,

with Culex pipiens/restuans representing 90% of the positive


Despite studies of the WNV cycle in Europe, Asia, Africa,

Australia, and North America, much remains to be learned

about the ecology of the virus, especially the conditions that

lead to outbreaks, which remain fairly unpredictable.2 In light

of this, continued documentation of WNV activity is critical

for enhancing our understanding of the cycle in North

America. To better understand the WNV cycle in northern

Ohio and provide continued documentation of the rate of

WNV activity during 2003, we monitored mosquito populations

for WNV during the summer of 2003. The objective of

this study was to document mosquito species composition and

abundance, as well as the presence and minimum infection

rate (MIR) of WNV in mosquitoes during this period.


This study was conducted at seven sites within the city limits

of Oberlin in Lorain County, OH, from 19 June 2003 to 18

August 2003. Mosquitoes were collected using CDC Gravid

Traps (model 1712; J. W. Hock, Gainesville, FL) baited with

an infusion of water and grass, which was allowed to ferment

at least 10 days before trapping. Infusion or water was added

as needed. Holes were drilled in the sides of the basins, 3 in

below the top of the basin, to prevent rainwater from collecting

and obstructing mosquito entry into the trap. To prevent

larval emergence, the traps were treated monthly with Vectolex

(Valent Biosciences Corp., Libertyville, IL).

Collections were made at each of seven wooded lots

throughout the city as shown by Mans and others.1 At each

site, three traps were placed at least 50 m apart, in spots that

were free of overgrowth and adequately accessible for collection.

Traps were operated for three consecutive nights each

week. Each trap was powered by a 6-V battery, (model PS-

6200; PowerSonic Corp., San Diego, CA) running at 20

Amps/h. Traps were equipped with LCS-2 PhotoSwitches

(P/N 1.60; J. W. Hock) programmed to activate traps from

dusk to dawn. Collections were made the morning after each

three-night trapping session was complete.

Mosquitoes were aspirated from gravid traps with batterypowered,

mechanical aspirators (Hausherr’s Machine Works,

Toms River, NJ), returned to the laboratory, and placed in a

−20°C freezer for at least an hour. After freezing, the mosquitoes

were sorted to species and sex.3 Cx. pipiens and Cx.

restuans were identified as the species complex Cx. pipiens/

restuans because of difficulty of differentiating between the

two species by morphologic characteristics.

Mosquitoes were placed into pools of 50 or less. Males and

females were separated, and each trap was treated independently.

After placement into pools, mosquitoes were stored at

–70°C and shipped to the Vector-Borne Disease Program of

the Ohio Department of Health for real-time reverse transcriptase-

polymerase chain reaction (RT-PCR) testing as previously

described.1 Aedes triseriatus were stored for future

studies of LaCrosse encephalitis. MIR was expressed as the

number of pools infected per 1,000 mosquitoes tested.

To determine if temperature and rainfall contributed to

increased abundance of Cx. pipiens/restuans in this study

compared with 2002 as reported by Mans and others,1 we

conducted an independent sample t test to compare May–

August mean weekly temperature and rainfall between the 2

years. Weather data were collected at the A. J. Lewis Center

at Oberlin College. We used a Spearman rank correlation

coefficient to test for the effect of date on the percent of

positive pools, MIR, and abundance of Cx. pipiens/restuans.

To test for an effect of trap site on percent positive pools and

Cx. pipiens/restuans abundance, we used a _2 contingency



During the summer of 2003, we collected 12,055 mosquitoes

representing 17 species or species groups and 4 genera

(Table 1). The Cx. pipiens/restuans complex accounted for

* Address correspondence to Mary C. Garvin, Department of Biology,

Oberlin College, 119 Woodland St., Oberlin, OH 44074. E-mail:

Am. J. Trop. Med. Hyg., 75(2), 2006, pp. 346–349

Copyright © 2006 by The American Society of Tropical Medicine and Hygiene


11,761 (97.6%) of the mosquitoes collected. Of the 575 pools

tested, 10 were WNV positive. Nine (90%) of the pools were

comprised of female Cx. pipiens/restuans, representing 2.6%

of all female Cx. pipiens/restuans pools tested, and one was a

female Anopheles quadrimaculatus. The overall MIR for Cx.

pipiens/restuans was 0.78 (Table 2).

