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Prevalence of avian influenza infection in free-range mule ducks (a cross between Muscovy [Cairina moschata domesticus] and Pekin ducks [Anas platyrhychos domesticus]) is a matter of concern and deserves particular attention. Thus, cloacal swabs were collected blindly from 30 targeted mule flocks at 4, 8, and 12 wk of age between October 2004 and January 2005. They were stored until selection. On the basis of a positive H5 antibody detection at 12 wk of age with the use of four H5 antigens, the samples from eight flocks were selectively analyzed. Positive samples were first screened with a matrix gene-based real-time reverse transcriptase-polymerase chain reaction assay before virus isolation. Eight avian influenza subtypes (H5N1, H5N2, H5N3, H6N2, H6N8, and H11N9) and three avian paramyxovirus type 1 viruses were isolated. All 11 are characterized as low pathogenicity (LP) and avirulent, respectively, by in vivo tests and, when relevant, nucleotide sequencing of the hemagglutinin (or fusion [F]) protein cleavage site. Regarding H5 isolates, all of their eight genes belong to the avian lineage and some particular genetic traits were determined. H5 genes were fully sequenced and phylogenetically analyzed; they all belong to the Eurasian lineage, lack additional glycosylation sites, and do not cluster, suggesting separate introductions from the wild reservoir. None were grouped with the Asian isolates. The N1 gene (H5N1 isolate) was very close genetically to an Italian LP-H7N1 gene. Antigenic relationships between these H5 isolates and others were assessed comparatively by crossed hemagglutination inhibition tests. All these data are very useful to control the evolution of H5 viruses at the genetic and antigenic level to better understand the source of new outbreaks (new introductions from wild birds or the result of spread among poultry) and to assess the immunity afforded by available vaccines. These data are useful also to update antigens for avian influenza survey and to choose the most suitable vaccine in the case of preventive vaccination of ducks.

Control of H5/H7 low-pathogenic avian influenza (LPAI) virus circulation is a major issue regarding animal and public health consequences. To improve vaccines and to prevent vaccinated poultry from becoming infected and from shedding wild viruses, we initiated studies targeting prevention of H7 infection through DNA vaccines encoding H7 and M1 viral proteins from an Italian H7N1 LPAI virus isolated from poultry in 1999. More recently, we expressed recombinant H7 and M1 proteins in the baculovirus system to assess whether they might enhance immunity when given as a boost after DNA vaccination. The protection afforded by three vaccine combinations-DNA/DNA, DNA/protein, protein/protein-given 3 wk apart were experimentally compared in 20 specific-pathogen-free chickens per group. Ten days after the boost, chickens were challenged with a homologous (Italian H7N1 LPAI) or heterologous (French H7N1 LPAI isolated from mallards in 2001) virus. Tracheal and cloacal shedding was measured by a matrix gene (M)-based real-time reverse transcription polymerase chain reaction assay and compared with that displayed by unvaccinated infected controls. After the homologous challenge, chickens of every vaccinated group displayed a significant decrease in cloacal shedding, whereas tracheal shedding was not significantly reduced in the protein/protein group. After the heterologous challenge, only the DNA/DNA group showed a nonsignificant decrease in tracheal shedding. According to these two trials, prime-boost DNA/protein vaccination appeared be more advantageous. Further development could be aimed at improving protein expression, shifting subtype (H5), and assessing the interest of proteins as a boost of recombinant vaccines.

In Asia, domestic ducks have been shown to play a pivotal role in H5 high-pathogenicity avian influenza virus transmission. We have also observed that the same situation may exist for H5 low-pathogenicity avian influenza (LPAI) virus. No data are available regarding the protection afforded by commercial inactivated vaccines against H5 LPAI virus infection in ducks, and two preliminary experiments using commercial inactivated vaccines gave poor results. Virus-like particles (VLPs) have been shown to be immunogenic in different species. With regard to the influenza model, the matrix (M) protein has been shown to be necessary for the formation of VLPs. In order to attempt to develop a VLP influenza vaccine expressing hemagglutinin and neuraminidase (NA) of interest, we generated a triple recombinant baculovirus (rB) expressing three structural proteins: H5, N3, and M, derived from a recent French LPAI virus strain. Although the three proteins were successfully expressed in rB-infected cells and displayed the expected biological activity, no VLPs were observed. Despite this result, the protection afforded to ducks by rB-infected cell lysates was assessed and was compared with the protection afforded by an inactivated commercial H5N9 vaccine. For this purpose, specific-pathogen-free Muscovy ducks (15 per group) received rB-infected cell lysates (3 wk apart), while a second group received the H5N9 vaccine. Ten days after the boost, a homologous virus challenge was implemented. Both vaccines induced positive hemagglutination inhibition titers and M immune response, whereas lysates of rB-infected cells elicited NA immune response. Tracheal and cloacal sheddings were measured using M-based real-time-reverse transcription-polymerase chain reaction and were compared with the sheddings of vaccinated and unvaccinated infected controls. Lysates of rB-infected cells afforded a significant decrease of cloacal shedding and a delayed peak of tracheal shedding, whereas the inactivated commercial vaccine afforded a significant decrease of tracheal shedding only.