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Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

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  • Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

    Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers
    Yu Yamamoto, Kikuyasu Nakamura, Masatoshi Okamatsu, Ayako Miyazaki, Manabu Yamada, and Masaji Mase
    Author affiliation: National Institute of Animal Health, Tsukuba, Japan

    To the Editor: Free-range domestic ducks can be a key factor in regional spreading of Asian subtype H5N1 avian influenza (AI) virus (1–3). Even asymptomatic domestic ducks can shed the virus continuously from the oral cavity and cloaca (3–5). Therefore, early detection of infected ducks that are shedding the virus would reduce the risk of spreading AI virus (H5N1) in a region where the virus has been endemic in domestic ducks. We previously reported that AI virus (H5N1) can replicate in feather epidermal cells in asymptomatic domestic ducks (6). Feathers are living tissues that are easily collectible from live birds with minimal damage. We now report the usefulness of feathers for virus detection in domestic ducks.

    An experimental infection study was conducted with Japanese domestic ducks (Anas platyrhynchos var. domestica) and influenza A virus (H5N1) A/chicken/Miyazaki/K11/2007 as previously described (6). Three 4-week-old domestic ducks (a–c) were inoculated intranasally with 107 50% egg infectious doses (EID50) of 0.1 mL. All experimental procedures were approved by the Ethics Committee of the National Institute of Animal Health in Japan.

    Inoculated ducks did not show any clinical signs except for persistent corneal opacity on day 3 or later. We collected 3–5 contour feathers, plucked from the body, and 2 sets of oropharyngeal and cloacal swabs from each duck at 24-hour intervals from days 2 through 10 postinoculation (pi). Samples were examined by rapid tests, virus isolation, and reverse transcription–PCR (RT-PCR). Feathers were also examined by immunohistochemical testing.

    On-site rapid tests were performed with a commercial kit, QuickVue Influenza A+B (Quidel Corp., San Diego, CA, USA), which can detect influenza virus nucleoprotein. The first set of swabs was used for rapid tests according to the manufacturer's instructions. We also tested 1–2 sticks of the feather calamus (≈15–30 mg per stick) for rapid tests (Appendix Figure, panel A). Briefly, we put the calamuses into the test tube containing attached reagent solution (340 μL) and chopped them into small pieces with an iris scissor and then placed the test strip in the tube. We obtained the following results: feathers tested positive for influenza A virus from days 3 through 6 pi in 1 duck (a), and on days 3 and 4 pi in 2 ducks (b and c), whereas all oropharyngeal and cloacal swabs were negative (Appendix Figure, panel B).

    For virus isolation, we used a second set of swabs placed in 1 mL of phosphate-buffered saline containing antimicrobial drugs and the remaining 2–3 feather calamuses. Virus titers of swabs and 10% (wt/vol) feather homogenate supernatants were calculated with 10-day-old embryonated chicken eggs and expressed as EID50/mL. Viruses were isolated from the oropharyngeal swabs, cloacal swabs, and feathers of all birds, and feathers tested positive for the virus for a longer period than did the swabs (Table). Although the feather samples used for virus isolation differed from those used in rapid tests, the higher virus titers of feathers in each bird corresponded to the positive period of feathers for rapid tests.

    One-step RT-PCR was performed on the total RNA extracted from the same samples as in virus isolation to detect the H5 AI virus gene (SuperScript One-Step RT-PCR System; Invitrogen, Carlsbad, CA, USA). The 1:10 dilution of RNA templates was used for feathers. The primers used were H5–248–270F and H5–671–647R; the expected product was 424 bp (7). The sensitivity of RT-PCR was slightly higher than that of virus isolation except for the results with cloacal swabs (Table).

    Immunohistochemical testing was performed to detect influenza virus nucleoprotein in the feather tissue by using a rabbit polyclonal antibody (ab22285; Abcam Ltd., Cambridge, UK). Virus antigens were detected in feather epidermal cells from days 3 through 6 pi, and a few stromal cells in the feather pulp on days 3 and 4 pi (Appendix Figure, panel C).

    Our results indicate that larger amounts of viruses can be isolated for a longer time from feathers than from swabs. Therefore, feathers can be considered useful samples for surveillance or diagnostic examination of AI virus (H5N1) in domestic ducks. The epidermis, the outer layer of the feather, is a tissue that has poor host immune response against viral replication (8). As has been observed in virus isolation, viruses may be able to survive longer in differentiated epidermal tissue such as contour feathers.

    The sensitivity of the rapid test was not adequate for swabs, a finding similar to that of other studies (9,10). However, positive results for rapid tests of feather samples only may shed light on the on-site field detection of AI (H5N1) in asymptomatic domestic ducks. When virus shedding from domestic ducks is maintained at a low level of viral load during the infection, selecting the sample with higher viral load and antigens in tissues, such as feathers, can increase the detection rate obtained from on-site examination. Our results show the potential of feathers as candidates for early AI virus (H5N1) detection

    http://www.cdc.gov/eid/content/14/10/1671.htm
    The salvage of human life ought to be placed above barter and exchange ~ Louis Harris, 1918

  • #2
    Re: Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

    > larger amounts of viruses can be isolated for a longer time from feathers than from swabs
    > viruses may be able to survive longer in differentiated epidermal tissue such as contour feathers

    they only consider this wrt. detection of H5N1, but it could also be important
    for spreading H5N1.
    is it similar for other flu-viruses ?

    it could go feather2feather2bill2intestine2feather..
    maybe even through feather-mites
    swans have a preen gland
    to treat their feathers
    I'm interested in expert panflu damage estimates
    my current links: [url]http://bit.ly/hFI7H[/url] ILI-charts: [url]http://bit.ly/CcRgT[/url]

    Comment


    • #3
      Re: Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

      http://www.eurekalert.org/pub_releas...-bfi062510.php


      > concentrates AIVs from water onto birds' bodies.
      > progressive virus "sticking" on feathers occurs because
      > flu-contaminated waters interact with the preen oil gland secretion.

      preened feathers attract flu-viruses swimming in water better
      than unpreened feathers ?

