Experimental infection of chickens as candidate sentinels for West Nile virus.

We evaluated the susceptibility, duration and intensity of viremia, and serologic responses of chickens to West Nile (WN) virus (WNV-NY99) infection by needle, mosquito, or oral inoculation. None of 21 infected chickens developed clinical disease, and all these developed neutralizing antibodies. Although viremias were detectable in all but one chicken, the magnitude (mean peak viremia <10,000 PFU/mL) was deemed insufficient to infect vector mosquitoes. WNV-NY99 was detected in cloacal and/or throat swabs from 13 of these chickens, and direct transmission of WNV-NY99 between chickens occurred once (in 16 trials), from a needle-inoculated bird. Nine chickens that ingested WNV-NY99 failed to become infected. The domestic chickens in this study were susceptible to WN virus infection, developed detectable antibodies, survived infection, and with one exception failed to infect cage mates. These are all considered positive attributes of a sentinel species for WN virus surveillance programs.

We evaluated the susceptibility, duration and intensity of viremia, and serologic responses of chickens to West Nile (WN) virus (WNV-NY99) infection by needle, mosquito, or oral inoculation. None of 21 infected chickens developed clinical disease, and all these developed neutralizing antibodies. Although viremias were detectable in all but one chicken, the magnitude (mean peak viremia <10 4 PFU/mL) was deemed insufficient to infect vector mosquitoes. WNV-NY99 was detected in cloacal and/or throat swabs from 13 of these chickens, and direct transmission of WNV-NY99 between chickens occurred once (in 16 trials), from a needle-inoculated bird. Nine chickens that ingested WNV-NY99 failed to become infected. The domestic chickens in this study were susceptible to WN virus infection, developed detectable antibodies, survived infection, and with one exception failed to infect cage mates. These are all considered positive attributes of a sentinel species for WN virus surveillance programs.
swabs and dipping the infected swabs in 0.5 mL of BA-1 before freezing at -70°C. All inoculated hens were observed twice daily during the first 7 dpi of infection for signs of clinical illness. A final serum sample (0.6 mL of whole blood) was taken at 14 dpi to test for seroconversion by plaque-reduction neutralization test (PRNT) (5). A sample that neutralized the challenge dose of WNV-NY99 by at least 90% was considered positive. Three hens were maintained until 28 dpi to monitor the development of neutralizing antibodies during this period.

Infection of Mosquitoes
Colonized Culex tritaeniorhynchus mosquitoes were infected by intrathoracic inoculation of 700 nL of a suspension containing 10 8.2 per mL WNV-NY99 (source Cx. pipiens pool #NY99-6480 collected in New York, 1999, 1 Vero passage, CDC accession no. B82123), and incubated for 7 to 10 days at 16:8 hours light:dark, 28°C, 80% relative humidity, before feeding on chickens. Successful infection of mosquitoes was confirmed by plaque assay of homogenates of whole mosquitoes (after incubation) or saliva extracted from mosquitoes after feeding (6).

Virus Titration and Identification
The concentration of WN virus infectious particles in fluids (including cloacal swabs, throat swabs, and blood samples) was evaluated by Vero plaque assay (5) of 10-fold serial dilutions. Plaques were counted after 3-5 days of incubation at 37°C, 5% CO 2 . Plaques from swabs were harvested and identified by neutralization using a standard antiserum available from the Centers for Disease Control and Prevention reference collection in Fort Collins, CO.

