Sera were tested for the presence find more of influenza A-specific anti-nucleoprotein and/or matrix protein (NP/M) antibodies by AGID tests as described elsewhere (10) with 1% Noble agar (Difco

Laboratories, Sparks, MD, USA) containing 8.5% NaCl (11). The antigen used for the AGID test was prepared from A/whistling swan/Shimane/35/80 (H6N3) (9). Sera from specific-pathogen-free chickens inoculated intramuscularly with the same antigen or with PBS were used as the positive and the negative control for reactions, respectively. The detection of anti-NS1-specific antibodies in sera was carried out with immunoblotting, as described previously (12). Briefly, recombinant influenza A NS1 expressed in Escherichia coli BL21 was separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (13) and transferred to Immobilon-P (Millipore, Billerica, MA, USA), then reacted with duck serum (diluted 1:100 with PBS, pH 7.4). After an incubation with goat anti-duck immunoglobulin (IgG)-horseradish peroxidase conjugate (Nordic Immunological selleck kinase inhibitor Laboratories, Tilburg, The Netherlands), reactions were visualized

with the ECL plus Western blotting detection system (GE Healthcare, Buckinghamshire, UK). Serum samples that tested positive for antibodies to both the NP/M and NS1 were tested further for the presence of subtype-specific anti-HA antibodies with HI tests using virus strains A/duck/Shimane/510/02 (H1N1), A/whistling swan/Shimane/31/97 (H2N3), A/whistling swan/Shimane/227/01 (H3N9), A/budgerigar/Hokkaido/1/77 (H4N6), A/whistling swan/499/83 (H5N3), A/whistling swan/Shimane/190/01 (H6N9), A/whistling swan/Shimane/42/80

(H7N7), A/turkey/Ontario/6118/68 (H8N4), A/turkey/Wisconsin/66 (H9N2), A/chicken/Germany/“N”/49 (H10N7), A/duck/Memphis/564/74 (H11N9), A/duck/Alberta/60/76 (H12N5), and A/gull/Maryland/704/77 (H13N6), and subtype-specific anti-NA antibodies with NI tests using strains A/swine/Iowa/15/30 (H1N1), A/turkey/Wisconsin/66 (H9N2), A/whistling swan/Shimane/499/83 Phosphatidylinositol diacylglycerol-lyase (H5N3), A/turkey/Ontario/6118/68 (H8N4), A/duck/Alberta/60/76 (H12N5), A/duck/Czechoslovakia/56 (H4N6), A/chicken/Germany/“N”/49 (H10N7), A/duck/Ukraine/1/63 (H3N8), and A/duck/Memphis/564/74 (H11N9). Non-specific HA inhibitors were removed by treating sera with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan) before carrying out HI tests. Serum samples showing HI and NI titers equal to or higher than 8 and 40, respectively, were defined as positive. Influenza A subtype H3N8 virus was isolated from throat and cloacae specimens from 13 ducks collected from two different farms in Vinh Phuc province (Table 1). Influenza A subtype H5N1 viruses were not isolated in the present study. In the AGID analysis, influenza A-specific anti-NP/M antibodies were detected in 29 (2.6%) of 1106 sera. Antibodies that recognized the recombinant NS1 were found in 15 of the 29 sera in the immunoblot analysis (Fig. 1 and Table 2).