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R E S E A R C H A R T I C L E Open Access
H5N2 Highly Pathogenic Avian Influenza
Viruses from the US 2014-2015 outbreak
have an unusually long pre-clinical period
in turkeys
Erica Spackman
*
, Mary J. Pantin-Jackwood, Darrell R. Kapczynski, David E. Swayne and David L. Suarez
Abstract
Background: From December 2014 through June 2015, the US experienced the most costly highly pathogenic
avian influenza (HPAI) outbreak to date. Most cases in commercial poultry were caused by an H5N2 strain which
was a reassortant with 5 Eurasian lineage genes, including a clade 2.3.4.4 goose/Guangdong/1996 lineage
hemagglutinin, and 3 genes from North American wild waterfowl low pathogenicity avian influenza viruses.
The outbreak primarily affected turkeys and table-egg layer type chickens. Three isolates were selected for
characterization in turkeys: the US index isolate from December 2014 (A/northern pintail/WA/40964/2014),
and two poultry isolates from April 2015 (A/chicken/IA/13388/2015 and A/turkey/MN/12528/2015).
Results: Four week old broad-breasted white turkeys were inoculated with one of three doses (10
2
,10
4
or 10
6
50% egg infectious doses [EID
50
] per bird) of each of the isolates to evaluate infectious dose and pathogenesis.
The mean bird infectious dose of A/northern pintail/WA/40964/2014 and A/turkey/MN/12528/2015 was 10
5
EID
50
per bird, but was 10
3
EID
50
per bird for A/chicken/IA/13388/2015, suggesting the latter had greater adaptation to
gallinaceous birds. All three isolates had unusually long mean death time of 5.3–5.9 days post challenge, and the
primary clinical signs were severe lethargy and neurological signs which started no more than 24 h before death
(the average pre-clinical period was 4 days). Infected turkeys also shed high levels of virus by both the
oropharyngeal and cloacal routes.
Conclusions: The unusually long mean death times, high levels of virus in feces, and increased adaptation
of the later viruses may have contributed to the rapidspreadofthevirusduringthepeakoftheoutbreak.
Keywords: Highly pathogenic avian influenza virus, Clade 2.3.4.4H5N2,Turkeydisease,Avianinfluenzaoutbreak,
Chicken disease
Background
An H5 HPAIV outbreak that began in December 2014
and lasted 6 months through June 2015 was the 5th
highly pathogenic avian influenza virus (HPAIV) out-
breakintheUSsincethe1920’sandwasthemost
geographically widespread. Direct and indirect costs to
the US economy were estimated to be near $3.3 billion
USD [1]. The hemagglutinin (HA) gene was determined
to belong to clade 2.3.4.4 of the goose/Guangdong/1996
(GS/GD/96) H5 lineage of HPAIV and was subsequently
named intercontinental group A (IcA) H5 [2, 3]. Wild
waterfowl are thought to have carried viruses of this
Eurasian lineage from Asia into North America during
migration over the Bearing sea route [2, 4–6]. The GS/
GD/96 lineage has circulated in wild and domestic birds
throughout Asia since 1996 with occasional incursions
into Europe and Africa. In December 2014, the IcA H5
HPAIVs were first discovered in poultry in Canada, and
subsequently in the US a few weeks later [4, 7].
In North America four variants of the IcA H5 viruses
were identified; an H5N8 with a genome that was
* Correspondence: Erica.spackman@ars.usda.gov
Southeast Poultry Research Laboratory, USDA-Agricultural Research Service,
934 College Station Rd, Athens, GA 30605, USA
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Spackman et al. BMC Veterinary Research (2016) 12:260
DOI 10.1186/s12917-016-0890-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
completely Eurasian in lineage (≥98% identity among
all gene segments with H5N8 virus isolates from South
Korea); and three reassortants with a mixture of North
American wild bird lineage genes, an H5N2, an H5N1
and an H5N8 [4, 8, 9]. The H5N2 was the variant that
caused the most cases in commercial poultry in the US
[10]. Turkey farms had the highest number of infected
premises (153 of 211) during this outbreak, but table-egg
layer chickens were the poultry type with the highest
numbers of birds affected [10]. The goal of these studies
was to characterize the pathobiology of the US index
H5N2 HPAIV isolate collected from a wild Northern
Pintail in December 2014 and to compare that with two
poultry isolates from later in the outbreak (April 2015)
in commercial broad breasted white turkeys.