      > Since waterbirds use to spread preen oil over their own (self-preening)
      > or other birds' (allo-preening) plumage, it is easily understandable
      > how these preening activities could facilitate the diffusion of the
      > viruses in nature.

      not so easy for me.
      These birds put their bill onto their preen gland(s?) to fetch the preen
      and then they put the preen onto their feathers with the bill ?
      In this precess they pick up flu-viruses from the (to be preen-improved)
      feathers and put them into their digestive tract or respiratory tract
      where they can replicate.
      Then the increased amount of flu-viruses is released into the water
      by poo where it hopes to attach to preened feathers of other
      birds who happen to swim nearby.

      The birds also put preeen on the feathers of other birds ?
      Their offspring, same species, also other species ?
      The same could be done by water, but maybe by bill it's more efficient.
      When on the feathers it must still go into the body-cells, maybe
      again by the bill, then breathing or swallowing.

      Birds may put viruses from their bill to food or drinking water,
      which then is also used by other birds

      That was for waterfowl, how about chickens,turkeys,sparrows,
      birds of prey, ... mammals

      What mutations make flu-viruses attract to preen ?
      Are these being lost in poultry,swine,human flu ?


      -----------------------------------


      http://www.plosone.org/article/info%...l.pone.0011315

      Can Preening Contribute to Influenza A Virus Infection in Wild Waterbirds?

      ...reading...

      > We consistently detected both viral RNA and infectious AIVs on swabs of
      > preened feathers of 345 wild mallards

      > feather samples (n = 5) and cotton swabs (n = 24) experimentally impregnated
      > with preen oil, when soaked in AIV-contaminated waters, attracted and
      > concentrated AIVs on their surfaces

      depends on the serotype ?
      hmm, hardly. Flu is enveloped, the envelope being stolen from the host-cell-membrane
      However the shape varies by the M-gene, it can be filamentous or spherical.

      > uropygial glands

      Bürzeldrüse

      > Virus characterization by serologic and sequence analyses showed that all isolates
      > were LPAIVs belonging to the H12, H10 (most prevalent), H9, H8, H5, H4, and H3 subtypes

      > consistent presence of viral RNA and infectious AIVs on birds' bodies

      maybe the glands extract viruses which then are put on the feathers
      by the infected birds themselves ?
      ----no----
      > absence of AIVs in uropygial glands of wild birds

      > feather swabs were 2.5 times more often AIV-positive than cloacal swabs
      > whereas VI percentages were 2.3 times higher in cloacal swabs than in feather swabs

      > Our study differs from previous reports on Eurasian H5N1 HPAI virus in domestic
      > duck feathers because we examined mature, rather than growing, feathers.
      I'm interested in expert panflu damage estimates
      my current links: [url]http://bit.ly/hFI7H[/url] ILI-charts: [url]http://bit.ly/CcRgT[/url]

      Comment


      • #4
        Re: Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

        >preened feathers attract flu-viruses swimming in water better
        >than unpreened feathers ?
        Since the preen and the viral envelope both consist of fatty acids, maybe freshly preened feathers bind better with the virus?

        >The birds also put preeen on the feathers of other birds ?
        >Their offspring, same species, also other species ?
        From watching wild birds, I've never seen one species preen another one. Usually species keep to themselves. I can't remember ever seeing 2 adults preening each other, but they must because it's talked about.

        >That was for waterfowl, how about chickens,turkeys,sparrows,
        >birds of prey, ... mammals
        Just about all birds have uropygial glands

        However, apparently what a bird eats dictates what fatty acids are in its preen. Maybe the preen of some types of ducks form a synergy or bind better with flu viruses?

        Just to show the difference, in this study about tropical birds' preen:
        * Thamnophilidae consisted of long chain acids, alcohols, esters, unsaturated hydrocarbons, and isoprenoids.
        * Formicaridae secretions contained exclusively squalene and its derivatives.
        * Pipridae, secretions were made up of complex long chain esters.
        * Dendrocolaptidae secretions consisted of long chain esters of both saturated and monounsaturated acids combined with mono-alcohols
        * Tyrannidae consisted of long chain esters of saturated, mono- and tri-unsaturated acids with mono- and di-ols that were less complex.

        Higher molecular weight saturated and unsaturated components in tropical species suggest that the volatility of the secretion components is correlated to environmental temperatures experienced by the birds.

        http://www.sciencedirect.com/science...3f6dafd57974d3
        The salvage of human life ought to be placed above barter and exchange ~ Louis Harris, 1918

        Comment


        • #5
          Re: Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

          Viral infectivity persisted in the feathers for 160 days at 4°C and
          for 15 days at 20°C.
          http://www.ncbi.nlm.nih.gov/pubmed/20581177
          I'm interested in expert panflu damage estimates
          my current links: [url]http://bit.ly/hFI7H[/url] ILI-charts: [url]http://bit.ly/CcRgT[/url]

          Comment

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