Susceptibility of Canada Geese (Branta canadensis) to highly pathogenic avian influenza virus (H5N1).
ABSTRACT 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.
- [show abstract] [hide abstract]
ABSTRACT: Outbreaks of highly pathogenic H5N1 avian influenza have occurred in Hong Kong in chickens and other gallinaceous poultry in 1997, 2001, twice in 2002 and 2003. High mortality rates were seen in gallinaceous birds but not in domestic or wild waterfowl or other wild birds until late 2002 when highly pathogenic H5N1 avian influenza occurred in waterfowl (geese, ducks and swans), captive Greater Flamingo (Phoenicopterus ruber) and other wild birds (Little Egret Egretta garzetta) at two waterfowl parks and from two dead wild Grey Heron (Ardea cinerea) and a Black-headed Gull (Larus ridibundus) in Hong Kong. H5N1 avian influenza virus was also isolated from a dead feral pigeon (Columba livia) and a dead tree sparrow (Passer montanus) during the second outbreak. The first waterfowl outbreak was controlled by immediate strict quarantine and depopulation 1 week before the second outbreak commenced. Control measures implemented for the second outbreak included strict isolation, culling, increased sanitation and vaccination. Outbreaks in gallinaceous birds occurred in some live poultry markets concurrently with the second waterfowl outbreak, and infection on a chicken farm was detected 1 week after the second waterfowl park outbreak was detected, on the same day the second grey heron case was detected. Subsequent virus surveillance showed the outbreaks had been contained.Avian Pathology 11/2004; 33(5):492-505. · 1.73 Impact Factor
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ABSTRACT: Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyer's patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.Veterinary Pathology 04/2007; 44(2):137-43. · 1.93 Impact Factor
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ABSTRACT: Susceptibilidad de la gaviota gallega (Larus atricilla) a los virus H5N1 y H5N3 de influenza aviar de alta patogenicidad. En este estudio se describen las manifestaciones clínicas, lesiones macro y microscópicas y distribución de los antígenos virales en gaviotas gallegas (Larus atricilla) jóvenes inoculadas por la vía intranasal con la cepa del virus de influenza A/tern/South Africa/61 (H5N3) ó la cepa A/pollo/Hong Kong/220/97 (H5N1), los cuales son considerados como altamente patogénicos en pollos. No se observó morbilidad ni mortalidad en ninguna de las de gaviotas inoculadas con los virus durante los 14 días de la duración del experimento. Las lesiones microscópicas ocasionadas por estos virus fueron leves, observándose solo aumento de la opacidad de los sacos aéreos (2/6), esplenomegalia (2/6) y máculas en el páncreas (1/6) en las aves inoculadas con el serotipo H5N3, mientras que el virus del serotipo H5N1 solo causó aumento de la opacidad de los sacos aéreos (2/8) y edema de la conjuntiva ocular (2/8). Las lesiones microscópicas en las aves inoculadas con el virus del serotipo H5N3 incluyeron inflamaciones leves y moderadas de los sacos aéreos con infiltración de heterófilos o de tipo linfoplasmacítica (6/6), neumonía intersticial leve o moderada (3/6), hepatitis y pancreatitis necrótica moderada a los 14 días después de la inoculación (2/6), y solo se encontraron antígenos virales asociados a las lesiones de hígado y páncreas, mediante la técnica de inmunohistoquímica. En las aves inoculadas con el serotipo H5N1 solo se encontraron lesiones inflamatorias en los sacos aéreos (3/8) y pulmones (3/8). Ambos virus fueron aislados en títulos bajos (<101.68 dosis letal embrionaria) a partir de hisopos oro-faríngeos y de cloaca hasta los 7 días después de la inoculación, a partir de muestras de pulmón y riñón tomadas de una de las aves inoculadas con el virus del tipo H5N3 a los 14 días después de la inoculación y a partir de muestras de pulmón de una de las aves inoculadas con el virus del tipo