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.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Recent outbreaks of highly pathogenic avian influenza (HPAI) in poultry have raised interest in the interplay between avian influenza (AI) viruses and their wild hosts. Studies linking virus ecology to host ecology are still scarce, particularly for non-duck species. Here, we link capture-resighting data of greater white-fronted geese Anser albifrons albifrons with the AI virus infection data collected during capture in The Netherlands in four consecutive winters. We ask what factors are related to AI virus prevalence and whether there are ecological consequences associated with AI virus infection in staging white-fronted geese. Mean seasonal (low pathogenic) AI virus prevalence ranged between 2.5 and 10.7 per cent, among the highest reported values for non-duck species, and occurred in distinct peaks with near-zero prevalence before and after. Throat samples had a 2.4 times higher detection frequency than cloacal samples. AI virus infection was significantly related to age and body mass in some but not other winters. AI virus infection was not related to resighting probability, nor to maximum distance travelled, which was at least 191 km during the short infectious lifespan of an AI virus. Our results suggest that transmission via the respiratory route could be an important transmission route of AI virus in this species. Near-zero prevalence upon arrival on their wintering grounds, in combination with the epidemic nature of AI virus infections in white-fronted geese, suggests that white-fronted geese are not likely to disperse Asian AI viruses from their Siberian breeding grounds to their European wintering areas.Proceedings of the Royal Society B: Biological Sciences 03/2010; 277(1690):2041-8. · 5.68 Impact Factor
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ABSTRACT: This paper takes a closer look at three interrelated areas of study: avian host biology, the role of human activities in virus transmission, and the surveillance activities centered on avian influenza in wild birds. There are few ecosystems in which birds are not found. Correspondingly, avian influenza viruses are equally global in distribution, relying on competent avian hosts. The immune systems, annual cycles, feeding behaviors, and migration patterns of these hosts influence the ecology of the disease. Decreased biodiversity has also been linked to heightened disease transmission in several disease systems, and it is evident that active destruction and modification of wetland environments for human use is impacting avian populations drastically. Legal and illegal trade in wild birds present a significant risk for introduction and maintenance of exotic diseases. After the emergence of HPAI H5N1 in Hong Kong in 1996 and the ensuing geographic spread of outbreaks after 2003, both infected countries and those at risk of introduction began intensifying avian influenza surveillance efforts. Several techniques for sampling wild birds for influenza viruses have been applied. Benefits, problems, and biases exist for each method. The wild bird avian influenza surveillance programs taking place across the continents are now scaling back due to the rise of other spending priorities; hopefully the lessons learned from this work will be preserved and will inform future research and disease outbreak response priorities.Animal Health Research Reviews 06/2010; 11(1):35-41.
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ABSTRACT: In the winter of 2010-2011, an outbreak of highly pathogenic avian influenza virus (HPAIV) infection occurred in wild and domestic birds in Japan. Tufted ducks were found dead in an urban area of Toyota City, Koriyama, Fukushima Prefecture. Two tufted ducks were examined histopathologically, immunohistochemically and molecularly. Gross findings included marked dark-red clotted blood in the pectoral muscles and multifocal hemorrhages on the serous membranes. Microscopically, non-suppurative meningoencephalitis, multifocal to coalescing pancreatic necrosis and severe pulmonary congestion were observed. HPAIV antigen was detected in the malacic areas, neuronal, glial and ependymal cells, pulmonary capillary endothelial cells and epithelium of pulmonary bronchioles, necrotic pancreatic acini and degenerated cardiac myocytes. The HPAIV isolate was genetically classified into clade 126.96.36.199 group A. The broad distribution of virus antigen in brain and pulmonary tissues associated with HPAIV spontaneous infection in tufted ducks might be useful in understanding its pathogenesis in nature.Journal of Veterinary Medical Science 05/2014; · 0.88 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.
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Address for correspondence: John Pasick, National Centre for Foreign
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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1827