Methods
Turkeys
Broad-breasted white turkeys were obtained from a com-
mercial turkey producer and were reared in a commercial
production environment from hatch until they were de-
livered to the Southeast Poultry Research Laboratory-
USDA-ARS (SEPRL) at 4 weeks of age. Each bird was
individually tagged for identification. The turkeys were
housed and cared for in accordance with procedures
approved by the SEPRL Institutional Animal Care and
Use Committee. Serum was collected from turkeys im-
mediately prior to challenge to confirm the absence of
antibody to type A influenza by commercial ELISA
(MultiS Screen, IDEXX Inc. Westbrook, ME).
Viruses
Three H5N2 HPAIV isolates were selected for evalu-
ation: the US index H5N2 HPAIV isolate from Decem-
ber 2014, A/Northern Pintail/Washington/40964/2014
(NOPI/40964); and two isolates from commercial poultry
operations collected in April 2015, A/turkey/MN/12582/
2015 (TK/12582) and A/chicken/IA/13388/2015 (CK/
13388). Viruses were provided by the National Veterinary
Services Laboratories-USDA-APHIS (Courtesy of Dr. Mia
Torchetti). Each isolate was passaged twice in embryonat-
ing chickens eggs (ECE) and titrated using standard proce-
dures [11]. Inocula were diluted to the appropriate dose in
brain heart infusion (BHI) broth.
Pathogenesis, infectious dose and transmission
To evaluate the infectious dose of each isolate and
transmission to non-inoculated hatch-mates housed in
the same isolator (contact exposed turkeys), three doses
of virus: 10
2
,10
4
and 10
6
50% egg infectious doses (EID
50
)
per bird, were administered to groups of five turkeys as re-
ported in Bertran et al. [12]. Virus was administered using
a simulated respiratory route, the intrachoanal route, in
0.1 ml per bird. The contact exposed turkeys (n=3) were
added to each dose group 24 h post challenge (PC).
Sixteen additional turkeys at the 10
6
EID
50
per bird
dose group were included to characterize the pathogen-
esis of the virus. Clinical signs and mortality were recorded
a minimum of daily and birds that were euthanized due
to severe illness were counted as dying the following
day for mean death time (MDT) calculations. Turkeys
were considered infected if virus was detected in oro-
pharyngeal (OP) or cloacal (CL) swabs at any time point
and/or if the bird seroconverted by the end of the experi-
ment (14 days PC).
The later experiments with TK/12582 and CK/13388
had a modified swab collection schedule from what was
done with NOPI/40964 because additional sample collec-
tion times were added to more precisely establish when
the turkeys started to shed detectable levels of virus. For
all three isolates, OP and CL swabs were collected from
the directly inoculated turkeys at 2, 4, 7, 10 and 14 days
PC, and OP and CL swabs were collected from contact
exposure turkeys at 1, 3, 6, 9 and 13 days PC. Additional
OP and CL samples collected from groups inoculated with
TK/12582 and CK/13388 were at 12, 24 and 36 h PC.
Virus titers in swabs were evaluated by quantitative real-
time RT-PCR. Sera were collected from all surviving
turkeys 14 days PC. Sera were tested for antibody by
homologous hemagglutination inhibition (HI) assay.
