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from 2002 through 2005 , raptors originating in colorado were bled by ulnar venipuncture and orally swabbed upon admission to the rocky mountain raptor program of colorado state university .
wnv was first detected in colorado in august 2002 , and testing of raptors was initiated in september ( oral swabs ) and october ( serum samples ) . in all other years , samples were collected from early to late april through mid to late october .
specimens were tested for wnv - neutralizing antibodies by plaque - reduction neutralization test ( prnt ) and for virus isolation by vero cell plaque assay ( 9 ) or wnv antigen by vectest wnv antigen detection assay ( medical analysis systems , ventura , ca , usa ) .
isolated viruses were identified as wnv by vectest . to confirm that antibody - positive adult raptors were recently infected , we evaluated 90% neutralization titers in acute - phase and convalescent - phase serum samples collected 3 weeks apart . a 4-fold increase in titer
cross - reactivity for another closely related north american flavivirus , saint louis encephalitis virus , was ruled out by comparing 90% neutralization titers .
a 4-fold greater titer for 1 of the viruses indicated that particular virus as the etiologic agent for the infection .
utility of wnv detection in raptors was evaluated in relation to other existing wnv surveillance techniques in northern colorado .
most of these ( 83% ) originated from weld and larimer counties , which represent an area of 6,639 square miles , larger than connecticut and rhode island combined . during the study , 38 raptors ( 11.8% ) tested positive for wnv .
some were positive by both oral swab and seroconversion , while others were positive according to only 1 of these .. usually , birds that were positive by oral swab only died before 1 or both blood samples could be collected , so we were unable to test for seroconversion . in 2002 , 17 raptors were tested ( blood by prnt and oral swab by plaque assay ) , 4 of which were seropositive for wnv between october 7 and november 15 . in 2003 , 52 birds were tested ( serum by prnt and oral swab by vectest ) , 7 of which seroconverted and 5 of which were oral swab  positive .
positive samples were detected between july 17 and september 1 . in 2004 , 113 birds were tested by plaque assay of oral swab ( no blood test ) , and 3 were found to be positive between july 28 and september 17 . in 2005 , 141 birds were tested ( serum by prnt and oral swab by plaque assay ) , of which 19 were positive ( 8 by seroconversion , 6 by virus isolation from swab , and 5 by both methods ; table ) .
* the proportion of positives for kestrel was significantly less than for all other species combined ( p = 0.0046 ,  = 0.0056 ; fisher exact test with bonferroni adjustment for 9 comparisons ) .
includes burrowing owl ( n = 2 ) , sharp - shinned hawk ( n = 2 ) , cooper 's hawk ( n = 1 ) , eastern screech owl ( n = 1 ) , merlin ( n = 1 ) , osprey ( n = 1 ) , prairie falcon ( n = 1 ) , and saw - whet owl ( n = 1 ) . to compare our test results with those from other surveillance systems for wnv
, we limited our data to specimens collected april 1october 15 , 2005 , from raptors originating in weld or larimer counties . in comparing the earliest date of detection for each of the surveillance methods in place in these counties ,
clinic - admitted raptor surveillance provided the earliest evidence of wnv activity ( april 8) , preceding all other wnv surveillance systems initial detections of wnv activity by nearly 14 weeks ( figure ) .
chronology of detections of west nile virus by various surveillance systems in place in larimer and weld counties , northern colorado , 2005 . confirmed human cases , mosquito , dead bird , and equine surveillance information provided by the centers for disease control and prevention s arbonet surveillance system through october 15 , 2005 .
the early detection of wnv in clinic - admitted raptors compared with other detections by surveillance systems in northern colorado during 2005 points to the potential utility of raptor rehabilitation centers for wnv surveillance .
although other active surveillance systems require significant allocations of human resources , clinic - admitted raptor surveillance is a passive system that takes advantage of existing resources outside the traditional public health infrastructure .
nationwide , about 1,000 wildlife rehabilitation facilities admit 10,000 birds annually ( p. redig , pers . comm . ) .
participation in surveillance efforts provides rehabilitators with valuable diagnostic information and can be accomplished at no cost to the rehabilitator , provided that provisions are supplied .
the detection of wnv in an oral swab of a great horned owl in early april in colorado was quite unexpected because of the early date .
this bird was an uninjured nestling that was brought to the clinic for nurturing until it could be replaced into its original nest .
the oral swab yielded a low number of infectious virus particles ( 2.5 pfu ) , and the nestling failed to develop clinical signs and failed to seroconvert .
we believe that the oral cavity may have been contaminated by a recent prey meal provided by the bird s parents shortly before admission .
although early spring transmission of wnv by mosquitoes to either the owlet or a prey animal is possible , persistent infection of the prey item is an alternative explanation .
experimentally infected hamsters develop chronically infected kidneys ( 10 ) , and birds may also maintain persistent visceral infections ( 2 ) .
if the early detection in the owl was an anomaly , the next earliest evidence of wnv activity from clinic - admitted raptors was june 28 , which also preceded all other detections .
the first confirmed human case of west nile fever in the study area developed symptoms on july 17 , and the first confirmed case of west nile neurologic disease occurred on august 6 ( figure ) .
although we have shown that a combination of serologic and oral swab testing increases the sensitivity of clinic - admitted raptor surveillance almost 2-fold , serolog testing has 3 important limitations : 1 ) blood sampling requires special training and expertise ; 2 ) evidence of seroconversion requires 2 samples spaced apart by at least 2 weeks , and therefore reporting of positive results is significantly delayed by several weeks after onset of infection ; and 3 ) neutralization tests can be prohibitively expensive and require extensive training , time , supplies , biosafety level-3 ( bsl-3 ) lab facilities , and expertise in interpreting results , which are complicated by cross - reactions with closely related viruses .
limiting sampling to oral swabs reduces sensitivity ; however , the savings in time and cost would permit a greater number of samples to be collected and tested . although we used plaque assay for detecting wnv in oral swabs , which also requires bsl-3 laboratory facilities , our samples could have been tested with high sensitivity and specificity for wnv - specific rna sequences by using reverse transcription 
, limited data from 1 small region of north america suggest that wnv surveillance using live raptor admissions to rehabilitation facilities should be considered along with other established surveillance methods already in use ( 12,13 ) .
clinic - admitted raptors are most useful for early detection or continued detection of wnv activity
in 2005 , 13.5% of clinic - admitted raptors in northern colorado tested positive for west nile virus ( wnv ) . 
 clinic - admitted  raptor surveillance detected wnv activity nearly 14 weeks earlier than other surveillance systems . 
 wnv surveillance using live raptor admissions to rehabilitation clinics may offer a novel surveillance method and should be considered along with other techniques already in use .