Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

Abstract

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.

Figure 1

Antibodies in falcons detected by ELISA and micro-virus neutralization test (VNT) during vaccination period. Figure 1A shows competition ELISA (ID Screen© WN competition ELISA, IDVet, Grabels, France) responses. Optical density at 450 nm is converted to signal/noise% (S/N%) ratio (S/N% = OD_sample/OD_{negative control}* 100), with values ≤ 40% considered positive, > 40% and ≤ 50% equivocal and > 50% negative. The threshold for positive results is indicated as a solid red line. Figure 1B displays neutralization antibody responses for all groups. Data are presented in a box-and-whisker plot, where the ends of the whiskers represent the minimum and maximum values, respectively. Outliers are represented as points instead of whisker-ends. The box includes 50% of the values of each group and the line in the middle of each group represents the median value of each group. The double immunization with inactivated vaccine (light blue) led to temporary seroconversion whereas the triple vaccination (dark blue) was more efficient. The recombinant vaccine generally induced only a slight seroconversion. With two shots of the recombinant vaccine (light green) low level and temporary seroconversion occurred, while three shots (dark green) provided a better result measureable by ELISA but not by VNT.

Figure 2

Oral and cloacal shedding of falcons during challenge. Cycle threshold values (Ct values) of oral (A) and cloacal (B) swabs for all groups are displayed. Data are presented in a box-and-whisker plot, where the ends of the whiskers represent the minimum and maximum values, respectively. Outliers are represented as points instead of whisker-ends. The box includes 50% of the values of each group and the line in the middle of each group represents the median value of each group. The amount and duration of oral shedding was significantly reduced in the group vaccinated three times with Duvaxyn® (dark blue, only amount reduced) and in the groups vaccinated with Recombitek® (light and dark green), but not in the group vaccinated twice with Duvaxyn® (light blue). In addition, the amount of cloacal shedding was significantly reduced in all groups. Duration of cloacal shedding was not shortened in any of the groups.

Figure 3

Viraemia detected by qRT-PCR and virus titration. Cycle threshold values (Ct values) of whole blood for all groups during the challenge period are displayed in Figure 3A. Results of blood titration for all groups during the challenge period are given in Figure 3B in log₁₀ tissue culture infection dose 50 per mL (TCID₅₀/mL) whole blood. Data are presented in a box-and-whisker plot, where the ends of the whiskers represent the minimum and maximum values, respectively. Outliers are represented as points instead of whisker-ends. The box includes 50% of the values of each group and the line in the middle of each group represents the median value of each group. The level of viraemia measured in Ct values was significantly lower in all vaccinated groups than in the control group (yellow). In contrast, duration of viraemia was only shortened significantly in the group vaccinated three times with Recombitek® (dark green).

Figure 4

Antibodies detected by ELISA and micro-virus neutralization test (VNT) during challenge in falcons. In Figure 4A antibodies of all groups detected by the ID Screen© WN competition ELISA (IDVet, Grabels, France) during the challenge period are displayed. Optical density at 450 nm is converted to signal/noise% (S/N%) ratio (S/N% = OD_sample/OD_{negative control} * 100), with values ≤ 40% considered positive, > 40% and ≤ 50% equivocal and > 50% negative. The threshold for positive results is indicated as a solid red line. In Figure 4B antibody titers of all groups determined by VNT against homologous challenge virus (neutralization titer) during the challenge period are displayed. Data are presented in a box-and-whisker plot, where the ends of the whiskers represent the minimum and maximum values, respectively. Outliers are represented as points instead of whisker-ends. The box includes 50% of the values of each group and the line in the middle of each group represents the median value of each group. Seroconversion occurred at 3 to 6 dpi.

Figure 5

WNV antigen dectection by immunohistochemistry in different tissues of falcons after virus challenge. A: Brain/Telencephalon (region lateral of the lateral ventricle) of a challenged falcon (F6) included in a previously published study [12] at day 10 post infection. B: Spleen of a bird at day 8 post infection without vaccination (F51). Antigen was detected in necrotic material of follicle arteries. C: Myocard of a falcon vaccinated twice with the inactivated vaccine (F17, necropsied at day 9 post infection). Antigen staining in cardiocytes (arrows). All results were obtained using the monoclonal antibody 15R4 (kindly provided by Petra Emmerich, Bernhard-Nocht Institute, Hamburg, Germany).