H5N1 a los 7 días después de la inoculación. Se detectaron anticuerpos específicos contra el virus de influenza mediante la técnica de precipitación en agar gel a los 14 días después de la inoculación en solo dos de las aves inoculadas con el virus del serotipo H5N3, sin que los mismos pudieran ser detectados en las aves inoculadas con el virus del serotipo H5N1. Abbreviations: AGP = agar gel precipitin; AI = avian influenza; BHI = brain–heart infusion; BSL-3Ag = biosafety level 3 agriculture; chicken/HK = A/chicken/Hong Kong/220/97 (H5N1); DPI = days postinoculation; ELD50 = mean embryo lethal dose; H&E = hematoxylin and eosin; HPAI = highly pathogenic avian influenza; IHC = immunohistochemistry; i.n. = intranasally; LPM = live poultry market; M = matrix protein; NP = nucleoprotein; OP = oropharyngeal; SEPRL = Southeast Poultry Research Laboratory; tern/SA = A/tern/South Africa/61 (H5N3)Avian Diseases 01/2009; · 1.73 Impact Factor
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1821 1821
Migratory birds have been implicated in the long-range
spread of highly pathogenic avian infl uenza (HPAI) A virus
(H5N1) from Asia to Europe and Africa. Although sampling
of healthy wild birds representing a large number of spe-
cies has not identifi ed possible carriers of infl uenza 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 mi-
gratory 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 periagri-
cultural and periurban settings and thus may be of potential
epidemiologic importance if H5N1 subtype were to establish
itself in North American wild bird populations.
ognized as the natural reservoirs for all infl uenza type A
viruses (1). Spread from such wild birds to domestic poul-
try and various mammalian species occurs intermittently.
Most viruses that initially infect domestic poultry will rep-
licate only within respiratory or digestive tracts and cause
ild aquatic birds belonging to the orders Anseri-
formes and Charadriiformes have long been rec-
no or very mild disease, referred to as low-pathogenic
avian infl uenza (LPAI) (2). However, once introduced into
domestic poultry, some viruses of the H5 and H7 hemag-
glutinin (HA) subtypes can mutate to a highly pathogenic
form, producing a systemic infection referred to as highly
pathogenic avian infl uenza (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 unprece-
dented 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 mi-
gratory 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 migrato-
ry birds could also introduce H5N1 subtype into the West-
ern Hemisphere. We examined the susceptibility of Canada
S usc eptibility of Canada Geese
(Branta c anadensis) to Highly
Pathogenic Avian Infl uenza
V irus (H5N1)
John Pasick,* Yohannes Berhane,* Carissa Embury-Hyatt,* John Copps,* Helen Kehler*
Katherine Handel,* Shawn Babiuk,* Kathleen Hooper-McGrevy,* Yan Li,† Quynh Mai Le,‡
and Song Lien Phuong§
*Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada;
†Public Health Agency of Canada, Winnipeg, Manitoba, Canada;
‡National Institute of Hygiene and Epidemiology, Hanoi, Vietnam;
and §National Center for Veterinary Diagnosis, Hanoi, Vietnam
geese (Branta canadensis) to infection with an HPAI vi-
rus (H5N1) and the effect that pre-exposure to an LPAI
virus (H5N2) has on clinical disease, pathology, and virus
Materials and Methods
The infl uenza viruses used in this study included A/
chicken/Vietnam/14/2005 (H5N1) and A/mallard/Brit-
ish Columbia/373/2005 (H5N2). Vietnam/05 stocks were
grown and titrated on Japanese quail fi brosarcoma (QT-35)
cells. This isolate bears a PQRERRRKR/GLF HA0 cleav-
age site (GenBank accession no. EF535027), has an intra-
venous pathogenicity index of 2.97, and produced a 100%
mortality rate in oronasally inoculated leghorn chickens re-
ceiving 105, 104, and 103 PFU by 3, 4, and 6 days postinfec-
tion (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 HA0 cleavage site (GenBank accession no.
DQ826532) typical for LPAI viruses.
