corvids collected as part of the wnv dead bird surveillance programs in manitoba and ontario , canada , in 2001 and 2002 were used .
laboratories in each province received dead birds , collected oropharyngeal or cloacal swabs or tissues , performed the antigen - capture assay , and shipped tissues or swabs to the national microbiology laboratory for confirmatory testing . in both laboratories , only birds lacking signs of obvious decay or decomposition were included for testing .
if specimens could not be shipped on the date of collection , most were held at 4c . on rare occasions , birds were frozen at 20c by submitters before shipment or by the diagnostic laboratories before testing . however , most specimens were held at approximately 4c for 1 to 4 days before processing .
thirty - one american crows ( corvus brachyrhynchos ) and 28 blue jays ( cyanocitta cristata ) , collected from june to december in 2001 and held at 20c were tested in march and april 2002 .
swabs were taken from the oropharyngeal cavity and cloaca of each bird . in 2002 ,
testing was restricted to crows ( n = 222 ) from which only oropharyngeal swabs were collected and tested by vectest .
polyester swabs ( vwr canlab , mississauga , ontario ) were applied to the oropharyngeal cavity and the cloaca .
swabs were placed in microcentrifuge tubes containing 1 ml of ba-1 diluent ( similar to that described by nasci et al . ) .
the antigen - capture assay was carried out according to the manufacturer s instructions except that the aliquot of swab eluate was 125 l , not 250 l . to confirm the infection status of birds ,
approximately 25-mg pieces of brain and kidney from each bird were pooled in microcentrifuge tubes and stored at 80c before shipment to the national microbiology laboratory . at that laboratory , 1 ml of ba-1 diluent and a tungsten bead ( qiagen , inc .
[ canada ] , mississauga , ontario ) were added to each tube , and tissues were homogenized on a retsech mixer mill ( qiagen ) .
beginning in mid - august , 2002 , a total of 109 american crows , 31 blue jays , 6 common ravens ( corvus corax ) , and 4 black - billed magpies ( pica pica ) were examined .
two cloacal swabs ( one each from a common raven and an american crow ) and one oropharyngeal swab from a blue jay were not taken ; hence swabs from both sites were available for a total of 147 birds .
specimens were collected by the procedures described above . however , after the 125-l aliquots of swab eluate were removed , the remainder of the sample was frozen at 80c and shipped to the national microbiology laboratory for confirmatory rt - pcr testing .
tissues were removed and tested for wnv by using rt - pcr on 42 ( 29 crows , 9 blue jays , 3 common ravens , and 1 magpie ) of the 150 birds . in both laboratories ,
swabs were incubated at 37c for 30 min and shaken intermittently ; if debris was present , tubes were centrifuged for 5 min at 4,000 rpm .
aliquots of 125 l of the manufacturer s grinding solution and of each swab sample were mixed in a microcentrifuge tube .
this mixture was centrifuged by using a vortex mixer ( vwr international , mississauga , ontario ) , and test strips were inserted into the supernatant fluid for 15 min , then removed and examined . during this study ,
two and five different persons from the manitoba and ontario laboratories , respectively , interpreted or read the test strips . as illustrated by nasci et al .
( 4 ) , presence of wnv was confirmed by the formation of a reddish purple line on the test strip , corresponding to the wnv - positive location and the development of a test control line .
real - time taqman rt - pcr assays to detect wn viral rna , as described by lanciotti et al . ( 1 ) , were conducted on all swabs and representative tissue samples from manitoba and all tissue samples from ontario . briefly , rna was extracted from 150 l to 200 l of swab eluates or tissue homogenates by using qiaamp viral rna kits ( qiagen ) .
a final volume of 70 l of eluted rna was stored at 80c until used .
five microliters of rna was combined with the appropriate primers and probes in buffer with the taqman rt - pcr ready - mix kit ( pe applied biosystems , foster city , ca ) .
samples were subjected to 40 amplification cycles in an abi prism 7700 sequence detection system instrument ( pe applied biosystems ) , according to the manufacturer s protocol for taqman rt - pcr cycling conditions .
all samples were screened with primers and probe specific to the 3 ntr , and positive extracts were confirmed by reamplification with the env primers and probe ( 1 ) .
swabs or tissues were considered positive only if positive results were obtained with both primer and probe sets .
sensitivity and specificity of the antigen - capture assay applied to oropharyngeal and cloacal swabs from corvids were calculated by using the results of taqman rt - pcr assays on swab or tissue samples as the  true  outcome ( 6 ) . to establish how storage conditions and duration of storage affected the sensitivity of the antigen - capture assay , cultures of egypt 101 strain of wnv ( approximately 4 x10 pfu / ml )
polyester swabs were then dipped into each virus solution , placed into 1 ml of manufacturer s grinding solution , and held at 20c , 4c , or room temperature ( approximately 18c ) .