Two to six birds at the 10
6
EID
50
per bird dose group
exposed to each virus were necropsied for tissue collection
when clinical signs were apparent. A full set of tissue sam-
ples (lungs, bursa, kidneys, adrenal gland, thymus, bursa,
brain, liver, heart, proventriculus, pancreas, intestine,
spleen, trachea, Harderian gland, beak, and thigh muscle)
were collected for microscopic evaluation and immuno-
histochemistry (IHC). Tissues were fixed in 10% neutral
buffered formalin, sectioned, paraffin embedded, and
stained with hematoxylin-and-eosin. Serial sections were
stained by IHC methods [13] to visualize influenza antigen
in individual tissues. An identical tissue set was collected
from two non-inoculated hatch-mates to serve as negative
controls.
Sera were collected from all surviving turkeys 14 days
PC to evaluate infection status by antibody detection with
homologous HI assay.
The infectious dose was calculated by the Reed-Muench
method [14], using the criteria that turkeys were consid-
ered infected if they had clinical signs, died, shed virus or
were positive for antibody 14 days PC.
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR targeting the influenza
M gene including the RNA extraction, was conducted as
previously described [15]. The standard curve was run in
duplicate using RNA from the same virus stock used to
Spackman et al. BMC Veterinary Research (2016) 12:260 Page 2 of 9
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prepare the inocula. Virus quantity was reported as
equivalents to infectious titer.
Hemagglutination inhibition assay
The HI assay was conducted using standard procedures
[11]. Homologous isolates were used as antigens and
were inactivated with 0.1% beta-propiolactone. A titer of
1:16 (2
4
) or above was considered positive.
Statistics
Virus titers shed by time point for the same swab type
(OP or CL) were tested among the viruses with Kruskal-
Wallis H test (Prism 7, GraphPad Software, La Jolla, CA).
Apvalue of ≤0.05 was considered significant.
Results
Infectious dose and transmission
Infection status was determined based on virus shed,
clinical disease, mortality and seroconversion. Turkeys
which survived to 14 days were not infected as indi-
cated by lack of virus shed and seroconversion. At each
dose and virus combination either 100% of the turkeys
were infected or none were infected (Table 1). The
50% turkey infectious dose (TID
50
) and 50% turkey le-
thal doses (TLD
50
) were the same for each isolate and
were approximately 10
5
EID
50
/bird for NOPI/40964
and TK/12582 but was two log
10
lower for CK/13388
which was 10
3
EID
50
/bird (Table 1).
Infection rates in the contact exposure groups were the
same as the inoculated groups to which they were ex-
posed; 100% of the contact exposure turkeys, with all
three isolates, at 10
6
EID
50
/bird dose group were infected.
The only lower dose group where the contact turkeys
were infected was the 10
4
EID
50
/bird dose group exposed
to CK/13388, where 100% (3 of 3) were infected. All the
infected contact exposed birds presented with clinical
signs similar to the inoculated turkeys and died.
Virus shed
No turkeys exposed to 10
2
EID
50
/bird shed detectable
levels of virus at any time. Only one group exposed to
10
4
EID
50
/bird shed detectable levels of virus: the CK/
13388 exposed group (Table 1). In this group, virus was
only detected in OP swabs from 3 of 5 turkeys and in
CL swabs from 2 of 5 turkeys, although the two turkeys
from which virus was not detected both presented with
clinical signs consistent with HPAIV and died (one at
24 h PC and the other at 5 days PC).