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 con-
sisted of 11 adult (6 male + 5 female) and 11 young-of-year
(6 male + 5 female) birds. The latter were estimated to be
≈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 106 50% egg infectious dose
(EID50) of British Columbia/05 applied to the nares, oral
cavity, and cloaca. Twenty-eight days later, pre-exposure
and naïve groups were challenged with 1.7 × 105 PFU of
Vietnam/05 applied to the nares, oral cavity, and eye. The
control group received a sham inoculum of minimal essen-
tial medium. Timed necropsies involving 1 juvenile and 1
adult bird from pre-exposure and naïve groups were per-
formed 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.
ELISA and Hemagglutination-Inhibition (HI) Assays
Group A specifi c nucleoprotein (NP) antibodies were
detected with a competitive ELISA as described previous-
ly (9). H5-specifi c antibodies were detected by microtiter
plate HI test that used 4 HA U of A/duck/British Colum-
bia/26–6/2005 (H5N2) and chicken erythrocytes.
Virus Neutralization Assay
We incubated 200 EID50 of Vietnam/05 with an equal
volume of 2-fold serially diluted test serum (1:4 to 1:512),
incubated for 60 min at 37°C, and then used it to inoculate
9-day-old chicken embryos through the allantoic cavity.
Egg deaths and HA titers were monitored and virus neu-
tralization titers determined.
Real-Time Reverse Transcription–PCR (RT-PCR)
Specimens were stored at –70°C before RNA was
extracted. Total RNA was extracted from 0.5 mL of 10%
(wt/vol) tissue emulsions or clarifi ed swab specimens by
using an RNeasy Mini Kit (QIAGEN, Mississauga, On-
tario, Canada). A semiquantitative real-time RT-PCR (10)
that targets the M1 gene of infl uenza A virus segment 7
was conducted. Full-length, in vitro transcribed segment 7
RNA, serially diluted in buffer, was run with each assay to
give a semiquantitative estimate of the viral load in each
Formalin-fi xed, deparaffi nized, and rehydrated 5-μm
tissue sections were quenched for 10 min in aqueous 3%
H2O2, rinsed in MilliQ water, and placed into Tris-buffered
saline plus Tween (TBST) buffer for 5 min. Sections were
pretreated with proteolytic enzyme (DakoCytomation, Car-
pinteria, CA, USA) for 15 min, rinsed twice with TBST,
and incubated for 1 h with a monoclonal antibody specifi c
for infl uenza A nucleoprotein (Clone 1331, Biodesign, Sas-
co, ME, USA) at a dilution of 1:5,000. The sections were
washed with TBST, then incubated for 30 min with the
Envision + anti-mouse (horse radish peroxidase–labeled)
polymer kit (DakoCytomation), followed by a TBST rinse.
Diaminobenzidine was used as the substrate chromagen,
and slides were counterstained with Gill’s hematoxylin.
A/mallard/British Columbia/373/2005 (H5N2)
Upon arrival, 12 of 12 juvenile geese tested negative
and 10 of 12 adult geese tested positive for infl uenza A
virus NP antibodies (Table 1). To determine the HA sub-
type specifi city of the seropositive birds, HI assays were
1822 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007
Canada Geese, Highly Pathogenic Avian Infl uenza
run with 4 HA U of the following antigens: H1N1 (A/Ck/
BC/3/98); H2N9 (A/Pintail/AB/293/77); H4N6 (A/Dk/
BC/14/99); H5N2 (A/mallard/BC/373/05); H6N1 (A/Tk/
ON/844–2/04); and H7N3 (A/Ck/BC/514/04). All tests
were negative, indicating that the birds did not appear to
have pre-existing H5-specifi c antibodies. Real-time RT-
PCR–negative cloacal swab specimens indicated that the
birds were also not actively infected.
