Virological and serological investigation of avian influenza in black-headed gulls captured on a rubbish dump in Zagreb, Croatia

A total of 142 black-headed gulls (BHGs), Chroicocephalus ridibundus, were captured during February and March 2009 at the Zagreb city rubbish dump (45.45 N 16.01 E) in order to collect cloacal swabs and serum samples. Cloacal swabs were tested by virus isolation in embryonated chicken eggs, which resulted in isolation of one avian influenza virus (AIV) that was of the H16 subtype. The collected sera were tested by blocking ELISA for avian influenza antibodies, resulting in 28.2% positive samples, which were retested by haemagglutination inhibition (HI) using H5 and H7 subtype antigens. Only one serum sample was positive for H5 and none for H7 antibodies. Statistically, no significant difference was found between the ages of AIV seropositive birds (chi(2) = 2.08, df = 1, P = 0.15). In contrast, regarding seroprevalence in different months of capture, a higher proportion of positive gulls was found during March than during February (chi(2) = 4.53, df = 1, P = 0.03), especially in younger birds (chi(2) = 7.67, df = 1, P = 0.006). This finding suggests that BHGs might contract AIV infection during their aggregations in large numbers on rubbish dumps or similar feeding sites during the winter. Although only one of the 142 tested birds was positive for H5 subtype antibodies, this finding cannot be neglected seeing that apparently healthy BHGs can carry highly pathogenic AIV of the H5N1 subtype. Nevertheless, the results of our study have shown that BHGs are more often infected with other AIV subtypes and therefore are most likely not the primary carriers of H5 AIV. To our knowledge this is the first detection of H16 AIV in Croatia.