Oro-pharyngeal and CL shed titers for groups exposed
to 10
6
EID
50
/bird are shown in Fig. 1. Oro-pharyngeal
shedding was detectable at all times and peaked 3–4days
PC for all three isolates with titers exceeding 10
6
EID
50
/ml
from birds exposed to TK/12528 and CK/13388. Although
Table 1 Mortality, mean death time, 50% infectious dose, 50% lethal dose and number of birds shedding for three H5N2 highly
pathogenic avian influenza viruses in 4-week old directly inoculated and contact exposed broad-breasted white turkeys
Isolate Dose
(EID
50
/bird)
Mortality Mean death
time (days)
Number of turkeys
shedding
Seroconversion 50% Turkey
infectious dose
e
Inoculated Contact
exposed
Inoculated Contact
exposed
Inoculated Contact
exposed
Inoculated Contact
exposed
A/northern pintail/WA/
40964/2014
10
2
0/5
a
0/3
a
NA
b
NA 0/5
d
0/3
d
0/5
i
0/3
i
10
5
EID
50
f
10
4
0/5 0/3 NA NA 0/5 0/3 0/5 0/3
10
6
5/5 3/3 5.3 (4–7)
gh
7.6 (7–8)
cg
5/5 3/3 NA NA
10
6
16/16 NA NA 16/16 NA NA NA
A/turkey/MN/12582/2015 10
2
0/5 0/3 NA NA 0/5 0/3 0/5 0/3 10
5
EID
50
10
4
0/5 0/3 NA NA 0/5 0/3 0/5 0/3
10
6
5/5 3/3 5.9 (3–10) 8.0 (6–10) 5/5 3/3 NA NA
10
6
16/16 NA NA 16/16 NA NA NA
A/chicken/IA/13388/2015 10
2
0/5 0/3 NA NA 0/5 0/3 0/5 0/3 10
3
EID
50
10
4
5/5 3/3 5.4 (1–7) 9.0 (6–10) 3/5 3/3 NA NA
10
6
5/5 3/3 5.6 (3–13) 7.6 (2–13) 5/5 3/3 NA NA
10
6
16/16 NA NA 16/16 NA NA NA
a
Number dead/total
b
NA not applicable. Either no mortality or no birds survived for antibody testing
c
Calculated from the day of placement with the inoculated turkeys
d
Number shedding/total
e
50% infectious dose was equal to the 50% lethal dose for all isolates
f
EID
50
= 50% egg infectious dose
g
Mean death time days (range of mortality in days). For inoculated birds includes data from all 21 turkeys exposed to 10
6
EID
50
/bird
h
Mean death times are combined for directly exposed turkeys in the same dose group for each isolate
i
Number seroconverted/total
Spackman et al. BMC Veterinary Research (2016) 12:260 Page 3 of 9
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titers from birds exposed to NOPI/40964 were about one
log
10
lower the difference was not significant. Cloacal
shedding also peaked at 3 days PC from birds exposed to
TK/12528 and CK/133388, but was later for NOPI/40964
exposed birds where the highest titers were at 7 days PC.
Turkeys in the 10
6
EID
50
/bird dose group that survived to
10 days PC were still shedding virus. There were no differ-
ences in the numbers of turkeys shedding among the iso-
lates for the 10
6
EID
50
/bird dose group (all were 100%).
Shed titers were compared among all three isolates for
all turkeys exposed to 10
6
EID
50
/bird by swab type at 2
and 4 days PC and between TK/12582 and CK/13388 at
12, 24, 36 h and 3DPC (at time points where five or
fewer birds were shedding detectable levels of virus stat-
istical analysis was not performed). At 36 h PC the OP
titers from TK/12528 were significantly lower than CK/
13388. Titers of shed among the viruses were only sig-
nificantly different at two other times; CK/13388 OP
shed titers were significantly lower than either NOPI/
40964 or TK/12582 at 2 days PC, and CK/13388 titers
were again significantly lower than either other virus
with CL at 4 days PC.
Each group of contact exposed turkeys shed virus
similarly to the inoculated groups with which they were
Fig. 1 Oropharyngeal (OP) and cloacal (CL) shed determined by real-time RT-PCR for each virus by time post challenge with 10
6
50% egg infectious
doses (EID
50
) per bird. Data are absent from the A/northen pintail/WA/40964/2014 isolates at 12, 24, 36 h and 3 days because the samples were not
collected, at 10 and 14 days because no turkeys survived to these time points. Thick bars indicate the mean of the group and error bars represent one
standard deviation
Spackman et al. BMC Veterinary Research (2016) 12:260 Page 4 of 9
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housed. In the groups housed with the 10
6
EID
50
/bird
dose group all (3 of 3) contact exposure turkeys shed
virus. In the 10
4
EID
50
/bird dose group for CK/13388,
which was the only middle or lower dose group to be in-
fected; all three turkeys shed virus (data not shown).
