Am. J. Trop. Med. Hyg., 75(2), 2006, pp. 346-349
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
WEST NILE VIRUS IN MOSQUITOES OF NORTHERN OHIO, 2003
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, Columbus, Ohio
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ABSTRACT
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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.
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INTRODUCTION
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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detected in 24 Ohio counties, and by the end of 2002, it could be found in all 88 counties (Ohio Department of Health, unpublished data).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 pools.
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.
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MATERIALS AND METHODS
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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 battery-powered, 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 tran-scriptase-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 analysis.
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RESULTS
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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 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).
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TABLE 1 Summary of mosquitoes collected, pooled, and assayed for WNV in Northern Ohio from 19 June to 18 August 2003
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TABLE 2 West Nile virus infection rates in female Cx. pipiens/restuans mosquitoes in Oberlin, Ohio from 19 June to 18 August 2003
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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.
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FIGURE 1. Minimum infection rate of West Nile virus in Cx. pipiens/restuans collected in gravid traps from June to August 2003.
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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).
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DISCUSSION
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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 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.
* Address correspondence to Mary C. Garvin, Department of Biology, Oberlin College, 119 Woodland St., Oberlin, OH 44074. E-mail: mary.garvin{at}oberlin.edu 
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.Garvin{at}oberlin.edu.
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ABSTRACT
INTRODUCTION