After inoculation with 106 EID50 of British Colum-
bia/05, all birds remained clinically normal. The juvenile
birds gained weight, but 3 of 5 adult birds had a 6%–10%
loss of bodyweight after infection. Cloacal swabs from ju-
venile birds were real-time RT-PCR positive at 3 dpi; swabs
from adult birds were negative (oropharynegeal swabs not
tested). At 6 and 10 dpi, cloacal and oropharyngeal swabs
from both juvenile and adult birds were real-time RT-PCR
negative, indicating that viral shedding was brief. Although
most of the British Columbia/05 infected birds developed
H5-specifi c HI antibody titers (Table 1), these sera did not
neutralize Vietnam/05 in a chicken embryo–based neutral-
A/chicken/Vietnam/14/2005 (H5N1) Challenge
Twenty-eight days after pre-exposure to British Co-
lumbia/05, birds in the pre-exposure and naïve groups were
challenged with Vietnam/05. Juvenile birds were estimated
to be 13 weeks of age at this time. Adult birds in the British
Columbia/05 pre-exposure group exhibited mild decreases
in feed consumption and mild depression 5–7 dpc. Except
for 1 bird with a positive oropharyngeal swab sample at
6 dpc, oropharyngeal and cloacal swab specimens for the
adults tested real-time RT-PCR negative at 2, 3, and 6 dpc.
Juvenile birds in the British Columbia/05 pre-exposure
group exhibited clinical signs similar to those of the adults
with the addition of transient nervous signs manifested as
repetitive jerking head movements. Viral shedding, as de-
termined by real-time RT-PCR and confi rmed by isolation,
was detected at 3 dpc in oropharyngeal swab samples in
3 of 5 birds and in a cloacal swab sample in 1 of 5 birds.
Complete necropsies showed no gross lesions in juvenile
or adult birds at 3, 6, 11, and 21 dpc. The cerebrum, brain
stem, and spinal cord of juvenile birds exhibited low levels
of viral nucleic acid at 11 and 21 dpc (online Appendix
Table, available from www.cdc.gov/EID/content/13/12/
1821-appT.htm). Other organs were weakly positive by
real-time RT-PCR to varying degrees.
In contrast, juvenile birds in the naïve group showed
100% morbidity after Vietnam/05 challenge; clinical signs
included severe depression, inappetence, bright yellow di-
arrhea, ruffl ed feathers, hunched posture, repetitive jerking
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1823
Table 1. NP and H5 antibody levels in juvenile and adult Canada geese*
0 dpi cELISA
(NP % inhibition)
852S/27R Neg (20)
853S/28R Neg (13)
856S/31R Neg (21)
858S/33R Neg (22)
859S/34R Neg (19)
851S/26R Neg (24)
854S/29R Neg (23)
855S/30R Neg (22)
860S/35R Neg (24)
861S/36R Neg (20)
857S/32Y Neg (18)
842S/42Y Pos (93)
844S/44Y Neg (23)
845S/45Y Pos (58)
846S/46Y Pos (76)
847S/47Y Pos (74)
840S/40Y Pos (45)
841S/41Y Neg (22)
843S/43Y Pos (78)
848S/48Y Pos (39)
849S/49Y Pos (93)
850S/50Y Pos (85)
*NP, nucleoprotein; cELISA, competitive ELISA; dpi, days postinfection; Neg, negative (<30% inhibition); Pos, positive (?30% inhibition); HI,
hemagglutinin inhibition; ND, not determined.
†4 hemagglutinin units of A/duck/British Columbia/26–6/2005 (H5N2) used in assay.
‡Euthanized or died before day 20–21 postinoculation with virus (H5N1).
0 dpi H5 HI
14 dpi (H5N2) cELISA
(NP % inhibition)
21 dpi (H5N2)
H5 HI assay†
20–21 dpi (H5N1)
20–21 dpi (H5N1)
H5 HI assay†
Euthanized or died‡
Pos (46% inhibition)
Pos (48% inhibition)
Pos (46% inhibition)
Pos (63% inhibition)
Pos (99% inhibition)
Pos (98% inhibition)
Pos (98% inhibition)
head movements, weakness, staggering gait, distressed vo-
calization, wing droop, and terminal coma. All birds died
or were humanely euthanized by 5 dpc. Viral nucleic acid
was detected in the oropharyngeal swab specimens col-
lected at all time points before euthanasia or death; cloacal
swab specimens were not as consistently positive. Adult
birds also showed 100% morbidity but with clinical signs
and viral shedding less pronounced than that observed in
juveniles. Necropsies were performed on 2 adults on days 3
and 5; the remaining 3 birds survived until 20 dpc.
