Matos et al. Vet Res (2016) 47:69
DOI 10.1186/s13567-016-0350-0
Open Access
RESEARCH ARTICLE
The outcome of experimentally
induced inclusion body hepatitis (IBH) by fowl
aviadenoviruses (FAdVs) is crucially influenced
by the genetic background of the host
Miguel Matos1*, Beatrice Grafl1, Dieter Liebhart1 and Michael Hess1,2
Abstract
In the present study, inclusion body hepatitis (IBH) was experimentally induced by oral inoculation of two groups of
specific pathogen-free (SPF) broilers and two groups of SPF layers at day-old with either a fowl aviadenovirus (FAdV)-D
or a FAdV-E strain. A substantial variation in the degree of susceptibility was observed with mortalities of 100 and 96%
in the FAdV-E and D infected SPF broiler groups, respectively, whereas in the groups of infected SPF layers mortalities
of only 20 and 8% were noticed. Significant changes in clinical chemistry analytes of all infected birds together with
histopathological lesions indicated impairment of liver and pancreas integrity and functions. Furthermore, significantly lower blood glucose concentrations were recorded at peak of infection in both inoculated SPF broiler groups,
in comparison to the control group, corresponding to a hypoglycaemic status. High viral loads were determined in
liver and pancreas of SPF broilers already at 4 days post-infection (dpi), in comparison to SPF layers, indicating a somewhat faster viral replication in the target organs. Overall, highest values were noticed in the pancreas of SPF broilers
independent of the virus used for infection. The actual study provides new insights into the pathogenesis of IBH, a
disease evolving to a metabolic disorder, to which SPF broilers were highly susceptible. Hence, this is the first study to
report a significant higher susceptibility of SPF broiler chickens to experimentally induced IBH in direct comparison to
SPF layers.
Introduction
Fowl aviadenoviruses (FAdVs) belong to the genus Aviadenovirus within the family Adenoviridae, being further
divided into five species designated FAdV-A to E [1].
Throughout the years, many reports established a causality between strains from species FAdV-A, FAdV-C and
FAdV-D together with FAdV-E with specific diseases
in chickens, such as adenoviral gizzard erosion (AGE),
hydropericardium hepatitis syndrome (HHS) and inclusion body hepatitis (IBH), respectively [2].
In the last 10 years IBH outbreaks have been reported
in different geographic regions emphasizing the wide
*Correspondence: miguel.matos@vetmeduni.ac.at
1
Clinic for Poultry and Fish Medicine, Department for Farm Animals
and Veterinary Public Health, University of Veterinary Medicine Vienna,
Veterinaerplatz 1, 1210 Vienna, Austria
Full list of author information is available at the end of the article
distribution of FAdVs throughout the world [3–8]. In the
field, IBH has been reported essentially from commercial
broiler flocks (meat-producing chickens), being responsible for serious economic losses due to increased mortality combined with reduced performance within flocks
[2]. However, experimental in vivo studies were predominantly conducted in specific pathogen-free (SPF) white
leghorn layers (egg-producing chickens), which are the
experimental model for infection studies.
In a recent study we were able to demonstrate the influence of virulent FAdV-D and E field strains on different
enzyme systems and metabolite concentrations in the
plasma of orally inoculated day-old SPF layer chickens
due to the infection of liver and pancreas as target organs
[9]. Consequently, it can be hypothesized that hosts with
different metabolic activities vary in their susceptibilities
towards infection. Therefore, the aim of the present study
© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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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.
Matos et al. Vet Res (2016) 47:69
was to characterize and compare the susceptibility of SPF
broiler and SPF layer chickens to experimentally induced
IBH by FAdV-D and E field strains.
Materials and methods
Viruses
The FAdV strains used in the present study—08/18 926
and 13/18 153—were isolated from liver samples of broilers during recent IBH outbreaks in Europe and they were
genotyped as belonging to species FAdV-D and E, respectively [4, 8]. The viruses were plaque purified three times
and propagated in primary chicken embryo liver (CEL)
cell cultures as described elsewhere [10]. The titers were
determined according to the method of end point titration [11] and a titer of 107 median tissue culture infective dose (TCID50) per mL was used to infect the birds. A
polymerase chain reaction (PCR) and a reverse transcription-PCR were performed to confirm the absence of contaminations by chicken anaemia virus and avian reovirus,
respectively. The strains’ pathogenicity was characterized
in vivo by inoculating SPF white leghorn chickens at dayold [9].
