The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×102 vs. 2.3×104 vs. 8.7×104; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.

Natural infections with different subtypes of low pathogenicity avian influenza viruses (LPAIVs) are very common in wild duck populations. Recent outbreaks of high pathogenicity avian influenza virus (HPAIV) H5N1 in Eurasian and African countries stimulated monitoring activities in aquatic wild bird populations. Surveillance mainly focused on virus detection. Only a few serologic investigations have been conducted so far, although such data may retrospectively elucidate epidemiologic patterns of different AIV subtypes in the populations under study. To better understand the immunologic and serologic reactions of mallards after infection with LPAIV, we investigated the AIV type- and subtype-specific antibody dynamics in mallards after different LPAIV infections by hemagglutination inhibition, competitive enzyme-linked immunosorbent assay, and western blot analysis, as appropriate. Four groups of mallard ducks were used: 1) naturally infected birds, 2) birds that were experimentally infected with LPAIV, 3) birds that were immunized with inactivated virus preparations, and 4) negative control birds. Ducks were monitored for up to 15 mo, and serum samples were investigated every 1-4 wk. It could be shown that infection with LPAIV in mallards can be traced serologically over prolonged periods of time.

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In this study, an indirect Newcastle disease virus enzyme-linked immunosorbent assay (ELISA) for waterfowl was evaluated concerning its efficiency and its suitability to monitor the antibody response in Muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) following vaccination with a commercial inactivated NDV vaccine for chickens. Three weeks after vaccination seroconversion was already evident in the ELISA. Comparison of the ELISA results with those of the haemagglutination inhibition (HI) test provided a positive linear correlation between both tests (Pearson's product-moment correlation; r00.652; PB0.001). However, a discrepancy of test results was evident in weeks 7 and 10, with 10 sera of vaccinated birds evaluated negative by HI test but positive by ELISA. Eight of these sera were confirmed to yield avian paramyxovirus specific reactivity by western blot analysis. Relative diagnostic sensitivity and specificity were determined to be 100.0% and 91.7% for the ELISA, compared with 91.1% and 97.2% for the HI test. Thus, the established ELISA represents a suitable alternative to the HI test in the monitoring of the immune response of waterfowl after vaccination, particularly for the analysis of high sample numbers. Further on, the results emphasize the immunogenicity of the inactivated Newcastle disease virus vaccine in domestic geese and Muscovy ducks.

In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species*mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)*and two goose species*domestic goose (Anser anser var. domestica) and redbreasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.