Pathogenesis
The pathogenesis for all three isolates was similar. Mean
death times for the inoculated turkeys were between 5.3
and 5.9 days and were between 7.6 and 9 days post
placement for the contact exposed turkeys (Table 1).
Clinical disease was not apparent until 24 h or less prior
to death (turkeys that could not reach food or water
were euthanized). With all three isolates, an average of
50% of the turkeys died without exhibiting any clinical
signs within 12 h prior to death (i.e. they appeared nor-
mal at the late afternoon/evening observation period
and were dead at the morning observation period).
The only clinical signs observed were neurological
signs and lethargy. Lethargic birds tended to rest with
ruffled body feathers. The severity of neurological signs
and lethargy was similar among all three isolates.
Neurological signs consisting of torticollis (Fig. 2),
tremors or ataxia were observed in 28 and 23% of the
turkeys infected with NOPI/40964 and TK/12582, and
50% of the turkeys infected with CK/13388.
Turkeys were selected for necropsy because they were
presenting with clinical signs (neurological and/or leth-
argy). Tissues were collected from six turkeys exposed to
NOPI/40964 (4 DPC) and from two turkeys exposed to
TK/12582 and two turkeys exposed to CK/13388 (3DPC).
Gross lesions were typically absent, however petechial
hemorrhages were observed in the skeletal muscle of two
turkeys: one infected with TK/12582 and CK/13388
each. The CK/13388 infected turkey with the muscle
hemorrhage also had swollen kidneys. A third turkey,
infected with TK/12582, also had swollen kidneys.
Other lesions were non-specific and were consistent
with anorexia and lethargy (i.e. approximately 10% of
the turkeys had empty alimentary tracts). In addition,
any turkeys dying or that were euthanized were necrop-
sied, but no tissues were taken. Gross lesions were all
similar to those observed in the turkeys from which tis-
sues were collected.
Tissues were examined for microscopic lesions and
viral antigen staining (Table 2 and Fig. 3). Microscopic
lesions were similar among the eight birds examined,
with only minor variations in severity. The most severe
lesions were found in the nasal cavity, brain, heart, ad-
renal gland and pancreas. Moderate to severe rhinitis
and sinusitis with multifocal necrosis of the nasal epi-
thelium was common in all birds. Mild tracheitis and
bronchitis, and mild to moderate interstitial pneumonia
were also present. Mild to severe, randomly dissemi-
natedfociofneuronalandglialcellnecrosiswereob-
served in the cerebrum and cerebellum of all turkeys
examined (Fig. 3a). Microgliosis, edema and mono-
nuclear perivascular cuffs were also commonly ob-
served in malacic areas of the brain. In the heart, mild
to moderate multifocal myocyte necrosis was com-
monly observed (Fig. 3b). Moderate to severe multifocal
necrosis of the pancreatic acinar epithelium was
present in all birds (Fig. 2c). In the adrenal gland, mild
to moderate multifocal to confluent areas of vacuolar
degeneration to necrosis of corticotropic cells, and less
commonly, chromaffin cells was observed in all but one
turkey (Fig. 2d). Thymus, bursa and mucosa-associated
lymphoid tissue had moderate to severe lymphoid de-
pletion with apoptosis to necrosis in remaining lympho-
cytes (Fig. 2e). Mild to moderate depletion of the white
pulp with multifocal lymphocytic necrosis was observed
in the spleen. Mild to moderate necrosis of the epithelia of
the Harderian glands and nasal glands was observed in
most turkeys. Infrequently, the kidneys presented mild
focal tubular necrosis, and the liver mild multifocal fibrin-
oid necrosis. Epithelial cell necrosis of the proventricular
gland was observed in three turkeys. No lesions were ob-
served in the intestine, eyelid, snood, or skeletal muscle.
Immunohistochemical staining was used to visualize
virus distribution in tissues. All three viruses demon-
strated similar tissue tropism where the highest levels
of viral staining relative to other tissues were observed in
the brain, heart, pancreas, and adrenal glands (Table 2).