Results
All of the 21 WNV-NY99 parenterally inoculated hens developed neutralizing antibodies and 20 of these had detectable viremia ( Table 1). One of 16 in-contact hens had a transient WNV-NY99 viremia of magnitude 10 2.4 PFU/mL on the third day after its cage mate had been injected, and seroconverted. None of nine orally inoculated hens developed WNV-NY99 viremia or antibodies. None of the 46 hens exposed to WNV-NY99 demonstrated overt clinical illness attributable to WN virus.
Three mosquito-infected hens were sampled more frequently (approximately twice per week) after the first week of infection to monitor the pattern of antibody response within 28 dpi (Figure). Neutralizing antibody was detected in one of the three birds as early as 7 dpi (reciprocal 90% neutralization titer = 10), and in all three at 10 dpi (titers = 40, 40, and 80). The titers increased steadily throughout this period, reaching 320, 80, and 160, respectively, by 28 dpi.
We determined the duration and magnitude of WNV-NY99 viremia in the 21 parenterally inoculated hens    (Table 1). All five hens inoculated by needle had detectable viremias that endured 2 to 3 days with mean peak viremia of 10 3.9 PFU/mL (range 10 3.4 -10 5.0 ). Of the 16 hens inoculated by mosquito, 15 had detectable viremias that endured 2 to 3 days with mean peak viremia of 10 3.4 (max 10 4.1 ). No virus was detected in blood samples collected 6 and 7 dpi (data not shown). Cloacal shedding of WNV-NY99 was observed in 12 of 21 (57%) parenterally inoculated hens ( Table 2). All 5 of the needle-inoculated birds and all 5 of the 60-week-old mosquitoinoculated birds shed, whereas only 2 of 11 (18%) 17-week-old mosquito-inoculated birds shed. Positive cloacal swabs were observed 2-6 dpi. Peak cloacal swab positivity was 3-5 dpi. Shedding in oral exudates was observed in two of six 17-weekold hens. In these six birds, the number of plaques detected from throat swabs was generally less than that from cloacal swabs ( Table 2). Viruses detected in swabs were identified as WN virus by PRNT and were reisolated from a subset of the positive swabs for confirmation of results. To evaluate the viability and stability of WNV-NY99 in fecal material outside the host, fecal urates of chickens were mixed with 100 PFU WNV-NY99. No negative effect of the fecal material was observed when compared with BA-1 diluent. However, viability was reduced by 99% after 24 hours at ambient temperature (data not shown).

Discussion
This study evaluated WNV-NY99 sentinel criteria for chickens by monitoring their response to experimental infection in captivity. We report for the first time quantitative data about WNV-NY99 viremias in chickens inoculated by mosquito bite. Turell (7,8) reported that chicks were infected with WN virus by mosquito bite, but data from these evaluations were not presented. The response of several bird species (including chickens, turkeys, and geese) to needle inoculation of this North American strain of WN virus has been documented (9-11). However, mosquito inoculation has been shown to elicit a different response to infection compared with needle inoculation in several vertebrate-virus systems (12,13).
Three central criteria for an arbovirus sentinel bird are susceptibility to infection, development of detectable antibodies, and survival. Birds that do not survive infection may be lost to surveillance programs designed to detect antibodies as a marker for infection. We found that all the chickens inoculated parenterally in our study, as in other WN virus infection studies in chickens (14,15), became infected, and survived to develop detectable neutralizing antibodies. Evaluation of alternative serodiagnostic assays for immunoglobulin M and hemagglutination-inhibiting antibodies are under way.
Birds used as sentinels for arbovirus surveillance should not contribute to the local arbovirus transmission cycle if they become infected. Detectable viremia in mature chickens (>3 weeks) is unusual for WN virus strains that have been studied previously (14,15), although young chicks do develop viremia >10 5 PF/mL (14, 7). Senne et al. (9) reported that WNV-NY99 needle-inoculated 7-week-old hens had viremia sufficient to infect mosquitoes, based on data from an experimental infection study of an African mosquito, Cx. univittatus, using an African strain of WN virus (cited in 16). However, new data do not support this statement. A study of vector competence of Cx. pipiens collected in New York and infected with WNV-NY99 suggests that the maximum viremia that we observed in the needle-inoculated hens (10 5 PFU/mL) is sufficient to infect about 17% of these mosquitoes; 2% will be able to transmit the virus in a subsequent bloodmeal (7). The maximum viremia detected in mosquito-inoculated hens reached 10 4.1 and is probably well below the level required to maintain the Cx. pipiens transmission cycle. Although other species of mosquitoes may have lower thresholds of infection, Cx. pipiens is recognized as the important vector in the avian transmission cycle in the northeastern United States (17). Thus, our data imply that chickens are incompetent to retransmit WNV-NY99 to Cx. pipiens in New York. However,  0  N T  5  0  1108  20  N  NA  NT  NT  200  0  NT  0  0  1110  20  N  NA  NT  NT  28  95  NT  3  0  2019  20  N  NA  NT  NT  3  8  NT  0  0  2027  20  N  NA  NT  NT  10  5 NT  1126  60  M  1  0  0  6  5  0  NT  NT  1128  60  M  1  0  0  0  0  1  NT  NT  1132  60  M  1  0  0  3  11  18  NT  NT  1134  60  M  1  0  3  9  24  3 NT NT a Data presented are from cloacal swabs unless otherwise indicated. b Cloacal swab/nasopharyngeal swab. N = needle; NA = not applicable; NT = not tested; M = mosquito.