We found no significant difference between mean weekly

temperature (t _ 0.608, df _ 34, P _ 0.547) or rainfall (t _

−0.02, df _ 34, P _ 0.984) between 2002 and 2003.

West Nile virus was first detected in Cx. pipiens/restuans on

June 23. Percent of positive pools and MIR correlated

positively with date (r2 _ 0.82, P _ 0.007 and r2 _ 0.64,

P _ 0.044, respectively, Figure 1). All weeks, except the last

two in July, produced at least one positive pool of Cx. pipiens/

restuans. We also found a significant effect of date on

abundance of Cx. pipiens/restuans (r2 _ −0.67, P _ 0.035,

Figure 2). Cx. pipiens/restuans abundance was highest

during the week of 7 July, with 3,338 specimens collected,

and declined throughout the rest of the summer. The single

An. quadrimaculatus positive pool was collected on

28 July.

Trap location had no significant effect on the percentage of

positive pools (_2 _ 3.67, df_ 6, P _ 0.721, Table 2). Every

trap location except Site 1 had at least one positive pool,

whereas Sites 3, 4, and 7 produced two positive pools each.

Site 7 had the highest percentage of positive pools with 4.7%.

MIR at each trap location ranged from 0 at Site 1 to 2.15 at

Site 5. Sample size limitations precluded statistical analysis of

these data. We also did not find a significant association between

site and abundance of Cx. pipiens/restuans (_2_10.28,

df _ 6, P _ 0.113).


Overall, we found that only 2.6% of Cx. pipiens/restuans

pools tested in 2003 were positive for WNV, a reduction from

the 34% reported at this site in 2002.1 This reduction is remarkable

given a > 7-fold increase in the number of Cx. pipiens/

restuans captured and tested. The increased abundance of

this species group is likely caused by the increased trapping

effort given that neither mean weekly temperature nor rainfall

varied between 2002 and 2003. However, similar to the

2002 study, this species group comprised 90% of the total

WNV-positive pools and was the most abundant mosquito

collected. This decreased virus activity reflects trends found

FIGURE 2. Abundance of Cx. pipiens/restuans collected in gravid

traps from June to August 2003.


Summary of mosquitoes collected, pooled, and assayed for WNV in

Northern Ohio from 19 June to 18 August 2003


No. pools tested

(no. mosquitoes tested)

No. female

positive pools (%)

Total no.

mosquitoes Female Male

Ae. albopictus 1 (1) 0 (0) 0 1

Ae. cinereus 1 (1) 0 (0) 0 1

Ae. vexans 30 (53) 2 (2) 0 55

An. barberi 1 (1) 0 (0) 0 1

An. punctipennis

9 (9) 12 (14) 0 23

An. quadrimaculatus

13 (16) 8 (21) 1 (7.7%) 37

Cx. pipiens/

restuans 344 (11,591) 61 (170) 9 (2.6%) 11,761

Ae. canadensis 6 (6) 0 (0) 0 6

Ae. grossbecki 3 (3) 0 (0) 0 3

Ae. japonicus 1 (1) 0 (0) 0 1

Ae. sticticus 1 (1) 0 (0) 0 1

Ae. stimulans 5 (10) 0 (0) 0 10

Ae. triseriatus 60 (132) 1 (2) 0 134

Ae. trivittatus 12 (17) 0 (0) 0 17

Ae. spp. 2 (2) 0 (0) 0 2

Ae. spp. 1 (1) 0 (0) 0 1

Ps. ferox 1 (1) 0 (0) 0 1

Total 491 (11,846) 84 (209) 10 (2.04%) 12,055


West Nile virus infection rates in female Cx. pipiens/restuans mosquitoes

in Oberlin, Ohio from 19 June to 18 August 2003



positive pools

(total pools tested)



tested MIR

1 0 (32) 657 0

2 1 (75) 3,327 0.30

3 2 (61) 2,249 0.89

4 2 (54) 1,851 1.08

5 1 (30) 465 2.15

6 1 (51) 1,766 0.57

7 2 (41) 1,276 1.57

Total 9 (344) 11,591 0.78

FIGURE 1. Minimum infection rate of West Nile virus in Cx. pipiens/

restuans collected in gravid traps from June to August 2003.