Gross pathologic lesions included congestion of the
mucosal surface of the trachea, edema and multifocal pin-
point hemorrhages on the serosal surface of the pancreas,
splenomegaly, hemorrhage within the ceca, conjunctivitis,
congestion of the meninges and cerebral blood vessels,
and hemorrhages on the surface of the brain. Virtually all
tissues collected from juvenile birds in the naïve group
were real-time RT-PCR positive; heaviest viral loads were
found in cerebrum, brain stem, and spinal cord. Adult bird
841S/41Y, which required euthanasia at 5 dpi, also had
levels of viral nucleic acid in the central nervous system
(CNS) comparable to those found in naïve juveniles. This
was one of the adult birds with no pre-existing NP antibod-
ies at the beginning of the acclimation period (Table 1).
Viral nucleic acid was found in the CNS of a second adult
(840S/40Y), euthanized at 20 dpc, but at levels that were
5–7 logs lower than those found in juveniles or the adult
bird euthanized at 5 dpc.
Specifi c infl uenza A virus immunolabeling was found
in all tissues collected from naïve juvenile birds (Table 2).
The most consistently affected tissues were the brain, spi-
nal cord, parasympathetic ganglia of the gastrointestinal
tract, heart, and pancreas (Figures 1, 2). Within the small
intestine and cecum, the strongest and most consistent im-
munolabeling involved the parasympathetic ganglia of the
submucosal and myenteric plexi (Figure 1, panel D) with
only the occasional scattered smooth muscle and vascular
endothelial cell within the gut mucosa positive for viral
antigen. In the 3 birds in which the proventriculus was af-
fected, viral antigen was detected in numerous cell types,
including both surface columnar and glandular epithelium,
smooth muscle cells of the muscularis mucosa, vascular
smooth muscle, and the parasympathetic ganglia (Figure 2,
panel C). In the lungs, antigen could be identifi ed in a few
capillary endothelial cells. Positive immunolabeling within
trachea, liver, kidney, and breast muscle was minimal and
observed in only a few birds. Immunohistochemical analy-
sis of tissues collected from naive adult birds detected spe-
cifi c immunolabeling in only 1 bird (841S/41Y) euthanized
at 5 dpc; tissues and cells affected were similar to those
observed in naive juveniles.
Deaths of mute (Cygnus olor) and whooper (C. cyg-
nus) swans have signaled the arrival of HPAI virus (H5N1)
in Europe (11,12). The affected swans had nervous signs
1824 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007
Table 2. Distribution of influenza virus antigen in tissues of naïve juvenile Canada geese tissues after challenge with influenza virus
dpi 3* dpi 4* dpi 5* dpi 5*
IHC-positive cell types
Vascular endothelium, mononuclear cells
Epithelium, Vascular smooth muscle, Smooth
muscle of muscularis externa, Mucous glands
Epithelium (columnar, glandular), muscularis
mucosa, vascular smooth muscle,
Parasympathetic ganglia, mucosal smooth
muscle, vascular endothelium
Exocrine acinar cells
Vascular smooth muscle, mononuclear cells
Vascular smooth muscle
Neurons, glial cells, ependymal cells, choroid
Ependymal cells, neurons, glial cells,
Spinal cord +++ ++++++
*Numbers of immunohistochemically positive cells: +, few; ++, moderate; +++, numerous; –, virus antigen negative; dpi, days postinfection.
