Hugh D. Lothrop1, Sarah S. Wheeler1, Ying Fang, Brian Carrol1, Susanne Kluh2, William K. Reisen1.
1Center for Vectorborne Disease Research, School of Veterinary Medicine,
University of California, Davis, CA 95616, [email protected]
2greater Los Angeles County Vector Control District, 12545 Florence Ave., Santa Fe Springs, CA 90670
ABSTRACT: To detect West Nile Virus RNA expectorated by sugar feeding mosquitoes, vials baited with a 60% sucrose solution scented with 10 µl of phenylacetaldehyde and plugged with a 1 cm segment of dental wick were used at field sites in Southern California. Sampling was conducted at three to seven sites in the Coachella Valley from January 1 to September 19, and resulted in 14 positive samples from 930 sample-weeks with 12 positives occurring in August. These results overlapped, but did not match temporally, with the results from current surveillance methods of mosquito pools and sentinel chickens.
INTRODUCTION
Exploiting sugar feeding behavior for mosquito- borne disease surveillance and control is a growing area of research. Expectoration of Ross River Virus by sugar feeding mosquitoes collected by CO2-baited traps was previously documented (Hall-Mendelin et al. 2010) indicating that sugar bait stations could supplement sentinel chicken flocks to detect arbovirus transmission and require far less infrastructure and maintenance. Sampling at five sites during 2011 in the Coachella Valley resulted in 27 West Nile virus (WNV) positives from 400 samples (Lothrop et al. 2012). In 2012 the bait formula was slightly modified, and two more sampling sites were added in the area where there was no indication of Culex tarsalis Coquillett presence. Less extensive sampling was conducted in Los Angeles and Kern counties, but results were limited to a single positive from Kern; therefore, only results for the Coachella Valley are presented in this report.
MATERIALS AND METHODS
laboratory Protocols. A 1.5 ml microcentrifuge vial (VWR, Radnor, PA) was loaded with 1.2 ml of 60% sucrose solution. Thirty microliters of phenylacetaldehyde (PAA) (Alfa Aesar, Ward Hill, MA) solution (33% in denatured absolute ethanol) containing 10 µl of PAA was added, and the vial plugged with a 1 cm segment of dental wick. Caged colony Culex quinquefasciatus Say were presented with baited vials containing 0.03 g/l Tinopal® SFP
(BASF, Ludwigshafen am Rhein, Germany) dye to test for feeding inhibition due to the denatured ethanol solvent portion of the bait. To test for RNA recovery, five baited wicks were spiked with 10 µl of a 10 fold dilution series of 107.7 plaque forming units (PFU)/ml
of the NY99 strain of WNV. The lowest titer, a 50 PFU dose, was our estimate of a single infected mosquito expectoration (Colton et al. 2005, Reisen et al. 2005). Vials were held at -80 ºC until tested for WNV RNA using qRT-PCR using previously published methods (Lanciotti et al. 2000).
Field Protocols. Vials were prepared without Tinopal following the laboratory protocol and deployed at field sites inverted in holders fabricated from PVC pipe fittings fastened to 3 foot wooden stakes; five holders with vials were placed at each field site (Lothrop et al. 2012). Samples were retrieved weekly and held at -80 ºC until shipped on dry ice to the Center for Vectorborne Diseases where they were tested using qRT-PCR. To compare sampling in rural habitats dominated by Cx. tarsalis with urban habitats dominated by Cx. quinquefasciatus, the bait stations were set at four rural sites around the margin of the Salton Sea and at three sites in the urbanized upper valley where Cx. tarsalis was absent. Sampling began on January 6 at three sites on the north shore of the Salton Sea where surveillance has generally detected virus activity the earliest in the season. Sampling at a fourth site started on April 12 on the west shore and at three additional on May 16: the upper Coachella Valley, Palm Desert and Cathedral City. Sampling was terminated at all sites on September 19.
RESULTS AND DISCUSSION
laboratory Observations. No feeding inhibition was detected for caged mosquitoes offered vials baited with 30 µl of 33% phenylacetaldehyde [PAA] solution compared to approximately 10 µl of 95% PAA. West Nile Virus RNA was recovered from spiked wicks with qRT-PCR Ct scores, from highest titer to lowest, of 21.4, 26.5, 26.6, 28.7 and 34.5, respectively. This finding assured us that we could recover WNV RNA from wicks saturated with the alcohol formulation in an amount representing a single mosquito expectoration.
Field Observations. As in 2011 there appeared to be dissociation between the conventional surveillance methods (sentinel chickens and mosquito pools) and baited sugar wicks (Figure 1). Mosquito pool results are exaggerated in the figure because during weeks 25 through 33 there were only between three and nine pools tested per week. In both years most of the positive sugar wicks were recovered in August, but in 2012 WNV positive sentinel chickens and mosquito pools preceded most of the positive sugar wicks. In Contrast, in 2011 the results were reversed. Of the 12 positive sugar wicks from August 2012, six were from urbanized and six from rural sites indicating similar sensitivity for Cx.
quinquefasciatus and Cx. tarsalis habitats. Continuation
of this research will include comparing the current dispersed deployment of bait stations along ecotones with clustered deployment around a scent emitter station. The latter protocol may improve the sensitivity of the bait stations without the repellant effect of placing a high concentration of scent in the stations themselves.
ACKNOWLEDGMENTS
This work was funded by the Coachella Valley Mosquito and Vector Control District and the National Institutes of Health.
REFERENCE LIST
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Figure 1. Percent sugar
bait (Bait), sentinel chicken seroconversions (Flock) and mosquito pool (Mosq. Pool) samples testing positive for West Nile Virus at seven sites in Coachella Valley, 2012.