Staining for virus antigen was present in areas of necrosis
in many tissues including brain, pancreas, adrenal gland
lymphoid tissues, liver, and spleen. Virus antigen was
found in parenchymal cells of organs including microglial
Fig. 2 Four week-old broad-breasted white turkey with torticollis
after exposure to A/northern pintail/WA/40964/2014 H5N2 highly
pathogenic avian influenza virus
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Table 2 Distribution of avian influenza virus antigen visualized by immunohistochemical staining in tissues by isolate
Bird
ID
Detection of avian influenza virus antigen in tissues
Nasal epithelium Nasal glands Trachea Lung Heart Brain Liver Kidney Adrenal gland Spleen Intestine Pancreas Harderian gland Thymus Bursa Proventric
A/Northern Pintail/WA/40964/2014
1043 + + −+ ++ +++ + + +++ + + +++ −−+−
1045 + ++ −+ + +++ −− +++ −+ +++ ++ ++ + −
1047 + + −+ + +++ + + +++ + + +++ + + ++ +
1052 + ++ −+−+++ + ++ ++ −− +++−
1053 + ++ −−++ +++ + + ++ −++ +++ + + ++ −
1056 ++ ++ + ++ ++ +++ + ++ +++ −+++ ++ + ++ ++ +
A/turkey/MN/12528/2015
1093 ++ +++ −+ ++ +++ −+−−−++ + + + −
1097 + ++ + + +++ +++ ++ + ++ + −++ +++ + + +++
A/chicken/IA/13388/2015
1040 + + −+ +++ +++ + + +++ + −+++ + −+−
1050 + ++ −+ ++ +++ −+++ −− +++ −+++−
Turkeys were selected for examination because they were presenting with clinical illness at 4 days post challenge (DPC) (A/Northern Pintail/WA/40964/2014) or 3DPC (A/turkey/MN/12528/2015
and A/chicken/IA/13388/2015)
−= no positive cells; + = single positive cells; ++ = scattered groups of positive cells; +++ = widespread staining
Proventric. = proventriculus
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cells and neurons, cardiac myocytes, pancreatic acinar
cells, adrenal corticotropic and chromaffin cells
(Fig. 3a-d), hepatocytes, and kidney tubular epithelial
cells. Viral staining was common in resident and infil-
trating phagocytes of the thymus, bursa and spleen
(Fig. 3e). Viral antigen was also present in epithelial
cells and macrophage in the nasal turbinates, trachea,
Harderian gland (Fig. 3f), nasal glands (Fig. 3g); and
proventricular gland cells (Fig. 3h). No viral antigen
was observed in vascular endothelial cells.
Discussion
The pathogenesis of three H5N2 HPAIV isolates from the
US 2014–2015 outbreak were characterized in commer-
cial broad breasted white turkeys. These H5N2 isolates,
which were first isolated in North America in Canada,
represent novel AIV reassortants with genes from
Eurasian viruses: a GS/GD/96 derived HA, Eurasian PB2,
PA, M and NS genes, and North American wild bid virus
lineage PB1, NP, NA genes [7]. The US index isolate,
NOPI/40964, was selected to represent the earliest intro-
ductions detected in the US and the H5N2 HPAIV
variants which are mostly likely to be adapted to wild
birds. Two later isolates from the Midwest, TK/12582 and
CK/13388, were selected in real-time during the outbreak
to represent isolates that had likely been passaged in
gallinaceous poultry based on a relatively severe clinical
presentation in the field and initial epidemiological infor-
mation. Based on subsequent sequence analysis and
field epidemiology, the TK/12582 isolate was probably
an introduction either directly from waterfowl or soon
thereafter and may not have been passaged extensively
in gallinaceous poultry (unpublished data). In contrast,
based on the same analysis, CK/13388 may have circu-
lated in poultry longer. This would corroborate the
infectious dose data where the TK/12582 and NOPI/
40964 isolates each required a TID
50
2log
10
higher
dose than the CK/13388.