Canada Geese, Highly Pathogenic Avian Infl uenza
that included somnolence, incoordination, and ataxia (11)
and gross pathology that included multifocal hemorrhagic
necrosis in the pancreas, pulmonary congestion and edema,
and subepicardial hemorrhages (13). Recent studies ad-
dressing the susceptibility of North American waterfowl
species to HPAI virus (H5N1) have shown wood ducks
(Aix sponsa) and laughing gulls (Larus atriculla) to be
highly susceptible, while mallards (Anas platyrhnchos),
northern pintails (A. acuta), blue-wing teals (A. crecca) and
redheads (Aythya Americana) to be refractory (14,15). Pre-
vious reports from Asia (4) and Europe (13) have indicated
that HPAI virus (H5N1) can produce deaths in naturally in-
fected Canada geese. Our study supports these observations
and further demonstrates this susceptibility to be dependent
on the age and immunologic status of the animal.
Adult birds were generally more resistant to Viet-
nam/05 than juveniles, regardless of which experimental
group they belonged to. Although results of this study in-
dicate that prior infection with a North American LPAI vi-
rus (H5N2) protects juvenile Canada geese against a lethal
H5N1 subtype challenge, the mechanism responsible is
unresolved. Although HI titers in poultry strongly corre-
late with protection against virulent challenge from viruses
expressing the same HA subtype (16), the ability of Brit-
ish Columbia/05 H5-specifi c antibodies to neutralize Viet-
nam/05 in vitro was not demonstrated. British Columbia/05
and Vietnam/05 have 84% amino acid similarity in their
HA1 subunits. The receptor binding domain (17), which
comprises an α-helix (190-helix, HA1 188–190) and 2 loop
structures (130-loop, HA1 134 to 138, and 220-loop, HA1
221 to 228) in addition to residues Tyr96, Trp153, and His183
is remarkably conserved for both viruses. Multiple amino
acid differences that cluster around the receptor-binding
domain (data not shown) may explain the inability of Brit-
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1825
Figure 1. Immunohistochemical staining for infl uenza virus nucleoprotein in central and peripheral nervous system of naive juvenile Canada
geese tissues after challenge with infl uenza virus (H5N1). A) Cerebrum. Positive immunolabeling of neurons, glial cells, ependymal and
choroid plexus epithelial cells. B) Cerebellum. Extensive positive immunolabeling of Purkinje cells and neurons of the granular layer. C)
Spinal cord. Positive immunolabeling of ependymal cells of the central canal and adjacent neurons and glial cells. D) Small intestine.
Positive immunolabeling of neurons of the submucosal plexus.
ish Columbia/05 antisera to neutralize Vietnam/05 in vitro.
Recent reports (18,19) have suggested that prior infection
with viruses expressing heterologous HA subtypes can also
protect chickens against a lethal (H5N1) challenge. Protec-
tion against HPAI virus (H5N1) in chickens that were pre-
viously infected with an H9N2 subtype correlated with the
proportion of pulmonary CD8+ T cells expressing gamma
interferon (19). The hypothesis that cell-mediated immu-
nity may have played a role in affording protection to the
birds in this study is supported by the observation that even
though NP antibody–positive naive adults did not appear to
possess H5-specifi c antibodies, they were resistant to Viet-
The pronounced neurotropism that Vietnam/05 exhib-
ited for Canada geese is similar to that reported for other
susceptible wild bird species (13–15). A unique fi nding in
our study was the widespread involvement of gastrointesti-
nal parasympathetic ganglia. This has not been previously
reported for wild birds, to our knowledge, although viral
antigen within the parasympathetic ganglia of the small
intestine of experimentally infected ducks has been docu-
mented (14). The mechanism by which avian infl uenza vi-
ruses invade the CNS has been most thoroughly investigat-
ed with mouse models (20–22). These studies have shown
that after intranasal inoculation, neurotropic infl uenza A
viruses can invade the CNS of mice by spreading along
peripheral nerves; viral antigen is mainly detected in the
vagal and trigeminal nuclei of the brainstem but not in the
cerebral cortex. A compartmentalized mouse dorsal root
ganglion neuron culture system (22) has further demon-
strated that infl uenza A viruses could infect the distal parts
of axons and reach the neuronal cell bodies by retrograde
axonal transport in a microtubule-independent fashion. The
involvement of the parasympathetic ganglia in our geese
1826 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007
Figure 2. Immunohistochemical (IHC) staining for infl uenza virus nucleoprotein in tissues of naïve juvenile Canada geese after challenge
with infl uenza virus (H5N1). A) Pancreas. Large areas of necrosis are surrounded by pancreatic acinar cells with strong positive intranuclear
and intracytoplasmic immunolabeling. B) Heart. Positive intranuclear and intracytoplasmic immunolabeling of myocytes. C) Proventriculus.