Animal trial
Embryonated SPF broiler eggs (Animal Health Service,
Deventer, The Netherlands) and SPF layer eggs (VALO,
Lohmann Tierzucht GmbH, Cuxhaven, Germany) were
incubated at our facilities. After hatch, the chicks were
individually tagged subcutaneously (Swiftack, Heartland Animal Health Inc., Fair Play, USA) and divided in
six groups: three groups of 27 SPF broiler chicks (groups
B0–2) and three groups of 20 SPF layer chicks (groups
L0–2). The groups were housed separately in isolator units
(Montair Andersen bv, HM 1500, Sevenum, Netherlands)
under negative pressure, where feed and water were available ad libitum throughout the animal experiment. At first
day of life, the body weight of all birds was measured and
birds from groups L1 and B1, and from groups L2 and B2
were orally inoculated with 0.5 mL of the 13/18 153 and
the 08/18 926 strains, respectively, while birds from groups
L0 and B0 were left uninoculated (Table 1). All birds were
daily monitored and an individual score was given based
on clinical signs: 0—active with no adverse clinical signs;
1—slightly weak with dropped wings; 2—depressed with
swollen crops; 3—weak, apathetic, with ruffled feathers and reluctant to move; 4—apathetic, unable to move
or stand, breathing intensely with eyes closed. Euthanasia was applied to birds clinically rated with the highest
score. The body weight of all birds was measured at 4, 7,
10, 14 and 21 days post-infection (dpi). Furthermore, at 4
dpi four randomly selected birds of each group were blood
sampled, euthanized and necropsied (Table 1). The same
procedure was performed at 7, 10, 14 and 21 dpi in groups
Page 2 of 10
L1, L2 and L0, whereas in groups B1 and B2 blood was
collected from five birds in poor condition, prior euthanasia and subsequent necropsy, between 5 and 7 dpi. In
group B0 five and eight randomly selected birds were sampled, euthanized and necropsied at 7, 10 and 14 or 21 dpi,
respectively.
The animal trial was discussed and approved by the
institutional ethics committee and the national authority according to §26 of the Law for Animal Experiments,
Tierversuchsgesetz 2012—TVG 2012, license number:
bmwf GZ 68.205/0041-WF/II/3b/2014.
Clinical chemistry
Preceding euthanasia, blood was collected from the jugular vein of the birds into heparin tubes (VACUETTE®,
Greiner Bio-One, Kremsmünster, Austria) and centrifuged at 1780 rcf for 12 min. Plasma was then separated
and the values of the following clinical chemistry analytes
were investigated by a fully selective clinical chemistry
analyzer (Cobas 501c®, Roche Diagnostics, Vienna, Austria): total protein, albumin, aspartate aminotransferase
(AST), glutamate dehydrogenase (GLDH), bile acids, uric
acid, lipase and glucose. All assays were applied according to manufacturer’s recommendations (Additional
file 1). The quality control was performed by analysing
two levels of control material before each run.
Post‑mortem examination
All euthanized and dead birds throughout the experiment
were examined by necropsy and gross lesions in liver,
pancreas, bursa of Fabricius and kidneys were recorded.
Specimens of these organs were further collected for
histopathological investigations. In addition, samples of
liver and pancreas were collected to determine the viral
load by real-time PCR.
Histopathology
Samples of liver, pancreas, bursa of Fabricius and kidney
were fixed in 4% neutral buffered formalin and embedded in paraffin blocks. Tissue sections with 4 μm of thickness were prepared using a microtome (Microm HM
360; Microm Laborgeräte GmbH, Walldorf, Germany),
mounted on glass slides and stained with haematoxylin
and eosin.
DNA extraction and determination of the viral load
DNA was extracted from 25 mg of liver and pancreas tissue from four birds of groups L1 and L2 at 4, 7 and 10
dpi, and from four and five birds at 4 and 5–7 dpi, respectively, of groups B1 and B2. For this, the DNeasy Blood
and Tissue Kit (Qiagen, Vienna, Austria) was used following the manufacturer’s instructions. The extracted
DNA was stored at −20 °C until use. A SYBR Green
Matos et al. Vet Res (2016) 47:69
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Table 1 Experimental design and mortality of birds after oral inoculation with FAdV isolates
Group
L1
B1
L2
B2
L0
B0
FAdV strain (species)
13/18 153 (FAdV-E)
13/18 153 (FAdV-E)
08/18 926 (FAdV-D)
08/18 926 (FAdV-D)
_c
_c
Chicken
type
Sampling scheme and mortality on the following days after inoculation
Layer
Broiler
Layer
Broiler
Layer
Broiler
4
5
6
7
8
9
10
14
21
Killed birds
4
_
_
3
_
_
4
4
2
Dead birdsa
_b
_
1
1
1
_
_
_
_
Killed birds
4
_
_
_
_
_
_
_
_
Dead birds
_
13
10
_
_
_
_
_
_
Killed birds
4
_
_
4
_
_
4
4
3
Dead birds
_
_
_
_
_
1
_
_
_
Killed birds
4
_
_
1
_
_
_
_
_
Dead birds
_
4
16
2
_
_
_
_
_
Killed birds
4
_
_
4
_
_
4
4
4
Dead birds
_
_
_
_
_
_
_
_
_
Killed birds
4
_
_
5
_
5
_
5
8
Dead birds
_
_
_
_
_
_
_
_
_
No.
of birds
20
27
20
27
20
27
Two groups of SPF broilers (B1–2) and two groups of SPF layers (L1–2) were inoculated orally at day-old with either a FAdV-D or -E strain, whereas one group SPF
broilers (B0) and one group of SPF layers (L0) were kept uninfected. Birds were routinely euthanized and sampled at 4, 7, 10, 14 and 21 dpi. In groups B1 and B2, five
birds with severe clinical signs were sampled between 5 and 7 dpi.
a
Birds found dead or had to be euthanized due to poor condition.
b
Not applicable.
c
Control group.
