Wild birds maintained in zoos and outdoor aviaries, as well as free-ranging species like California Condors (Chang et al. 2007
) and now Island Scrub-Jays (Boyce et al. 2011
), are routinely vaccinated for protection against WNV virus. However, there are no WNV vaccines approved specifically for use in birds, and there are few data on the efficacy of commercially available equine vaccines in birds. In our study, none of the three vaccines provided the same level of protection upon challenge as a naturally mounted immune response after acute infection with WNV, exemplified by the naturally infected birds in group 5. However, because WNV infection in highly susceptible species such as corvids is often fatal (Komar et al. 2003
, Reisen et al. 2005
), as seen in our nonvaccinated positive controls (group 4) of which 60% succumbed to infection, even a partially effective vaccine may be beneficial in vulnerable populations.
The Fort Dodge West Nile-Innovator DNA and pCBWN provided the best immune-priming and had the lowest peak viremias. Because the pCBWN vaccine group had low sample sizes, a direct comparison between groups 1 and 2 lacked statistical power. All of the birds vaccinated with Merial developed bilateral necrotic lesions in the pectoral muscle at the vaccination site. These lesions were apparent at gross necropsy ( and ) in all but one bird, including the bird (No. 6320) that was vaccinated but not challenged with WNV. On gross and microscopic examination, Merial-vaccinated birds had extensive coagulation necrosis extending deep into the pectoral muscle ( and ). These lesions were attributed to the Merial vaccine, because they were seen in all Merial-vaccinated birds, including the vaccinated nonchallenged bird (No. 6320), and were not seen in any other group. In mammalian hosts and poultry, recombinant vaccines created from Avipox
genera, such as fowlpox and canarypox, have been considered safe and immunogenic (Taylor et al. 1988
), and the WNV canarypox vaccine has been shown to be safe and effective in horses, dogs, and cats (Minke et al. 2004
, Karaca et al. 2005
). The lesions noted at the vaccine inoculation sites in our study may have been due to replication of the recombinant canarypox virus, and we recommend that the Merial vaccine be assessed carefully before its use in other avian species, especially passerines. Although some immune-priming was detected in this group and the overall viremia was somewhat lower than the nonvaccinated positive controls, the vaccine was not as immunogenic as the Fort Dodge vaccine. While pectoral lesions were also noted in the group 1 and 4 (), these lesions were predominately inflammatory and acute in nature. The inflammatory component was attributed to subcutaneous WNV challenge inoculation over the pectoral muscle, as seen in previous experimental WNV infection studies (L. Woods, personal observation).
Each vaccine was evaluated to discern whether it was protective against WNV infection and if vaccine-related tissue damage would affect survivability in free-ranging birds. Two birds vaccinated with Fort Dodge had lesions that may have affected survivability, one with a systemic vasculitis and moderately severe myocarditis and one with encephalitis. Encephalitis and systemic vasculitis also were detected in one bird vaccinated with pCBWN. Four birds vaccinated with Merial, which were sacrificed 14
dpi, had lesions in target tissues that were typical of WNV infection, including encephalitis, polyneuritis, splenitis, and myocarditis/myocardial degeneration. Although these birds did not die during our study, lesions detected in these birds may have impacted survival in nature. Three birds from the Merial group had a systemic vasculitis with fibrinoid necrosis in vessel walls in the heart, kidney, spleen, and mesentery. IHC did not reveal any deposition of antigen in the vessel walls, which suggests that the vasculitis may have been caused by a type III hypersensitivity immune complex reaction. These factors, coupled with pectoral muscle necrosis induced by the Merial vaccine, would certainly have had significant impact on the survivorship of these vaccinated birds.
We were unable to detect a postvaccination antibody response in any of the vaccinated birds before WNV challenge. These results differed from previous studies that utilized pCBWN with multiple vaccinations and lower PRNT cut-off values. In one study (Bunning et al. 2007
), where American Crows received two vaccinations at 21-day intervals, 80% of the birds were PRNT70
positive for WNV antibodies at a serum dilution of 1:10 six weeks postvaccination. However, by 9 weeks postvaccination the percent PRNT70
antibody positive dropped to 50%. In a second study (Turell et al. 2003
), where Fish Crows received a single vaccination, 56% of the birds developed a PRNT80
detectable antibody response at a serum dilution of 1:20 by 14 days postvaccination; however, by day 42 postvaccination antibodies were no longer detectable at PRNT80
. In agreement with our findings, American Robins vaccinated with the pCBWN vaccine also failed to produce detectable antibodies when given a single vaccination and tested by PRNT90
at a serum dilution of 1:10, 14 days postvaccination (Kilpatrick et al. 2010
). Likewise, antibodies were not detected in 10 free-ranging Island Scrub-Jays that were vaccinated a single time with the Fort Dodge West Nile-Innovator DNA vaccine (Boyce et al. 2011
). Jays may need multiple inoculations or “boosters” with DNA vaccines to elevate antibody titers to detectable levels, similar to what was observed when free-ranging Island Scrub-Jays were inoculated with a killed vaccine (Fort Dodge West Nile-Innovator). Boyce et al. (2011
) found that five jays vaccinated twice with the killed vaccine had detectable PRNT80
antibody titers >1:20, whereas antibodies were detected in only 1 of 13 jays that received a single dose of killed vaccine. The lack of sustained humoral responses in WNV-vaccinated Island Scrub-Jays (single dose), as opposed to the responses of mice and House Sparrows experimentally exposed to wild-type WNV (Nemeth et al. 2009
, Appler et al. 2010
), illustrates the challenge inherent in designing efficacious vaccines for certain vertebrate species. These results also stress the importance of immunogenic epitopes located on WNV nonstructural proteins and the propagation of competent virus to stimulate a long-lasting sterile immunity in birds.
Aside from the one bird (No. 6302) in group 1 that apparently developed sterilizing immunity and one bird (No. 6313) in group 2 that had a reduced viremia, all vaccinated birds produced viremias suitable to infect California Culex
vectors with WNV (Reisen et al. 2005
). Therefore, although vaccination with all three vaccines lowered the viremia compared with nonvaccinated positive controls, the viremia was not lowered sufficiently to preclude the vaccinated birds from participating in the WNV transmission cycle. These findings suggest that vaccination of free-ranging Island Scrub-Jays with the vaccines we evaluated would reduce, but not effectively interrupt WNV transmission.
In summary, none of the vaccines we tested elicited sterilizing immunity after one vaccination, and none were completely without side effects. The Fort Dodge and pCBWN vaccines provided the best protective immune priming with the least side effects. In light of the well-documented devastating effects of high morbidity and mortality associated with WNV infection in corvids, vaccination with either vaccine could increase the numbers of birds that survive an epizootic should WNV be introduced to Santa Cruz Island. Unfortunately, neither of these vaccines are readily available for use in wild birds. The Fort Dodge vaccine was removed from the commercial market in 2010 after we completed this study, and pCBWN experimental vaccine is only available in limited quantities. In contrast, the Merial vaccine remains commercially available, but caused unacceptable side effects and appeared to be less effective. We acknowledge the limitations of our study (especially sample sizes), and encourage additional work to evaluate the efficacy and side effects of WNV vaccines for use in wild birds.