Strong positive immunolabeling of compound tubular gland epithelium. D) Splenic arteriole. Positive IHC staining of vascular smooth
Canada Geese, Highly Pathogenic Avian Infl uenza
suggests that CNS infection may occur by transmission of
infl uenza virus via autonomic nerves to their centers in the
brain stem. In contrast to the situation in mice, there is a
more diffuse infection of cortical and midbrain neurons as
well as choroid and ependymal epithelial cells. The latter
may indicate that a hematogenous route involving penetra-
tion of the blood–brain barrier with infection propagated to
glial cells and neurons (23) may also be involved.
Our work has demonstrated that Canada geese, and in
particular immunologically naïve, young-of-year animals,
may be suitable targets for dead bird surveillance activi-
ties. Based on our experiments, HPAI virus (H5N1) can
be expected to produce pronounced neurologic signs and
high deaths in this age group. CNS, pancreas, and heart
specimens can be used in PCR or immunohistochemical
diagnosis. However, prior exposure to North American lin-
eage H5 viruses specifi cally, or avian infl uenza viruses of
other HA subtypes more generally, may protect juvenile
and adult geese against a virulent H5N1 subtype challenge,
hence complicating detection. Determining the mechanism
responsible for this apparent cross-protection will require
We gratefully acknowledge the excellent technical assistance
provided by Lisa Manning, Estella Moffat, Shelly Ganske, Marlee
Ritchie, Kimberly Azaransky, Kevin Tierney, Shannon Toback,
Marsha Leith, Leanne McIntyre, and Julie Kubay.
This project was fi nancially supported by the Canadian Food
Dr Pasick is a veterinary virologist at the Canadian Food In-
spection Agency’s National Centre for Foreign Animal Disease
and has recently been appointed as a World Organization for
Animal Health reference laboratory expert for highly pathogenic
avian infl uenza. His primary research interests include avian in-
fl uenza diagnostics, pathogenesis, and ecology.
1. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka
Y. Evolution and ecology of infl uenza A viruses. Microbiol Rev.
2. Swayne DE, Halverson DA. Infl uenza. In: Saif YM, Barbes HJ, Glis-
son JR, Fadly AM, McDougald LR, Swayne DE, editors. Diseases of
poultry, 11th ed. Ames (IA): Iowa State Press; 2003. p. 135–60.
3. Banks J, Speidel EC, McCauley JW, Alexander DJ. Phylogenetic
analysis of H7 haemagglutinin subtype infl uenza A viruses. Arch Vi-
4. Ellis TM, Bousfi eld RB, Bissett LA, Dyrting KC, Luk GSM, Tsim
ST, et al. Investigation of outbreaks of highly pathogenic H5N1 avi-
an infl uenza in waterfowl and wild birds in Hong Kong in late 2002.
Avian Pathol. 2004;33:492–505.
5. Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang X-w, et al. Highly patho-
genic H5N1 infl uenza virus infection in migratory birds. Science.
6. Chen H, Smith GDJ, Zhang SY, Qin K, Wang J, Li KS, et al. H5N1
virus outbreak in migratory waterfowl. Nature. 2005;436:191–2.
7. Gilbert M, Xiao X, Domenech J, Lubroth J, Martin V, Slingen-
berg J. Anatidae migration in the western Palearctic and spread of
highly pathogenic avian infl uenza H5N1 virus. Emerg Infect Dis.
8. Kilpatrick AM, Chmura AA, Gibbons DW, Fleischer RC, Marra PP,
Daszak P. Predicting the global spread of H5N1 avian infl uenza.
Proc Natl Acad Sci U S A. 2006;103:19368–73.