based real-time PCR with primers annealing within the
highly conserved 52 K region was performed to determine the viral load, as described by Günes et al. [12]. The
real-time PCR was performed on a Rotor-Gene Q thermal cycler (Qiagen, Hilden, Germany), using the doublestranded DNA-binding dye method with a Rotor-Gene
SYBR Green PCR kit (Qiagen). During the annealing/
extension step data were collected being further analysed in the Rotor-Gene Q software 1.7 (Qiagen). Standard curves were obtained by preparing 10-fold serial
dilutions of a linearized plasmid containing the partial
52 K gene of a FAdV-D strain (SR49) and were run two
times in duplicate. During sample preparation and realtime PCR run, negative extraction control and no template control (NTC) were included to monitor possible
contaminations. The number of viral genome copies per
reaction was calculated by comparing threshold cycle
(CT) values of the investigated samples with the standard
curves. An assessment of the specificity of the real-time
PCR products was accomplished by analysing the melting curve together with the separation of the amplification products by electrophoresis.
Statistical analysis
A Shapiro–Wilk test was performed together with a visual inspection of histograms, normal Q–Q plots and an
assessment of skewness and kurtosis z-values to confirm
the normal distribution assumptions of the data within
each group. Viral load data were log transformed to meet
the normality assumptions. Survival curves were estimated by the Kaplan–Meier method, in which routinely
killed birds were censored. A pairwise comparison by the
log-rank test was performed to investigate the significance of differences in survival rates. Survival rate data
were presented in terms of cumulative mortality (1 minus
the survival rate). An unpaired t test was used to compare the body weight and the clinical chemistry results
from each infected group with their respective control
group at each time point. Statistical differences regarding the viral load in liver and pancreas between broilers and layers infected with the same strain at each time
point were investigated by a one-way ANOVA succeeded
by pairwise comparisons using the Gabriel post hoc test.
In all cases, significant differences were assumed when
P < 0.05. Data were analysed with the statistical software
package SPSS Version 22 (IBM SPSS Statistics; IBM Corporation, Armonk, New York, USA).
Results
Clinical signs, mortality and body weight
Specific pathogen-free broiler chickens from groups B1
and B2 showed severe clinical signs starting at 4 dpi, with
high clinical scores reached at 6 dpi (Figure 1A). The condition of the SPF broilers downgraded very quickly and
significant mortalities of 100 and 96% were recorded
between 5 and 6 dpi in group B1 and 5–7 dpi in group B2,
respectively (Figure 1B). Moreover, the body weight of
the inoculated SPF broilers was found to be significantly
Matos et al. Vet Res (2016) 47:69
Page 4 of 10
lower at 5–7 dpi compared with the body weight of SPF
broilers from the control group (B0) (Figure 1C).
Specific pathogen-free layer chickens showed milder
clinical signs in comparison to the broilers, from 6 to 9
dpi, and reached a peak at 7 dpi in both groups L1 and
L2 (Figure 1A). In these groups, mortalities of 20 and 8%
were recorded between 6 and 8 or at 9 dpi, respectively,
being significantly lower when compared to both groups
B1 and B2 (Figure 1B). Furthermore, a significantly lower
body weight was already observed in birds from group
L1 at 7 dpi and in birds from both groups L1 and L2 at
10 and 14 dpi when compared to the control group (L0)
(Figure 1C).
No clinical signs and mortality were recorded in the
control groups (L0 and B0).
Gross pathology
Figure 1 Mean clinical score, cumulative mortality and mean
body weight difference. A Infected birds belonging to groups L1,
B1, L2 and B2 were individually scored based on the following clinical
signs: 0—active with no clinical signs; 1—slightly weak with dropped
wings; 2—depressed with swollen crops; 3—weak, apathetic, with
ruffled feathers and reluctant to move; 4—apathetic, unable to
move or stand, breathing intensely with eyes closed. An average
of each group’s clinical score was calculated at each time point. All
clinical signs were observed between 4 and 9 dpi. No clinical signs
were observed in the control birds. B Mortality rates (%), recorded in
groups L1, B1, L2, B2, L0 and B0 throughout the animal experiment.
Mortality curves with different lowercase letters are significantly different (P < 0.05). C Mean differences in body weight (%) of infected
birds belonging to groups L1, B1, L2 and B2 in comparison to the
respective control group (L0 or B0), at 1, 4, 7, 10, 14 and 21 dpi. Values
of groups B1 and B2 at 7 dpi correspond to pooled birds that were
euthanized and sampled between 5 and 7 dpi due to poor condition.
Asterisks indicate statistical significant difference (P < 0.05). From 7
dpi onwards there were no SPF broilers alive in groups B1 and B2.
In all SPF broiler chickens from groups B1 and B2 killed
at 4 dpi, swollen livers were the most prominent finding
during necropsy. Additionally, small necrotic foci were
present in the liver of one bird from group B2. At this
time point, no other lesions were recorded. Furthermore,
no pathomorphological lesions were observed in SPF layers. However, all dead and killed birds of the inoculated
groups between 5 and 9 dpi, regardless of host and virus
strain, presented swollen marble-like livers with a colour
ranging from yellow to brown. Moreover, swollen kidneys were observed during the necropsy of two SPF layer
chickens (one found dead and one killed) from group L1
at 7 dpi, whereas in SPF broilers no lesions were found in
kidneys.