West Nile Virus
we recognize that conditions may exist in which vector mosquitoes, including strains of Cx. pipiens, could theoretically have lower transmission thresholds that permit them to acquire WNV-NY99 infection from mature chickens. Birds used as sentinels for arbovirus surveillance should not spread arbovirus infections directly to flock mates, because a finding of birds that are seropositive as a result of direct transmission (in the absence of mosquito vectors) would lead to misinterpretation of the true risk for mosquito-borne transmission. In our study, we observed one such transmission event (out of 16 trials). This transmission originated from a needle-inoculated hen. Experimental direct transmission (from needle-inoculated birds) has been observed with other WNV-NY99-infected species, including domestic goslings (11) and American Crows (R.G. McLean, pers. comm.), but not chicken pullets and turkey poults (9,10). The importance of direct transmission of WNV-NY99 among birds in nature remains unknown.
The means by which WNV-NY99 direct transmission among birds occurs may include inhalation of infectious aerosols due to viral shedding in bodily fluids such as fecal material and saliva, ingestion of contaminated food, or contact with viremic blood. The possibility of oral ingestion of WNV-NY99 was tested in nine chickens with negative results. We did, however, document the presence of infectious WNV-NY99 in oral exudates and feces. WNV-NY99 has been reported previously in cloacal swabs of needle-inoculated chicken pullets and turkey poults (9,10) but not goslings (11), and in oropharyngeal swabs of turkeys and goslings (10,11). We observed that the quantity of virus collected in swabs was relatively low (not exceeding 200 infectious virus particles in our preliminary evaluation) and that stability of WNV-NY99 in avian fecal material outside the host was reduced dramatically after 24 hours, suggesting that risk of transmission from infected feces decreases as the time outside the host increases. Because WN virus infection in humans exposed to viral shedding in birds has not been documented, the actual risk is unknown and can be reduced through proper recommended animal-handling techniques, such as the use of disposable gloves and HEPA-filtered masks.
Chickens should be evaluated as sentinels for detecting and monitoring enzootic WN virus transmission. Chickens have been used extensively for surveillance of Kunjin virus (a subtype of WN virus) in Australia (R. Russell, pers. comm.). Pre-existing flocks of domestic chickens were naturally exposed to WN virus in Bucharest in 1996 (37% seropositive) (18), New York City in 1999 (63%) (19), and eastern Suffolk County, NY, in 1999 (30%, S. Campbell, pers. comm.). Thus, based on these data, chickens would seem to be strong candidates for use as sentinels for WN virus.
In summary, we present the first experimental infection study of WNV-NY99 in chickens in which mosquito and oral transmission routes are evaluated. We found that WNV-NY99 viremia in chickens is probably insufficient to infect the primary epiornitic vector, Cx. pipiens. The observation of transmission to a hen in contact with a needle-inoculated WNV-NY99-infected hen requires further study on the risk of direct transmission among chickens and to their handlers by contaminated bodily fluids. This experimental infection study provides data that, in part, justify chickens as candidates for WN virus sentinels in North America.