Susceptibility of Canada geese to highly pathogenic avian influenza virus

Emerging Infectious Diseases, Dec, 2007 by John Pasick, Yohannes Berhane, Carissa Embury-Hyatt, John Copps, Helen Kehler, Katherine Handel, Shawn Babiuk, Kathleen Hooper-McGrevy, Yan Li, Quynh Mai Le, Song Lien Phuong

Migratory birds have been implicated in the long-range spread of highly pathogenic avian influenza (HPAI) A virus (H5N1) from Asia to Europe and Africa. Although sampling of healthy wild birds representing a large number of species has not identified possible carriers of influenza virus (H5N1) into Europe, surveillance of dead and sick birds has demonstrated mute (Cygnus olor) and whooper (C. cygnus) swans as potential sentinels. Because of concerns that migratory birds could spread H5N1 subtype to the Western Hemisphere and lead to its establishment within free-living avian populations, experimental studies have addressed the susceptibility of several indigenous North American duck and gull species. We examined the susceptibility of Canada geese (Branta canadensis) to HPAI virus (H5N1). Large populations of this species can be found in periagricultural and periurban settings and thus may be of potential epidemiologic importance if H5N1 subtype were to establish itself in North American wild bird populations.

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Wild aquatic birds belonging to the orders Anseriformes and Charadriiformes have long been recognized as the natural reservoirs for all influenza type A viruses (1). Spread from such wild birds to domestic poultry and various mammalian species occurs intermittently. Most viruses that initially infect domestic poultry will replicate only within respiratory or digestive tracts and cause no or very mild disease, referred to as low-pathogenic avian influenza (LPAI) (2). However, once introduced into domestic poultry, some viruses of the H5 and H7 hemagglutinin (HA) subtypes can mutate to a highly pathogenic form, producing a systemic infection referred to as highly pathogenic avian influenza (HPAI) (2). The hypothesis that HPAI H5 and H7 viruses emerge from low-pathogenic precursors only after the H5 and H7 LPAI precursors have been introduced into domestic poultry has been supported by work demonstrating that HPAI viruses do not appear to form separate phylogenetic lineages in waterfowl (3). Except for A/tern/South Africa/1961 (H5N3), no evidence existed before 2002 that an HPAI virus could cause deaths or be maintained within wild bird populations.

In late 2003, an HPAI (H5N1) outbreak of unprecedented magnitude began in Southeast Asia. Approximately 1 year before this, a high mortality rate attributed to HPAI virus (H5N1) was observed in waterfowl and other wild birds in Hong Kong (4). This led to speculation that wild birds may have contributed to the virus spread. In the spring of 2005, mass dieoffs of wild birds occurred at Qinghai Lake, People's Republic of China (5,6), an event heralded as the beginning of the long-range spread of HPAI (H5N1) from Asia into Europe and subsequently Africa, with migratory birds implicated as playing a role (7,8). Identifying which species of birds were involved in this spread is not only of academic interest but also of practical importance to surveillance activities because of concerns that migratory birds could also introduce H5N1 subtype into the Western Hemisphere. We examined the susceptibility of Canada geese (Branta canadensis) to infection with an HPAI virus (H5N1) and the effect that pre-exposure to an LPAI virus (H5N2) has on clinical disease, pathology, and virus shedding.

Materials and Methods

Viruses

The influenza viruses used in this study included A/ chicken/Vietnam/14/2005 (H5N1) and A/mallard/British Columbia/373/2005 (H5N2). Vietnam/05 stocks were grown and titrated on Japanese quail fibrosarcoma (QT-35) cells. This isolate bears a PQRERRRKR/GLF [HA.sub.0] cleavage site (GenBank accession no. EF535027), has an intravenous pathogenicity index of 2.97, and produced a 100% mortality rate in oronasally inoculated leghorn chickens receiving [10.sup.5], [10.sup.4], and [10.sup.3] PFU by 3, 4, and 6 days postinfection (dpi), respectively. British Columbia/05 stocks were grown and titrated in 9-day-old chicken embryos. Prior characterization of this isolate demonstrated that it has a PQRETR/GLF [HA.sub.0] cleavage site (GenBank accession no. DQ826532) typical for LPAI viruses.

Animals

Twenty-two Canada geese were captured with the permission of Environment Canada (Canadian Wildlife Service permit no. CWS06-M009) and were handled and cared for in accordance with Canadian Council on Animal Care guidelines and the animal use protocol approved by the Institutional Animal Care Committee. The geese consisted of 11 adult (6 male + 5 female) and 11 young-of-year (6 male + 5 female) birds. The latter were estimated to be [approximately equal to] 40 days of age at capture. Adult and juvenile birds were randomly assembled into 3 experimental groups, and each group subsequently housed in separate Biosafety Level-3 biocontainment cubicles: 1) a control group comprising 1 juvenile + 1 adult bird, 2) a pre-exposure group comprising 5 juvenile + 5 adult birds, and 3) a naive group comprising 5 juvenile + 5 adult birds.

After a 3-week acclimation period, the pre-exposure group was inoculated with [10.sup.6] 50% egg infectious dose ([EID.sub.50]) of British Columbia/05 applied to the nares, oral cavity, and cloaca. Twenty-eight days later, pre-exposure and naive groups were challenged with 1.7 x [10.sup.5] PFU of Vietnam/05 applied to the nares, oral cavity, and eye. The control group received a sham inoculum of minimal essential medium. Timed necropsies involving 1 juvenile and 1 adult bird from pre-exposure and naive groups were performed on days 3 and 6 postchallenge (dpc). All remaining birds were either humanely euthanized when moribund or allowed to survive until 20 or 21 days if they showed mild disease or remained clinically normal.