In fact, the TID
50
and TLD
50
were the only differences
observed among the three isolates. All three primarily pre-
sented with neurological signs and severe lethargy during
the 24 h prior to death, and virus distribution among the
tissues was similar. Microscopic lesions produced by all
three isolates were typical for HPAIV in turkeys and other
gallinaceous birds. Importantly, a high level of virus was
observed in the brain by IHC, which could account for in-
jury to the brain resulting in neurological signs.
Although the clinical presentations among all three
isolates were similar, the pathogenesis of these isolates
had some unusual characteristics for HPAIV. First, the
MDT’s, which were between 5.3 and 5.9 days, and the
pre-clinical period were atypically long and mortality
was distributed over a 10 day period with some turkeys
dying as late as 13 days PC. By contrast, most HPAIV
produce MDTs ranging between 2 and 4 days and death
has been associated with replication of virus in blood
vessel endothelial cells with vascular thrombosis or em-
bolism and multi-organ failure [16, 17]. However, in this
study, neural pathogenesis has been associated with dir-
ect replication of virus in neurons and neuropil support
cells without vascular endothelial cells involvement, and
accompanied by replication in other critical organ par-
enchymal cells leading to multi-organ failure, as has
been shown with some previous HPAIV infections [17].
The even longer MDTs with the contact exposed turkeys
Fig. 3 Histological lesions and immunohistochemical detection of
viral antigen in 4-week-old turkeys intranasally inoculated with H5N2
HPAI viruses. Tissues collected at 4 days post-inoculation. Virus
antigen is stained in red. Magnifications 40×. aCerebellum.
Vacuolation of the molecular and granular layers of the cerebellum
with necrosis of the Purkinge neurons. Inset: viral antigen in neurons
and glial cells. bHeart. Focal hyalinization and fragmentation of cardiac
myocytes. Inset: viral antigen in cardiac myocytes. cPancreas. Diffuse
pancreatic necrosis. Inset: viral staining in acinar epithelium. dAdrenal
gland. Confluent necrosis of corticotrophic and chromaffin cells. Inset:
viral antigen in adrenal corticotrophic and cromaffin cells. eBursa de
Fabricious. Lymphoid depletion with apoptosis to necrosis in
remaining lymphocytes. Inset: viral antigen in phagocytic c and
necrotic cells. fHarderian gland. Viral antigen present in epithelial
cells and infiltrating phagocytes. gNasal gland. Viral antigen present in
glandular epithelial cells and infiltrating phagocytes. hProventriculus.
Viral antigen present in glandular epithelial cells
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are most likely because it took a day or two for the tur-
keys to become infected. Also, since the environment is
artificial (e.g. grate floor isolators limiting coprophagy,
high rate of airflow, etc.) the results with the contacts
cannot be extrapolated directly to the field, and only
provide a relative measure of transmissibility among iso-
lates tested in similar ways.
Secondly, each of these viruses was shed at relatively
high titers by the cloacal route. Highly pathogenic
AIV is more often shed at the highest titers by the
oral/respiratory route in gallinaceous birds [16]. It is
possiblethatthelateonsetofmorbidityandmortal-
ity, and high virus titers in manure contributed to the
rapid spread of the virus in the Midwest, especially in
light of the relatively high TID
50
. Essentially the turkeys
are infected and are shedding substantial amounts of virus
in their manure and orally as early as 24–36 post infection
for several days, but don’tappearsick.
One aspect of the field situation that was not replicated
in the laboratory was the age of the turkeys. During the
outbreak 85% of the infected turkey flocks were at least
9 weeks of age (57% of affected turkeys were 12 or greater
weeks of age) [10]. Unfortunately, facilities which can
work with HPAIV often cannot accommodate broad-
breasted white turkeys in isolation cabinets over about 7
weeks of age because of their size, so it was not possible
to determine if turkey age affected susceptibility for
biological reasons or whether older birds have a higher
chance of exposure because they have been around for
a longer period of time (or a combination of both) as
animal facilities with the appropriate biosafety level for
floor pen studies were not available.