No gross lesions were observed in other organs. Furthermore, no macroscopical changes were present in
organs from birds of the control groups (L0 and B0).
Histopathology
Although microscopical lesions were already observed at
4 dpi, most severe histological changes in infected birds
were recorded at 5–7 dpi, when large basophilic intranuclear inclusion bodies were observed in the hepatocytes and acinar cells of liver and pancreas, respectively,
together with large areas of cellular degeneration and
necrosis (Additional file 2). In coincidence with this,
areas of lymphocyte infiltration were found in liver and
pancreas of birds from groups L1 and L2. Lymphocyte
depletion in bursa of Fabricius was observed in all birds
from group B2 and in 1 and 2 birds from groups L1 and
B1, respectively, whereas signs of atrophy of the bursa of
Fabricius was observed in 1 bird each from groups B1, L2
and B2 at 5–7 dpi.
Matos et al. Vet Res (2016) 47:69
Page 5 of 10
Table 2 Clinical chemistry analytes. Means and standard deviations of total protein, albumin, AST, GLDH, bile acids, uric acid
and lipase measured in the plasma of orally inoculated SPF layers and SPF broilers from groups L1, B1, L2, B2, L0 and B0 at 4, 7, 10,
14 and 21 days post infection (dpi).
Dpia
4
7
Group
Total protein
(g/dL)
Albumin
(g/dL)
L1
3.05 ± 0.16
1.41 ± 0.13
182.25 ± 17.10
9.27 ± 4.38
48.00 ± 15.25
B1
2.48 ± 0.27
1.09 ± 0.09
226.50 ± 18.98
8.85 ± 0.76
53.75 ± 4.57
L2
2.84 ± 0.22
1.35 ± 0.13
164.50 ± 3.32c
8.47 ± 2.44
41.75 ± 12.69
B2
1.93 ± 0.12
0.84 ± 0.06
256.75 ± 82.43
21.01 ± 21.18
51.00 ± 17.80
13.65 ± 5.57
6.00 ± 0.71
L0
3.04 ± 0.16
1.28 ± 0.31
228.25 ± 35.72
8.51 ± 2.50
54.00 ± 6.98
12.48 ± 3.34
6.75 ± 1.78
B0
2.16 ± 0.43
1.01 ± 0.20
211.67 ± 56.54
13.44 ± 19.43
49.67 ± 26.10
12.33 ± 3.84
9.75 ± 7.66
L1
2.91 ± 0.44
0.99 ± 0.08c
1304.00 ± 1648.35
92.34 ± 148.87
49.75 ± 45.98
8.38 ± 4.00
78.50 ± 102.60
B1b
1.96 ± 0.29c
0.64 ± 0.04c
2382.40 ± 817.10c
35.88 ± 8.43c
272.40 ± 129.75c
8.34 ± 3.42
36.25 ± 11.50c
c
c
5.45 ± 1.06
35.33 ± 12.66c
10.38 ± 2.55
271.40 ± 132.48c
L2
3.14 ± 0.11
b
B2
10
14
21
2.53 ± 0.37
AST (U/L)
1.13 ± 0.12
c
GLDH (U/L)
425.25 ± 153.27
6.21 ± 2.34
c
0.84 ± 0.25
4319.67 ± 2970.77
166.50 ± 19.49
Bile acids
(µmol/L)
Uric acid (mg/
dL)
6.73 ± 0.88c
15.48 ± 8.24
7.95 ± 0.95c
c
167.75 ± 68.27
c
268.59 ± 274.16 197.00 ± 158.42
Lipase (U/L)
5.75 ± 0.83
7.25 ± 0.43
5.75 ± 1.09
L0
3.23 ± 0.18
1.54 ± 0.1
4.39 ± 2.07
24.00 ± 6.98
6.58 ± 0.77
5.75 ± 1.09
B0
2.34 ± 0.22
1.25 ± 0.13
203 ± 8.09
13.19 ± 4.60
29.80 ± 7.53
8.60 ± 1.72
7.20 ± 0.98
L1
3.15 ± 0.22
1.29 ± 0.12
237.00 ± 18.13c
30.60 ± 22.86
40.25 ± 12.50c
7.68 ± 2.31
7.00 ± 0.82c
c
L2
3.07 ± 0.36
1.29 ± 0.27
278.75 ± 129.96
9.80 ± 6.75
42.50 ± 8.39
6.55 ± 1.16
20.25 ± 19.14
L0
2.52 ± 0.42
0.70 ± 0.49
149.08 ± 26.61
5.52 ± 2.63
23.50 ± 9.20
6.87 ± 1.28
5.50 ± 0.50
B0
2.32 ± 0.31
0.62 ± 0.44
182.17 ± 24.60
7.78 ± 4.50
30.83 ± 10.17
9.59 ± 2.25
8.80 ± 3.82
L1
3.18 ± 0.20
1.33 ± 0.07
166.75 ± 6.99
4.17 ± 2.37
26.00 ± 9.83
6.53 ± 3.20
7.75 ± 3.03
L2
3.35 ± 0.21c
1.47 ± 0.04
225.00 ± 31.55c
66.35 ± 94.58
32.25 ± 2.63c
5.55 ± 1.50
5.33 ± 0.47
L0
2.60 ± 0.42
0.84 ± 0.53
158.25 ± 29.93
5.10 ± 1.82
19.88 ± 5.96
6.78 ± 1.88
7.50 ± 1.12
B0
2.46 ± 0.41
0.74 ± 0.47
162.31 ± 28.62
6.05 ± 4.54
21.92 ± 8.14
9.57 ± 1.42
5.80 ± 0.40
L1
3.07 ± 0.30
1.27 ± 0.06
164.50 ± 4.95
4.61 ± 0.14
21.00 ± 4.24
5.10 ± 0.00
6.00 ± 0.00
L2
3.06 ± 0.28
1.43 ± 0.09
168.00 ± 21.63
3.12 ± 1.88
29.33 ± 7.57
5.47 ± 1.86
5.50 ± 0.50
L0
3.22 ± 0.64
1.44 ± 0.40
182.00 ± 24.54
4.11 ± 1.46
26.25 ± 7.80
7.53 ± 1.69
6.50 ± 0.50
B0
2.75 ± 0.17
1.23 ± 0.08
199.50 ± 18.61
10.29 ± 9.45
16.25 ± 11.35
7.81 ± 0.85
5.75 ± 0.97
From 10 dpi onwards there were no SPF broilers alive in groups B1 and B2. All dead and killed birds at each investigated time point of each group (Table 1) were
included.