the test strips were applied to aliquots of each solution 0 , 3 , 5 , 7 , 10 , and 14 days later , as described previously . to assess virus viability in grinding solution ,
50 l of each tested grinding solution and virus dilution was placed into 450 l of ba-1 diluent , and aliquots of 200 l of this mixture were added to vero cells reared on standard nutrient medium ( i.e. , dulbecco s modified eagle medium and 10% fetal bovine serum [ fbs ] ) at 37c and 5% co2 . after a 1-h adsorption , 2 ml of nutrient medium ( same as above but 5% fbs ) was added to tissue culture wells , which were incubated as above for up to 7 days .
positive ( wnv in ba-1 as above ) and negative ( media only ) controls were maintained under the same conditions , and cells in culture were observed daily for cytopathic effect ( cpe ) .
cells from positive controls and the highest concentration of virus used ( i.e. , 4 x 10 pfu / ml ) in grinding solution for each temperature and sampling date were acetone fixed , exposed to wnv - specific antibodies ( produced in rabbits and supplied by h. weingartl , canadian food inspection agency , winnipeg , manitoba ) and conjugated goat anti - rabbit immunoglobulin ( ig ) g ( kirkegaard & perry , gaithersburg , md ) and assessed for fluorescence under a uv microscope .
thirty - one american crows ( corvus brachyrhynchos ) and 28 blue jays ( cyanocitta cristata ) , collected from june to december in 2001 and held at 20c were tested in march and april 2002 .
swabs were taken from the oropharyngeal cavity and cloaca of each bird . in 2002 ,
testing was restricted to crows ( n = 222 ) from which only oropharyngeal swabs were collected and tested by vectest .
polyester swabs ( vwr canlab , mississauga , ontario ) were applied to the oropharyngeal cavity and the cloaca .
swabs were placed in microcentrifuge tubes containing 1 ml of ba-1 diluent ( similar to that described by nasci et al . ) .
the antigen - capture assay was carried out according to the manufacturer s instructions except that the aliquot of swab eluate was 125 l , not 250 l . to confirm the infection status of birds ,
approximately 25-mg pieces of brain and kidney from each bird were pooled in microcentrifuge tubes and stored at 80c before shipment to the national microbiology laboratory . at that laboratory , 1 ml of ba-1 diluent and a tungsten bead ( qiagen , inc .
[ canada ] , mississauga , ontario ) were added to each tube , and tissues were homogenized on a retsech mixer mill ( qiagen ) .
beginning in mid - august , 2002 , a total of 109 american crows , 31 blue jays , 6 common ravens ( corvus corax ) , and 4 black - billed magpies ( pica pica ) were examined .
two cloacal swabs ( one each from a common raven and an american crow ) and one oropharyngeal swab from a blue jay were not taken ; hence swabs from both sites were available for a total of 147 birds .
specimens were collected by the procedures described above . however , after the 125-l aliquots of swab eluate were removed , the remainder of the sample was frozen at 80c and shipped to the national microbiology laboratory for confirmatory rt - pcr testing .
tissues were removed and tested for wnv by using rt - pcr on 42 ( 29 crows , 9 blue jays , 3 common ravens , and 1 magpie ) of the 150 birds . in both laboratories ,
swabs were incubated at 37c for 30 min and shaken intermittently ; if debris was present , tubes were centrifuged for 5 min at 4,000 rpm .
aliquots of 125 l of the manufacturer s grinding solution and of each swab sample were mixed in a microcentrifuge tube .
this mixture was centrifuged by using a vortex mixer ( vwr international , mississauga , ontario ) , and test strips were inserted into the supernatant fluid for 15 min , then removed and examined . during this study ,
two and five different persons from the manitoba and ontario laboratories , respectively , interpreted or read the test strips . as illustrated by nasci et al .
( 4 ) , presence of wnv was confirmed by the formation of a reddish purple line on the test strip , corresponding to the wnv - positive location and the development of a test control line .
real - time taqman rt - pcr assays to detect wn viral rna , as described by lanciotti et al .
