AVIAN MORTALITY SURVEILLANCE FOR WEST NILE VIRUS IN COLORADO NICOLE M. NEMETH,* SUSAN BECKETT, ERIC EDWARDS, KACI KLENK, AND NICHOLAS KOMAR Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado; Centers for Disease Control and Prevention, Arbovirus Diseases Branch, Fort Collins, Colorado Abstract. We tested 1,549 avian carcasses of 104 species to identify targets for West Nile virus (WNV) surveillance in Colorado, determine species affected by WNV, compare virus isolation versus RNA detection applied to hearts and oral swabs from carcasses, and compare the VecTest WNV Antigen Assay (VecTest) to standard assays. Forty-two species tested positive. From June to September 2003, 86% of corvids, 34% of non-corvid passerines, and 37% of raptors tested positive. We developed the Target Species Index, which identified American crows as the most important avian indicator species. However, testing multiple species maximizes detection, which may be important early and late in the transmission season. This index may benefit surveillance for other zoonotic pathogens, such as highly pathogenic avian influenza H5N1 virus. VecTest using oral swabs was most sensitive for American crow, black-billed magpie, house finch, house sparrow, and American kestrel. Wildlife rehabilitation centers should be recruited to enhance WNV surveillance. INTRODUCTION Avian mortality surveillance has been established as a use- ful and sensitive early detection system for West Nile virus (WNV; genus Flavivirus, family Flaviviridae) activity. 1–3 WNV-positive birds have often provided the earliest indica- tion of WNV activity and frequently precede human WNV case reports. 4 The American crow (Corvus brachyrhynchos) has proven to be a sensitive avian sentinel for WNV surveil- lance in the northeast United States 5–7 and southern Califor- nia, 8 but reports documenting the use of crows and other species for surveillance are lacking from other regions. The primary value of testing avian carcasses for WNV infection is the documentation of local WNV activity, which can be quan- tified for risk determination using a variety of other surveil- lance systems, including mosquito collection and testing, sen- tinel animals, or dead bird cluster analyses. 9–11 American crow populations have been reduced in some areas because of WNV-related die-offs. 12–15 In regions where crows are sparse, whether naturally or because of WNV- related depletion, carcasses of other bird species may be use- ful WNV surveillance tools. Furthermore, the VecTest WNV Antigen Assay (VecTest; Medical Analysis Systems, Cama- rillo, CA) has been shown to be a sensitive diagnostic tool in testing oral and cloacal swabs from American crows and some other corvids, but few data exist on the use of VecTest for rapid WNV detection in samples from non-corvids. 16–20 To make recommendations on appropriate avian mortality surveillance techniques for WNV in a High Plains state (Col- orado), we tested carcasses of multiple bird species to 1) de- termine the spectrum of avian species affected by WNV in Colorado, 2) compare WNV detection rates in carcasses of the American crow and other birds in Colorado, 3) compare WNV detection assays (virus isolation versus TaqMan reverse transcriptase-polymerase chain reaction [RT-PCR]) and WNV detection in samples (heart versus oral swab) from avian carcasses, and 4) evaluate VecTest applied to oral swabs from corvid and non-corvid bird species. MATERIALS AND METHODS Sampling and origin of dead birds. Dead birds were col- lected during 2002–2005, mostly from private citizens through local health departments, wildlife rehabilitators, and the Cen- ters for Disease Control and Prevention, Arbovirus Diseases Branch in Fort Collins, CO. In 2002, dead bird collection began in mid-August, after WNV was first detected by the Colorado Department of Public Health and Environment, and collection was carried out until November. In subsequent years, dead birds were collected from April until October. American crows were not included in the study in 2002, and corvids were not included in 2004 because they were tested exclusively by the state health laboratory for their own sur- veillance purposes. The vast majority of birds in this study were from Colorado. However, several raptors were submitted that originated from New Mexico, Wyoming, or Nebraska. Although birds from numerous counties were tested in all years, in 2003– 2005, the study focused mostly on two northern Colorado counties (Larimer and Weld). Birds deemed dead  48 hours were refrigerated and sampled the same day when possible or were placed at -20°C and thawed for sampling within 2–4 days. After thawing, the oropharyngeal cavity was swabbed with a cotton-tipped ap- plicator, and several pieces of heart were removed aseptically with a sterile surgical blade. The heart was deemed a conve- nient tissue for sampling and has consistently proven to be one of the most reliable tissues for WNV detection in birds. 21–26 In all years except 2003, oral swabs were broken off into 1 mL BA-1 diluent (Hank’s M-199 salts, 1% bovine serum albumin, 350 mg/L sodium bicarbonate, 100 units/mL penicillin, 100 mg/L streptomycin, 1 mg/L Fungizone in 0.05 mol/L Tris, pH 7.6); in 2003, oral swabs were submerged in 1 mL of VecTest grinding buffer. In all years, heart samples were placed in cryovials with 1 mL BA-1 diluent and BB pellets. Heart samples (0.5 cm 3 ) were ground in a Qiagen mixer mill (Qiagen, Valencia, CA) at 25 cycles/s for 5 minutes and clari- fied by centrifugation (12,000 × g for 3 minutes). Oral swab samples were tested by VecTest immediately after sampling, after which oral swab and heart samples were held at -80°C until further testing was performed. Plaque assay. Virus isolation was performed on heart ho- mogenates (2002–2005) and oral swabs (2002 and 2004–2005) by Vero cell plaque assay as previously described. 23 * Address correspondence to Nicole M. Nemeth, Department of Mi- crobiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail: nnemeth@colostate.edu Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 431–437 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene 431