a
Day post infection.
b
Data from pooled birds, which were euthanized between 5 and 7 dpi due to poor condition.
c
Statistical significant difference—P < 0.05 when compared to the respective control group (L1 and L2 compared to L0, and B1 and B2 compared to B0).
At 10 dpi, areas of lymphocyte infiltration in liver and
pancreas were seen in all birds from groups L1 and L2,
with very few birds presenting small areas of necrosis in
liver and pancreas.
No histological changes were observed in the kidney.
Furthermore, no microscopical lesions were present in
organs from birds of the control groups (L0 and B0).
Clinical chemistry
The clinical chemistry results are presented in Table 2
and Figure 2. Total plasmatic protein was significantly
changed in group B1 at 5–7 dpi and in group L2 at 14
dpi, being lower at the former and higher at the latter sampling point, when compared to their respective
control group. The levels of plasmatic albumin were
significantly lower at 5–7 dpi in all inoculated groups
when compared to the levels observed in the respective
control group.
Two liver enzymes were measured in the plasma of the
birds—AST and GLDH—and AST activity was significantly higher at 5–7 dpi in groups B1, L2, B2, in comparison to the respective control. At 7 dpi, high values of AST
were also observed in group L1, however, a significant
difference could not be found. At 10 and 14 dpi significantly higher values were recorded in groups L1 and L2,
respectively, compared to the control group (L0). GLDH
values were found to be very high at 5–7 dpi in groups L1,
B1 and B2, at 10 dpi in group L1 and at 14 dpi in group
L2. However, significant differences with the respective
control were only found at 5–7 dpi in group B1.
Matos et al. Vet Res (2016) 47:69
Page 6 of 10
birds from group L1 were higher in comparison to group
L2 at 4 and 7 dpi.
Figure 2 Blood glucose concentration. Means and standard
deviations of blood glucose concentration values (mg/dL) of SPF
layer and SPF broiler chickens from groups L1, B1, L2, B2, L0 and B0 at
4, 7, 10, 14 and 21 dpi. Values of groups B1 and B2 at 7 dpi correspond to pooled birds that were euthanized and sampled between
5 and 7 dpi due to poor condition. From 7 dpi onwards there were
no SPF broilers alive in groups B1 and B2. All dead and killed birds at
each investigated time point of each group (Table 1) were included.
Asterisks indicate statistical significant difference when compared to
the respective control group (L0 or B0) (P < 0.05).
Significant changes were observed in the levels of
metabolites measured in the plasma. Bile acids plasmatic
concentration was significantly increased at 5–7, 10 and
14 dpi in groups B1, L2 and B2, groups L1 and L2, and
group L2, respectively. Plasmatic uric acid levels were
significantly lower at 4 dpi in groups L1 and L2, in comparison to the control group L0.
Lipase activities were very high in all inoculated groups
at 5–7 dpi, being significantly increased in groups B1,
L2 and B2. At 10 dpi lipase activities were significantly
and non-significantly increased in groups L1 and L2,
respectively.
Levels of plasmatic glucose were non-significantly
increased at 4 dpi in group B2 and significantly decreased
at 5–7 dpi in groups B1 and B2, in comparison with the
control group B0 (Figure 2).
Viral load
At 4 dpi the viral load in the liver of birds from both
groups B1 and B2 was significantly higher in comparison
to group L2 and non-significantly higher than in group
L1 (Figure 3A). At 7 dpi, the viral load peaked in the liver
of birds from groups L1 and L2 and the viral genome copies per reaction in these groups were significantly higher
in comparison to groups B1 and B2 at 5–7 dpi.
