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.
Since 2000, surveillance for West Nile virus (WNV) in dead corvids has been the cornerstone of the early warning system for this virus in the Canadian public health system. During 2001 and 2002, WNV was detected in avian tissues by using real-time TaqMan reverse transcription–polymerase chain reaction (RT-PCR), as described by Lanciotti et al. (
The ultimate goal of this study was to determine whether the VecTest assay could serve as a suitable alternative testing procedure for WNV dead bird surveillance. This goal was achieved by quantifying the sensitivity and specificity of this antigen-capture assay to detect WNV in corvids collected as part of routine dead bird surveillance programs in Manitoba and Ontario. The effect of storage conditions and duration of storage of swabs in grinding solution on the sensitivity of the assay and viability of virus was also assessed.
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. Birds typically were shipped to the laboratories via courier. During submission, most animals were held within insulated coolers containing freezer packs. If specimens could not be shipped on the date of collection, most were held at 4°C. On rare occasions, birds were frozen at –20°C by submitters before shipment or by the diagnostic laboratories before testing. However, most specimens were held at approximately 4°C for 1 to 4 days before processing.
Thirty-one American Crows (
Beginning in mid-August, 2002, a total of 109 American Crows, 31 Blue Jays, 6 Common Ravens (
In both laboratories, swabs were tested for WNV with the VecTest assay (WNV/St. Louis encephalitis antigen panel), as described by Nasci et al. (
Real-time TaqMan RT-PCR assays to detect WN viral RNA, as described by Lanciotti et al. (
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 x107 PFU/mL) were serially diluted to 4 X 104 PFU/mL in BA-1 diluent. Polyester swabs were then dipped into each virus solution, placed into 1 mL of manufacturer’s grinding solution, and held at –20°C, 4°C, or room temperature (approximately 18°C). 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 μL of each tested grinding solution and virus dilution was placed into 450 μL of BA-1 diluent, and aliquots of 200 μL 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 37°C 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 107 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. Hence, the results obtained in the two laboratories are not directly comparable.
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 (
| Species tested (y) | N | Oropharyngeal swabs | Cloacal swabs | ||
|---|---|---|---|---|---|
| Sensitivity (%) | Specificity (%) | Sensitivity (%) | Specificity (%) | ||
| Ontario | |||||
| American Crows (2001) | 33 | 92.8 | 79 | 58.3 | 94.7 |
| Blue Jays (2001) | 28 | 60 | 100 | 40 | 100 |
| American Crows (2002) | 222 | 83.3 | 95.8 | NDa | ND |
| Manitoba | |||||
| American Crows | 109b | 83.9 | 93.6 | 83.1 | 97.9 |
| Blue Jays | 31c | 71.4 | 100 | 57.1 | 100 |
| Common Ravens | 6b | 100 | 100 | 100 | 0 |
| Black-billed Magpies | 4 | 0 | 100 | 66.7 | 100 |
aND, not done. bCloacal swabs were not available from one bird in each of these groups of birds. cAn 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 cannot 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. (
The “false-negative” 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 (
Regardless of the storage conditions of swab samples, VecTest strips always detected WNV at viral concentrations >4 X 106 PFU/mL but never at titers of #4 x 104 PFU/mL. The VecTest assay detected WNV at 4 X 105 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 105 PFU/mL when solutions were held at –20°C. Grinding solution appears to inactivate WNV. 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 –20°C to 18°C, without loss of sensitivity. As noted by Nasci et al. (
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. Campbell, Ontario Veterinary College, ON, pers. comm.). 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 (
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 (
We thank Kirti Dave for providing technical advice on the use of the VecTest assay; Kelly Nichol for providing assistance at the laboratory in Guelph; and Heather White for her help in preparing the manuscript.
Dr. Lindsay is a research scientist at the National Microbiology Laboratory of Health Canada in Winnipeg, Manitoba. His research interests focus primarily on vector-borne zoonotic diseases.