Relatively little data has been reported regarding experi-
mental infection of turkeys with HPAIV and none with
isolates from this lineage (clade 2.3.4.4 H5 isolates).
Experimental pathogenesis studies in birds have character-
ized H5N8 HPAIV isolates in chickens and ducks [18–23]
and the pathogenesis of NOPI/40964 H5N2 HPAIV in
chickens has been reported [12]. The 50% infectious dose
in chickens was similar at 10
5.7
EID
50
(versus 10
5.0
EID
50
in turkeys). Notably there were differences between the
pathogenesis of NOPI/40964 in chickens and turkeys:
the MDT was 3 days in chickens versus 5.3 days in tur-
keys and the clinical presentation was also different.
Neurological signs were more common in turkeys (only
1 of 10 chickens presented with neurological signs) and
there were lesions unique to chickens: 1) petechial hemor-
rhages and cyanotic combs and wattles (turkey snoods
appeared normal); and 2) infra-orbital swelling [12]. Dif-
ferences in lesions between chickens and turkeys were also
observed with H5N2 HPAI in the field in Canada [7] and
with a 1997 H5N1 HPAIV GS/GD/96 lineage virus [24].
Similar clinical presentation (i.e. neurological signs) and
gross lesions in turkeys in the field were observed in the
UK with a GS/GD/96 lineage H5N1 HPAIV [25] and
with the unrelated H5N8 HPAIV in Ireland in 1983
[26]. Based on limited information from field reports,
lack of respiratory signs is not uncommon in turkeys
with HPAIV [7, 25, 26], although respiratory signs were
reported with an H5N2 HPAIV in Italy in 1997 [27].
However, in field cases the involvement of secondary
bacterial infections cannot be ruled out.
Conclusions
High mortality is the most consistent sign of HPAIV in-
fection in turkeys as there are some biological variations
in the pathogenesis of the virus. In the case of these
three isolates, the long pre-clinical period, late MDT and
high titers of cloacal shed are unusual characteristics
and may have contributed to spread despite the initially
high exposure dose to produce infection; i.e. high TID
50
.
Also, neurological signs were the most common clinical
sign after severe lethargy observed in the turkeys, which
contrasts chickens, where hemorrhagic lesions are re-
ported more frequently. This and a shorter reported MDT
in chickens highlights the difference in pathogenesis of
HPAIV between chickens and turkeys. Finally, there are
many other factors involved in the spread of HPAIV in
poultry; attention to biosecurity should be a priority re-
gardless of the characteristics of the lineages involved.
Acknowledgements
The authors thank David Rives and Eric Gonder for providing turkeys; Carol
Cardona for information on isolates in field; and Mia Torchetti for providing
the viruses. We also thank: Scott Lee, Aniko Zsak, Diane Smith, Kira Moresco,
Roger Brock, Gerald Damron for technical assistance with this work.
Funding
This research was supported by US Department of Agriculture, ARS CRIS
Project 6612-32000-063-00D and with federal funds from the National
Institute of Allergy and Infectious Diseases, National Institutes of Health,
under IAA No. AAI12004-001-00001. Its contents are solely the responsibility of
the authors and do not necessarily represent the official views of the NIH.
Mention of trade n ames or commer cial products in this manuscript i s
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture. USDA is an equal opportunity provider and employer.
Availability of data and material
The datasets during and/or analysed during the current study available from
the corresponding author on reasonable request.
Authors’contributions
All authors contributed to the experimental design and editing the
manuscript. ES: conducted the bird work and related lab work, and
composed the manuscript. MPJ: evaluated the microscopic lesions and
ran the immunohistochemistry. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Spackman et al. BMC Veterinary Research (2016) 12:260 Page 8 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Ethics approval
This work did not involve human subjects. All animal work was approved in
advance by the Southeast Poultry Research Laboratory Institutional Animal
Care and Use committee.
Received: 9 July 2016 Accepted: 17 November 2016
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