In the pancreas, high loads of viral DNA were determined in both groups B1 and B2 at 4 and 5–7 dpi, being
significantly higher compared with groups L1 and L2
(Figure 3B). Furthermore, the viral load in pancreas of
Discussion
In a recent study we used five FAdV field strains belonging to species FAdV-D and E to orally inoculate separate
groups of day-old SPF white leghorn chickens and a panel
of biomarkers based on clinical chemistry was established, which correlated with the pathogenicity of FAdV
strains and the pathogenesis of IBH [9]. It was shown that
pathogenic FAdV strains are capable to interfere with
enzyme systems and metabolites concentration which are
related to liver and pancreas functions.
In continuity with the aforementioned investigations,
the aim of the present study was to compare and assess
the influence of the genetic background of the host on
the outcome of a FAdV infection considering the different metabolism and growth rate between meat-producing (SPF broilers) and egg-producing (SPF layers)
chickens. For this, two previously tested FAdV-D and E
field strains were chosen based on their pathogenicity in
SPF layers [9]. Independent of the virus used for infection in the present investigation, significant mortalities
approaching 100% together with severe clinical signs
were recorded in the inoculated SPF broilers, differing
substantially from the infection outcome in SPF layers.
However, the recorded mortality in SPF layers was considerably lower when compared with the results which
we previously reported, following the same inoculation
procedure [9]. In the previous study, the occurrence of
non-specific mortality in the first week of life together
with a lower body weight of the control birds throughout the animal experiment, in comparison to the control
SPF layers of the present study (data not shown), indicates a diminished quality and performance of the chicks,
which may have influenced the outcome of the infection.
Nonetheless, further investigations are needed to test this
hypothesis.
Previous pathogenicity studies were mostly performed in layer-type chickens and mortalities of 30–50%
were reported when birds were inoculated by natural
routes with a similar dose of a virulent IBH strain [5,
9, 13–15], hence considerably lower than the mortalities recorded in the inoculated SPF broilers’ groups of
the current study. Genetically different chickens were
used earlier to investigate IBH in experimental studies
[16–20]. However, in nearly all of these studies broilers
of commercial origin were used, whose serological status
regarding maternal antibodies against FAdVs was either
positive or unknown [16, 18–20]. Exceptional to this,
Cook [17] used different chicken breeds of SPF and nonSPF origin to study the influence of host, age and route
Matos et al. Vet Res (2016) 47:69
Page 7 of 10
Figure 3 Viral load in liver and pancreas. Means and standard deviations of the viral genome copies per reaction (log10) in (A) liver and (B)
pancreas of orally inoculated SPF layer and SPF broiler chickens from groups L1, B1, L2 and B2 at 4, 7 and 10 dpi quantified by real-time PCR. Values
of groups B1 and B2 at 7 dpi correspond to pooled birds that were euthanized and sampled between 5 and 7 dpi due to poor condition. All dead
and killed birds at each investigated time point from each group (Table 1) were included. Negative results are indicated with §. There were no SPF
broilers alive at 10 dpi (¥). Mean values with different lowercase letters at each time point are significantly different (P < 0.05).
of inoculation on the outcome of a FAdV infection. As a
result, SPF Light Sussex chickens were highly susceptible
to the infection in comparison to SPF Rhode Island Red
chickens, with mortalities of 67 and 33% being recorded,
respectively, following intraperitoneal inoculation at dayold. Nonetheless, no broiler chickens were used in the
investigation. Different to this, Alvarado et al. [20] used
broilers and layers of SPF origin together with broilers
from vaccinated breeders to investigate the pathogenicity
of a FAdV-E strain, and different mortalities in SPF broilers (40%) and SPF layers (20%) were reported, following
subcutaneous infection at week-old. However, the study
did not focus to elucidate the mechanisms of these differences in susceptibility, which would have been hampered
Matos et al. Vet Res (2016) 47:69
by the fact that subcutaneous infection route was used.
Thus, this is the first study to report a significant susceptibility of SPF broiler chickens following an oral infection
with FAdV strains belonging to species FAdV-D and E, in
direct comparison to SPF layers, and to unravel the background of this phenomenon.
Therefore, in the actual study, further investigations were
carried out to elucidate the pathogenesis of IBH in general
and the influence of the birds’ metabolism. Liver and pancreas are important target organs for FAdVs [9] and, therefore, the blood glucose concentration was determined in
all birds throughout the study, as the glucose metabolism
is a good indicator for liver and pancreas functions [21]. A
recent investigation demonstrated that chickens from lines
bred for high juvenile body weight have an impaired glucose homeostasis and a different pancreas physiology in
comparison to chickens with a low juvenile body weight
[22]. In the current investigation significantly lower blood
glucose concentrations were recorded in both inoculated
SPF broilers’ groups during the peak of infection, in comparison to the control group, corresponding to a hypoglycaemic status according to previously established reference
intervals [21, 23]. It seems that IBH, to which broilers are
highly susceptible, evolves to a metabolic disorder. Furthermore, significant changes in clinical chemistry analytes measured in the plasma of infected birds confirmed
tissue damage and functional impairment of both liver
and pancreas, which were further validated by histopathological studies. In a field investigation, Goodwin et al. [24]
reported the presence of adenoviral inclusion bodies in
liver, pancreas and small intestine of hypoglycaemic broiler
chicks from a flock suffering from spiking mortality, to
which the outcome of the present experimental study parallels. Glucose together with sodium bicarbonate and calcium were once effective as a supportive treatment during
an IBH outbreak, in which broiler chickens were suffering
from hypoglycaemia, metabolic acidosis and hypocalcaemia [25], highlighting the potential of FAdV infections to
unfold in a metabolic impairment. It can be hypothesized
that the blood glucose of the infected birds would decrease
due to apathy and anorexia together with malabsorption
related to the ongoing pancreatitis caused by the infection.
