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Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during the Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the possible animal health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall, 147 cloacal swabs were sampled in the Sardinia region from June 2009 to September 2011. Samples were obtained from 12 taxonomic orders, including 16 families and 40 species of birds. Based on the endangered host status or on the ecology of the host-virus interaction, samples were categorized into three groups of species: 1) endemic, endangered, or both (17 species); 2) potential reservoir (21 species); and 3) potential spillover (two species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus matrix gene amplification. Forty-one serum samples were tested by nucleoprotein-enzyme-linked immunosorbent assay (NP-ELISA) for antibodies against influenza A virus nucleoprotein and by hemagglutination inhibition assay for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, whereas two weak seropositive results were detected by NP-ELISA in a mallard (Anas platyrhynchos) and in a yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study suggests a naïve status in these avian populations. These data provide new information on AIV circulation in Sardinia's wild birds that could be applied to implement conservation strategies for threatened species.

Background: infectious low pathogenic avian influenza viruses (LPaIVs) have been recently detected on feathers of wild ducks. Laboratory trial results suggested that the preen oil gland secretion, covering waterbirds’ feathers, may attract and concentrate virus particles from aIV-contaminated waters to birds’ bodies. We evaluated whether ducks can become infected by the ingestion of preen oil-associated viral particles, experimentally smeared on their plumage. In addition, we compared virologic and serologic results obtained from mallards whose feathers were experimentally infected, with those from wild mallards naturally carrying aIVs on feathers.Methods: we experimentally coated 7 mallards (anas plathyrynchos) using preen oil mixed with a LPaIV (h10n7 subtype), and housed them for 45 days with a control, uncoated duck. cloacal, oropharyngeal and feather swabs were collected from all birds and examined for aIV molecular detection and isolation. Blood samples were also taken to detect influenza specific antibodies. In addition, sera from 10 wild mallards, carrying on feathers infectious LPaIV h10n7, were examined.Results: virologic and serologic results indicated that through self- and allopreening all the birds experimentally coated with the preen oil/aIV mix and the control duck ingested viruses covering feathers and became infected. Virus isolation from feathers was up to 32 days post-coating treatment. one out of 8 wild mallards showing antibodies against type a influenza virus was seropositive for h10 subtype too.Conclusions: our experimental and field results show evidences suggesting that uninfected birds carrying viruses on their feathers, including immune ones, might play an active role in spreading aIV infection in nature. For this reason, routine aIV surveillance programs, aimed at detecting intestinal and/or respiratory viruses, should include the collection of samples, such as feather swabs, enabling the detection of viruses sticky to preened birds’ bodies....

Cloacal swabs collected from mallard ducks (Anas platyrhynchos) experimentally infected with a H7N1 avian influenza strain were examined by Reverse Transcription Polymerase Chain Reaction to detect the influenza A virus. Reverse Transcription Polymerase Chain Reaction was compared with standard methods: inoculation of embryonated chicken eggs and inoculation of three established cell lines: Newborn Swine Kidney cells, Newborn Pig Trachea cells and Madine Darby Canine Kidney cells. Reverse Transcription Polymerase Chain Reaction was performed using a set of primers based on conserved regions of the matrix and nucleoprotein genes.

Since the “bird flu” incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002–2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived “in toto” from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.