9. Zhou E-M, Chan M, Heckert RA, Riva J, Cantin M-F. Evaluation of
a competitive ELISA for detection of antibodies against avian infl u-
enza virus nucleoprotein. Avian Dis. 1998;42:517–22.
10. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue
ML, et al. Development of a real-time reverse transcription PCR as-
say for type A infl uenza virus and avian H5 and H7 hemagglutinin
subtypes. J Clin Microbiol. 2002;40:3256–60.
11. Terregino C, Milani A, Capua I, Marino AMF, Cavaliere N. Highly
pathogenic avian infl uenza H5N1 subtype in mute swans in Italy. Vet
12. Nagy A, Machova J, Hornickova J, Tomci M, Nagl I, Horyna B, et
al. Highly pathogenic avian infl uenza subtype H5N1 in mute swans
in the Czech Republic. Vet Microbiol. 2007;120:9–16.
13. Teifke JP, Klopfl eisch R, Globig A, Starlick E, Hoffmann B, Wolf
PU, et al. Pathology of natural infections by H5N1 highly pathogen-
ic avian infl uenza virus in mute (Cygnus olor) and whooper (Cygnus
cygnus) swans. Vet Pathol. 2007;44:137–43.
14. Brown JD, Stallknecht DE, Beck JR, Suarez DL, Swayne DE. Sus-
ceptibility of North American ducks and gulls to H5N1 highly patho-
genic avian infl uenza viruses. Emerg Infect Dis. 2006;12:1663–70.
15. Perkins LE, Swayne DE. Susceptibility of laughing gulls (Larus
atricilla) to H5N1 and H5N3 highly pathogenic avian infl uenza vi-
ruses. Avian Dis. 2002;46:877–85.
16. Swayne DE, Beck JR, Perdue ML, Beard CW. Effi cacy of vaccines
in chickens against highly pathogenic Hong Kong H5N1 avian infl u-
enza. Avian Dis. 2001;45:355–65.
17. Stevens J, Blixt O, Tumpey TM, Taubeberger JK, Paulson JC, Wil-
son IA. Structure and receptor specifi city of the hemagglutinin from
an H5N1 infl uenza virus. Science. 2006;312:404–10.
18. Seo SH, Webster RG. Cross-reactive, cell-mediated immunity and
protection of chickens from lethal H5N1 infl uenza virus infection in
Hong Kong poultry markets. J Virol. 2001;75:2516–25.
19. Seo SH, Peiris M, Webster RG. Protective cross-reactive cellular
immunity to lethal A/goose/Guangdong/1/96-like H5N1 infl uenza
virus is correlated with the proportion of pulmonary CD8+ T cells
expressing gamma interferon. J Virol. 2002;76:4886–90.
20. Park CH, Ishinaka M, Takada A, Kida H, Kimura T, Ochiai K, et al.
The invasion routes of neurovirulent A/Hong Kong/483/97 (H5N1)
infl uenza virus into the central nervous system after respiratory in-
fection in mice. Arch Virol. 2002;147:1425–36.
21. Tanaka H, Park CH, Ninomiya A, Ozaki H, Takada A, Umemura T,
et al. Neurotropism of the 1997 Hong Kong H5N1 infl uenza virus in
mice. Vet Microbiol. 2003;95:1–13.
22. Matsuda K, Sibata T, Sakoda Y, Kida H, Kimura T, Ochai K, et al.
In vitro demonstration of neural transmission of avian infl uenza A
virus. J Gen Virol. 2005;86:1131–9.
23. Silvano FD, Yoshikawa M, Shimada A, Otsuki K, Umemura T. En-
hanced neuropathogenicity of avian infl uenza A virus by passages
through sir sac and brain of chicks. J Vet Med Sci. 1997;59:143–8.
Address for correspondence: John Pasick, National Centre for Foreign
Animal Disease, Canadian Food Inspection Agency, 1015 Arlington St,
Winnipeg, Manitoba, Canada R3E 3M4; email: firstname.lastname@example.org
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1827