However, during short-term fasting periods, the blood
glucose concentration in granivorous birds is maintained
by hepatic glycogenolysis, in which glucagon—a hormone
produced by pancreatic alpha-cells—plays an important
role as a regulator [21]. Therefore, severe lesions in liver
and/or pancreas can contribute towards disturbances in
the glucose homeostasis. Recently, it was demonstrated
that birds with extensive lesions in pancreas due to an
experimental infection with low-pathogenic avian influenza viruses (LPAIVs) experienced hyperglycaemia and
not hypoglycaemia as observed in our study [26, 27]. In
Page 8 of 10
comparison, FAdV induced IBH severely affects not only
the pancreas but also the liver of birds, preventing compensation mechanisms to occur. Nevertheless, further
studies would be of interest to fully address this issue.
In addition to hepatic and pancreatic changes, lesions
in kidneys have been described throughout the years
as a consequence of experimental FAdV infections [9,
15, 28–30]. Kidneys are mostly responsible for removing uric acid from the blood and, therefore, concentrations greater than 13 mg/dL are suggestive for impaired
renal function [21]. This was never the case in the present study and, in fact, plasmatic uric acid concentration was significantly lower only in the infected SPF
layers’ groups at 4 dpi when compared with the control
group. This can rather be interpreted as a consequence
of liver instead of kidney damage, since the liver is highly
responsible for uric acid production [21].Therefore, the
findings of the present study do not provide evidence
of kidney function impairment due to FAdV infection,
confirming our previously published data [9]. In agreement with this, no lesions were observed in the kidneys
during the histological investigation, despite of swollen
kidneys noticed during necropsy in two birds. Although
glomerulonephritis has been suggested as the underlying cause for the macroscopical changes in kidneys of
broilers suffering from IBH in the field [31], a direct connection between FAdVs and glomerulonephritis has not
been demonstrated, so far, in experimentally infected
birds. Assessing the glomerular size and cellularity in
kidneys of experimentally FAdV infected birds by histomorphometric studies would be of interest to test this
hypothesis.
In our previous studies we were aiming to establish a
link between viral load in target organs with histopathological and macroscopical lesions, clinical chemistry and
clinical signs, in chickens experimentally infected with
FAdVs, although different diseases were investigated and
not all parameters were investigated at the same time [9,
32]. In the present study, very high loads of viral DNA
were seen already at 4 dpi in both liver and pancreas of
infected SPF broilers, preceding the onset of severe clinical signs and high mortality together with severe changes
in clinical chemistry and histopathological lesions. Thus,
these findings provide evidence that FAdVs replicate
faster in both liver and pancreas of broilers in comparison to layers, harmonizing with the outcome of the infection. Furthermore, independent of the virus, much higher
viral loads were determined in the pancreas of broilers in
comparison to livers throughout the experiment, something not observed in layers. This highlights the importance of the pancreas as a target organ for FAdVs with
its crucial function in context of the noticed metabolic
derangement.
Matos et al. Vet Res (2016) 47:69
In conclusion, this is the first study to report a significant difference in susceptibility of SPF broiler chickens to
an oral infection with FAdV strains belonging to species
FAdV-D and E in direct comparison to SPF layers, underlining the importance of the genetic background of the host
on the outcome of a FAdV infection. Furthermore, during
the peak of infection SPF broilers suffered from hypoglycaemia, in which the severe lesions in liver and pancreas
seemed to play an important role. Therefore, we propose
that FAdV infections, to which broiler chickens are very
susceptible, can lead to metabolic disorders. Nevertheless,
further studies are needed to better understand the pathogenesis of IBH in broilers and the involvement of the liver
and pancreas in metabolic derangements by FAdVs.
Additional files
Additional file 1. Kits and methods used to investigate the clinical
chemistry analytes in the plasma of birds. Total protein, albumin,
aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH),
bile acids, uric acid and lipase were investigated in the plasma of birds at
different time points by a fully selective clinical chemistry analyzer (Cobas
501c®, Roche Diagnostics, Vienna, Austria).
Additional file 2. Histopathological findings in infected birds
at different time points. Histopathological lesions recorded in liver,
pancreas and bursa of Fabricius of infected birds from groups L1, B1, L2
and B2 at 4, 7 and 10 dpi. No histopathological changes were observed in
the kidney. Furthermore, no microscopical lesions were present in organs
from birds of the control groups.
Abbreviations
AGE: adenoviral gizzard erosion; AST: aspartate aminotransferase; CEL: chicken
embryo liver; CT: threshold cycle; FAdV: fowl aviadenovirus; GLDH: glutamate
dehydrogenase; HHS: hydropericardium hepatitis syndrome; IBH: inclusion
body hepatitis; NTC: negative template control; PCR: polymerase chain reaction; SPF: specific-pathogen-free; TCID50: median tissue culture infective dose.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MH, BG and MM participated in the design of the study. MM and BG performed the animal experiment. MM and DL carried out the histopathological
analysis. MM performed the viral load investigations by real-time PCR, statistical analysis and drafted the manuscript. All authors read and approved the
final manuscript.
