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Supplementary Information

Influenza A viruses of migrating wild aquatic birds in North America

Scott Krauss, David Walker, S. Paul Pryor, Larry Niles, Li Chenghong, Virginia S. Hinshaw, and Robert G. Webster

Vector Borne Zoonotic Dis. 2004 Fall;4(3):177-89.

Running Title: Influenza in wild aquatic birds
Address for correspondence:
Robert G. Webster
Division of Virology
Department of Infectious Diseases
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
FAX: (901) 523-2622
Email: robert.webster@stjude.org


Surveillance of North America's wild ducks and shorebirds for 26 and 16 years, respectively, revealed differences in the prevalence of orthomyxoviruses between these hosts. Shorebirds had a high frequency of influenza A virus isolation during their northern migration, while wild ducks had high virus isolation frequencies during their southern migration. Some subtypes of influenza occurred regularly in both hosts with a 2-year periodicity, whereas others rarely occurred. Hemagglutinin subtypes H1 through H12 occurred in both hosts; H13 occurred only in shorebirds; and H14, H15, and influenza B and C never were detected. Shorebirds manifested a broader range of subtypes suggesting that shorebirds are the leading source of some viruses (such as H5) which are isolated less frequently from wild ducks. The viruses reported in this study are available for genomic study to determine whether prediction of host range or pandemic potential is possible.

Materials and Methods

Wild duck species sampled

Surveillance of wild ducks in Alberta, Canada was initiated in 1976 in conjunction with the Canadian Wildlife Service (Prairie and Northern Region) from late July through early September. This period of months is the time when the breeding season is completed, many of the adult ducks have molted, and the birds are staging before they migrate south to their winter habitat. During the 26 years of the study 17592 cloacal swabs were collected from wild ducks.

The major species sampled were:

  • mallard ( Anas platyrhynchos ) 64.4%
  • northern pintail ( Anas acuta ) 17.7%
  • blue-winged teal ( Anas discors ) 10.7%
  • redhead ( Aythya Americana ) 2.3%
  • American green-winged teal ( Anas crecca ) 1.1%
  • American wigeon ( Anas Americana ) 1.1%.
  • The remaining 2.7% of the sampled birds was represented by:
  • black duck ( Anas rubripes )
  • bufflehead ( Bucephala albeola )
  • Canada goose ( Branta canadensis )
  • canvasback ( Aythya valisineria )
  • cinnamon teal ( Anas cyanoptera )
  • common goldeneye ( Bucephala clangula )
  • coot ( Fulica americana )
  • gadwall ( Anas strepera )
  • lesser scaup ( Aythya affinis )
  • ruddy duck ( Oxyura jamaicensis )
  • northern shoveler ( Anas clypeata )

Virus isolation and serological identification of subtype

The samples were thawed, and 0.3 mL of fluid from the vial was mixed with 0.3 mL PBS containing high concentrations of antibiotics (penicillin G, 4000 U/mL; streptomycin sulfate, 800 U/mL; polymyxin B, 400 U/mL; and gentamycin sulfate, 0.1 mg/mL). For each sample, the allantoic cavities of three 11-day-old embryonated chicken eggs were injected with 0.2 mL of inoculum. The eggs were incubated at 35 °C for 48 to 72 h, and the embryos examined for viability. The eggs then were chilled at 4 o C for 12 to 16 h (overnight). The allantoic fluid of each egg was tested for the presence of agglutinating virus by the hemagglutination assay. The positive samples were checked for bacterial contamination by streaking blood-agar plates and then incubating the plates at 37 o C for 48 h. Bacterial contamination was removed by passing the allantoic fluid through a 0.2- m M syringe filter.

To determine the hemagglutinin (HA) subtype, samples that were positive for hemagglutination were tested in hemagglutination inhibition (HAI) assays, and the neuraminidase (NA) subtype was determined by the neuraminidase inhibition (NI) assay. Hemagglutinating agents were identified on the basis of their antigenic characteristics (inhibition) by using monospecific antisera prepared against the surface antigens of reference influenza A viruses. When monospecific serum was not available, chicken or rabbit antiserum prepared against whole-virus antigen was used.

Mixing of birds and viruses: overlapping ranges

There are many common North American waterfowl species which have western and eastern palearctic distribution, at least seventeen (del Hoyo et al. 1992). With so many species having distributions throughout large portions of the northern hemisphere, it is difficult to imagine that regular mixing does not occur. Waterfowl banding over many years and more recently satellite telemetry have shown that many birds marked in North America move to other continents for some portion of the year. Probably most of the mixing occurs between Alaska and Northeastern Siberia since they are so close to each other. Some Northern Pintails marked with satellite radios in California have migrated to Siberia in the spring and returned to California in the fall (U.S.G.S.,2003). Some snow geese that breed on Wrangel Island off the coast of Siberia and on the mainland of Siberia were collared (a plastic neckband with a unique three digit code)on the breeding grounds and later observed in many areas of western North America (Kirbes et al. 1999). Most Snow geese from Wrangel Island, 50,000+, winter along the west coast of North America (Kirbes et al. 1999). Six of 20 sandhill cranes marked with a satellite radio in the spring of 1998 and 1999 in central Nebraska migrated to Siberia for the breeding season and returned to winter in the southern United States (U.S.G.S., 1998). Blue-winged teal banded by one of the authors in Alberta are regularly recovered in Central America, the Caribbean islands, Venezuela and Columbia. Birds that leave North America for some portion of the year regularly mix with North American waterfowl in staging areas during migration across North America and on the wintering areas in the southern United States. There are many more records of migratory birds moving between continents. Most waterfowl winter in large flocks, so mixing of viruses is inevitable.

del Hoyo, J, Elliott, A., & Sargatel, J., eds., 1992. Handbook of the Birds of the World, vol. 1, Lynx Edicions, Barcelona. Kerbes, R.H., Meeres, K.M., & Hines, J.E., eds., Distribution, survival and numbers of Lesser Snow Geese of the Western Canadian Arctic and Wrangel Island, Russia, 1999, Can Wildl Ser, Occas Pap, Number 98, Ottawa, Ontario U.S. Geological Survey. 2003. Discovery for recovery.

Western Ecological Research Center
U.S. Geological Survey. 1998. Operation crane watch.

Northern Prairie Wildlife Research Center – no longer available