( 1 ) , were conducted on all swabs and representative tissue samples from manitoba and all tissue samples from ontario .
briefly , rna was extracted from 150 l to 200 l of swab eluates or tissue homogenates by using qiaamp viral rna kits ( qiagen ) .
a final volume of 70 l of eluted rna was stored at 80c until used .
five microliters of rna was combined with the appropriate primers and probes in buffer with the taqman rt - pcr ready - mix kit ( pe applied biosystems , foster city , ca ) .
samples were subjected to 40 amplification cycles in an abi prism 7700 sequence detection system instrument ( pe applied biosystems ) , according to the manufacturer s protocol for taqman rt - pcr cycling conditions .
all samples were screened with primers and probe specific to the 3 ntr , and positive extracts were confirmed by reamplification with the env primers and probe ( 1 ) . swabs or tissues
were considered positive only if positive results were obtained with both primer and probe sets .
sensitivity and specificity of the antigen - capture assay applied to oropharyngeal and cloacal swabs from corvids were calculated by using the results of taqman rt - pcr assays on swab or tissue samples as the  true  outcome ( 6 ) . to establish how storage conditions and duration of storage affected the sensitivity of the antigen - capture assay , cultures of egypt 101 strain of wnv ( approximately 4 x10 pfu / ml )
polyester swabs were then dipped into each virus solution , placed into 1 ml of manufacturer s grinding solution , and held at 20c , 4c , or room temperature ( approximately 18c ) .
the test strips were applied to aliquots of each solution 0 , 3 , 5 , 7 , 10 , and 14 days later , as described previously . to assess virus viability in grinding solution ,
50 l of each tested grinding solution and virus dilution was placed into 450 l of ba-1 diluent , and aliquots of 200 l of this mixture were added to vero cells reared on standard nutrient medium ( i.e. , dulbecco s modified eagle medium and 10% fetal bovine serum [ fbs ] ) at 37c and 5% co2 . after a 1-h adsorption , 2 ml of nutrient medium ( same as above but 5% fbs ) was added to tissue culture wells , which were incubated as above for up to 7 days .
positive ( wnv in ba-1 as above ) and negative ( media only ) controls were maintained under the same conditions , and cells in culture were observed daily for cytopathic effect ( cpe ) .
cells from positive controls and the highest concentration of virus used ( i.e. , 4 x 10 pfu / ml ) in grinding solution for each temperature and sampling date were acetone fixed , exposed to wnv - specific antibodies ( produced in rabbits and supplied by h. weingartl , canadian food inspection agency , winnipeg , manitoba ) and conjugated goat anti - rabbit immunoglobulin ( ig ) g ( kirkegaard & perry , gaithersburg , md ) and assessed for fluorescence under a uv microscope .
the sensitivity and specificity of the vectest assay for detecting wnv in oropharyngeal and cloacal swabs are presented in the table .
data from ontario reflect the sensitivity and specificity of this antigen - capture assay by using swab eluates in relation to the infection status ( taqman rt - pcr on kidney and brain ) of corvids .
in contrast , with the exception of 42 birds in which tissue samples were used to establish wnv infection status , the manitoba results evaluate the sensitivity and specificity of the antigen - capture assay by using swab eluates in relation to the detection of virus in the same sample by taqman rt - pcr .
data from the ontario birds from 2001 need to be interpreted with caution since sample size was small and the birds were frozen for up to 10 months
. however , cloacal swabs appeared to be less sensitive than oropharyngeal swabs in detecting an infected bird .
the sensitivity and specificity of the antigen - capture assay , when oropharyngeal swabs from crows collected in 2002 were used , were 83.3% and 95.8% , respectively , indicating that this assay is acceptable for use as a field detection test in a wnv surveillance program .
the advantages of this assay compensate for lower sensitivity only in areas where infected crows are relatively common .
the manitoba data also indicate that this antigen - capture assay is sufficiently sensitive and specific to detect wnv in oropharyngeal and cloacal swabs from crows when compared to rt - pcr results .
sixty - two of 109 crows were positive by rt - pcr on either oropharyngeal or cloacal swab , although the number of birds that would have been positive using tissues was not determined .
all but three birds positive on oropharyngeal swabs were also positive on cloacal swabs . small sample sizes and the relatively small number of positive samples on rt - pcr make evaluation of the utility of this antigen - capture assay on blue jays , common ravens , and magpies difficult . in general , the test s specificity seems superior to its sensitivity ( table ) .
further evaluation is necessary before there can be confidence in this assay as a means of detecting wnv in these species .
cloacal swabs were not available from one bird in each of these groups of birds .  an oropharyngeal swab was not taken from one of the blue jays . in manitoba ,
the vectest assay was not evaluated against rt - pcr on tissue for all birds , and thus the sensitivity and specificity can not be related to the true infection status of each bird .
however , indirect evidence suggests that the manitoba antigen - capture results approximate those that would have be obtained had rt - pcr on tissues been used in the comparison . on the 29 manitoba crows in which tissues were used to establish infection status by rt - pcr , the sensitivity and specificity ( i.e. , 83.3% and 94.1% , respectively ) of the antigen - capture assay on oropharyngeal swabs were comparable to those from ontario .
likewise , the data from manitoba are similar to the data on sensitivity and specificity of the antigen - capture assay on swab eluates from crows in ontario and compatible with the high prevalence or titers of virus in oropharyngeal or cloacal samples described by komar et al .