throughout Ohio in 2003. Between 2002 and 2003, human

cases declined from 430 to 170 and horse cases from 644 to

106. Moreover, the percentage of positive live birds declined

from 17.4 to 5.5% during the 2-year period (Ohio Department

of Health, unpublished data). The endemic European

cycle of WNV seems to follow a similar pattern, whereby

outbreaks with 10 or more human cases are usually followed

by few cases in the next 2 consecutive years despite increased

surveillance.4 Acquired immunity of birds may be the most

reasonable explanation for the observed decline in WNV activity

in Cx. pipiens/restuans.5 Birds that survived initial infection

with WNV in the summer of 2002 may have developed

permanent immunity, precluding their serving as reservoir

hosts in the summer of 2003. In addition, because immunocompetence

is a heritable trait,6 offspring of immune birds

may have developed resistance to WNV. Furthermore, infection

may have resulted in increased mortality in the reservoir

host population given the mortality observed in captive blue

jays and crows.5,7 The combined effect of these factors could

have resulted in a reduction of the reservoir capacity of avian

populations and, therefore, less transmission than in the previous

year. Given that neither temperature nor rainfall varied

significantly between 2002 and 2003, we do not believe that

weather conditions contributed to the decreased activity observed

in 2003.

Although, because of the trapping methods used, we were

unable to capture and test mammalophilic mosquito species

that could serve as bridge vectors, others have speculated

about the possibility of members of the Cx. pipiens complex

serving this role given reports of nonavian feeding in both Cx.

pipiens and Cx. restuans.8–10 Even if only a small fraction of

the Cx. pipiens in our study area take blood meals from mammals,

the role of this species may be significant in the epizootic

cycle because of its relative abundance and vector competence.

11 Cx. restuans is less likely to be a major epidemic

vector because its early summer population peak does not

correspond to the peak activity in the epizootic cycle in the

late summer.12,13 However, given its ornithophilic feeding behavior,

it may have played a major role in amplification of the

virus early in the summer.

Abundance of Cx. pipiens/restuans did not positively correlate

with MIR. However, similar to the 2002 study, we

found a positive correlation between both the percentage of

positive pools and MIR and date.1 The late summer rise in

WNV activity in 2002 may have been the result of seasonal

increase in abundance of Cx. pipiens relative to Cx. restuans,

the more efficient of the two vector species.1,14,15 Environmental

conditions also may have played a role; ambient temperature

has been shown to influence the vector competence

of Cx. pipiens in the laboratory,16 and rainfall may have influenced

availability of breeding habitat. In addition, avian

demography may have played a role in the transmission dynamics

of the late summer. If many of the adult birds in our

study area had developed antibody-based immunity to WNV,

but did not transfer this immunity to offspring, we would

expect relatively more amplification in birds late in the summer

because of the abundance of immunonaive juveniles during

that time.17 These factors should be considered in future

attempts to study WNV transmission in nature.

Received April 27, 2005. Accepted for publication April 7, 2006.

Acknowledgments: The authors thank Richard Gary, Robert

Restifo, Steven Chordas (Zoonotic and Vector-borne Disease Program,

Ohio Department of Health), Scott Pozna, and Kenneth Pierce

(Lorain County General Health District) for logistical support of this

project. We also thank John Petersen (Oberlin College) for access to,

and assistance with, weather data.

Financial support: This work was supported by a grant from the

Mellon Foundation to Oberlin College and the Vector-Borne Disease

Program, Ohio Department of Health.

Authors’ addresses: Bradley J. White, Center for Tropical Disease

Research and Training, University of Notre Dame, 107 Galvin Life

Sciences Building, Notre Dame, IN 46556. David R. Andrew and

Mary C. Garvin, Department of Biology, Oberlin College, 117 Woodland

Street, Oberlin, OH 44074. Nicole Z. Mans, Department of Entomology,

University of California Davis, One Shields Avenue,

Davis, CA 95616. Ojimadu A. Ohajuruka, Vector-Borne Disease

Program, Ohio Department of Health, 900 Freeway Dr. N., Columbus,

OH 43229.

Reprint requests: Mary Garvin, Department of Biology, Oberlin College,

119 Woodland Street, Oberlin, OH 44074. E-mail: Mary


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