APPENDIX II-BQ: White, et
This appendix is copied from:
Copyright@2006 The American Society of Tropical Medicine and Hygiene
Am. J. Trop. Med. Hyg. 75(2), 2006, pp. 346-349.
BRADLEY J. WHITE, DAVID R. ANDREW, NICOLE Z. MANS, OJIMADU A. OHAJURUKA, AND MARY C. GARVIN*
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,
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all 88 counties (Ohio Department of Health, unpublished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
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.
MATERIALS AND METHODS
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
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)
positive pools (%)
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
9 (9) 12 (14) 0 23
13 (16) 8 (21) 1 (7.7%) 37
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
(total pools tested)
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.
WNV IN NORTHERN OHIO, 2003 347
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
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,
Reprint requests: Mary Garvin, Department of Biology, Oberlin College,
119 Woodland Street, Oberlin, OH 44074. E-mail: Mary
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