vectest samples had higher mean cycle threshold ( ct ) values ( i.e. , ct values are a measure of the overall virus titer in samples ; higher ct values indicate lower viral loads ) for both primer and probe sets ( i.e. , 26.1  4.5 , generic ;  26.0  4.9 , envelope ) compared to bona fide positives ( i.e. , 21.0  3.4 , generic ; 19.7  3.7 , envelope ) .
this observation suggests that the viral titers in the false - negative samples were below the threshold for consistent detection when the antigen - capture format was used .
similar but lower levels of sensitivity have been reported for this antigen - capture assay when it has been used to detect wnv in field - collected mosquitoes ( 4 ) . regardless of the storage conditions of swab samples , vectest strips always detected wnv at viral concentrations > 4 x 10 pfu / ml but never at titers of # 4 x 10 pfu / ml .
the vectest assay detected wnv at 4 x 10 pfu / ml when the grinding solutions were held at any of the three temperatures for up to 7 days ; thereafter wnv was only consistently detected at 4 x 10 pfu / ml when solutions were held at 20c .
none of the tissue cultures inoculated with grinding solution containing wnv had evidence of cpe or positive indirect fluorescent - antibody assay results , whereas all of the positive controls did .
based on these results , swabs can be placed in grinding solution for up to 7 days before the test strips are applied and held from 20c to 18c , without loss of sensitivity . as noted by nasci et al .
( 4 ) , once in the grinding solution , the titer of wnv is substantially reduced , as would be any potential biological hazard associated with handling or shipping the swab samples . in noncorvid species the vectest assay also has low sensitivity when compared with immunohistochemistry to detect viral antigen in fixed tissue and rt - pcr on frozen tissue .
the sensitivity of this antigen - capture assay was 46.7% for oropharyngeal swabs taken from 27 different raptors ( of various species ) in ontario during 2002 ( g.d .
this obviates the use of this assay as a reliable screening test for wnv infection in rehabilitation centers , veterinary clinics , zoos , animal disease diagnostic laboratories , and other settings where potential exposure to a biocontainment level 3 agent is of concern .
the advantages of this antigen - capture assay over conventional molecular - based diagnostic procedures such as real - time taqman rt - pcr include simplicity of procedures , no requirement for expensive and technically demanding instruments , and much shorter turn - around times for testing ( i.e. , results are available in 15 min ) .
in addition , testing swabs rather than tissues eliminates the need to dissect submitted birds , thus decreasing the processing time and cost of testing and the risk for lacerations to laboratory workers ( 7 ) .
the greatest limitation to the use of this system in wnv surveillance programs is the potential loss of information on early season wnv activity
. this loss could result from the lower sensitivity of the antigen - capture assay compared to the real - time rt - pcr taqman assay .
however , the loss of temporal sensitivity likely would be offset somewhat by the rapid turn - around times possible with the antigen - capture format .
in addition , in our experience , the vectest assay does not appear to work as effectively in noncorvid ( or all corvid ) species .
thus , its usefulness would be greatly diminished in jurisdictions that test all avian species as part of their wnv surveillance programs .
the poorer performance of the vectest assay in noncorvids may be related to species - specific differences ( and variability ) in virus titers in excretions and secretions at the time of death ( 3 ) .
the vectest assay will likely replace taqman rt - pcr assays as the front - line diagnostic test for future wnv dead bird surveillance programs in canada .
when this antigen - capture assay is used , only index cases of wnv infection in dead birds detected in a given jurisdiction such as a health unit or municipality need to be confirmed with rt - pcr assays before public notification of virus activity .
this procedure would compensate for the slightly lower specificity of the antigen - capture assay and should result in markedly decreased workloads for laboratories using molecular diagnostic procedures .
vectest positives obtained thereafter need not be confirmed by an alternative assay since the impact of false positives in a jurisdiction with previously confirmed virus activity should be negligible .
 the utility of the vectest antigen - capture assay to detect west nile virus ( wnv ) in field - collected dead corvids was evaluated in manitoba and ontario , canada , in 2001 and 2002 . 
 swabs were taken from the oropharynx , cloaca , or both of 109 american crows , 31 blue jays , 6 common ravens , and 4 black - billed magpies from manitoba , and 255 american crows and 28 blue jays from ontario . 
 the sensitivity and specificity of the antigen - capture assay were greatest for samples from american crows ; oropharyngeal swabs were more sensitive than cloacal swabs , and interlaboratory variation in the results was minimal . 
 the sensitivity and specificity of the vectest using oropharyngeal swabs from crows were 83.9% and 93.6% , respectively , for manitoba samples and 83.3% and 95.8% , respectively , for ontario birds . 
 the vectest antigen - capture assay on oropharyngeal secretions from crows is a reliable and rapid diagnostic test that appears suitable for incorporation into a wnv surveillance program . 