Acknowledgements
The authors would like to thank Prof. Dr. Ilse Schwendenwein from the Clinical
Pathology Platform, Department of Pathobiology, University of Veterinary
Medicine Vienna, for the clinical chemistry analysis.
Author details
1
Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz
1, 1210 Vienna, Austria. 2 Christian Doppler Laboratory for Innovative Poultry
Vaccines (IPOV), University of Veterinary Medicine Vienna, Veterinaerplatz 1,
1210 Vienna, Austria.
Received: 15 March 2016 Accepted: 2 June 2016
Page 9 of 10
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Matos et al. Vet Res (2016) 47:69
DOI 10.1186/s13567-016-0350-0
Open Access
RESEARCH ARTICLE
The outcome of experimentally
induced inclusion body hepatitis (IBH) by fowl
aviadenoviruses (FAdVs) is crucially influenced
by the genetic background of the host
Miguel Matos1*, Beatrice Grafl1, Dieter Liebhart1 and Michael Hess1,2
Abstract
In the present study, inclusion body hepatitis (IBH) was experimentally induced by oral inoculation of two groups of
specific pathogen-free (SPF) broilers and two groups of SPF layers at day-old with either a fowl aviadenovirus (FAdV)-D
or a FAdV-E strain. A substantial variation in the degree of susceptibility was observed with mortalities of 100 and 96%
in the FAdV-E and D infected SPF broiler groups, respectively, whereas in the groups of infected SPF layers mortalities
of only 20 and 8% were noticed. Significant changes in clinical chemistry analytes of all infected birds together with
histopathological lesions indicated impairment of liver and pancreas integrity and functions. Furthermore, significantly lower blood glucose concentrations were recorded at peak of infection in both inoculated SPF broiler groups,
in comparison to the control group, corresponding to a hypoglycaemic status. High viral loads were determined in
liver and pancreas of SPF broilers already at 4 days post-infection (dpi), in comparison to SPF layers, indicating a somewhat faster viral replication in the target organs. Overall, highest values were noticed in the pancreas of SPF broilers
independent of the virus used for infection. The actual study provides new insights into the pathogenesis of IBH, a
disease evolving to a metabolic disorder, to which SPF broilers were highly susceptible. Hence, this is the first study to
report a significant higher susceptibility of SPF broiler chickens to experimentally induced IBH in direct comparison to
SPF layers.
Introduction
Fowl aviadenoviruses (FAdVs) belong to the genus Aviadenovirus within the family Adenoviridae, being further
divided into five species designated FAdV-A to E [1].
Throughout the years, many reports established a causality between strains from species FAdV-A, FAdV-C and
FAdV-D together with FAdV-E with specific diseases
in chickens, such as adenoviral gizzard erosion (AGE),
hydropericardium hepatitis syndrome (HHS) and inclusion body hepatitis (IBH), respectively [2].
In the last 10 years IBH outbreaks have been reported
in different geographic regions emphasizing the wide
*Correspondence: miguel.matos@vetmeduni.ac.at
1
Clinic for Poultry and Fish Medicine, Department for Farm Animals
and Veterinary Public Health, University of Veterinary Medicine Vienna,
Veterinaerplatz 1, 1210 Vienna, Austria
Full list of author information is available at the end of the article
distribution of FAdVs throughout the world [3–8]. In the
field, IBH has been reported essentially from commercial
broiler flocks (meat-producing chickens), being responsible for serious economic losses due to increased mortality combined with reduced performance within flocks
[2]. However, experimental in vivo studies were predominantly conducted in specific pathogen-free (SPF) white
leghorn layers (egg-producing chickens), which are the
experimental model for infection studies.
In a recent study we were able to demonstrate the influence of virulent FAdV-D and E field strains on different
enzyme systems and metabolite concentrations in the
plasma of orally inoculated day-old SPF layer chickens
due to the infection of liver and pancreas as target organs
[9]. Consequently, it can be hypothesized that hosts with
different metabolic activities vary in their susceptibilities
towards infection. Therefore, the aim of the present study
© 2016 The Author(s). 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.
Matos et al. Vet Res (2016) 47:69
was to characterize and compare the susceptibility of SPF
broiler and SPF layer chickens to experimentally induced
IBH by FAdV-D and E field strains.
Materials and methods
Viruses
The FAdV strains used in the present study—08/18 926
and 13/18 153—were isolated from liver samples of broilers during recent IBH outbreaks in Europe and they were
genotyped as belonging to species FAdV-D and E, respectively [4, 8]. The viruses were plaque purified three times
and propagated in primary chicken embryo liver (CEL)
cell cultures as described elsewhere [10]. The titers were
determined according to the method of end point titration [11] and a titer of 107 median tissue culture infective dose (TCID50) per mL was used to infect the birds. A
polymerase chain reaction (PCR) and a reverse transcription-PCR were performed to confirm the absence of contaminations by chicken anaemia virus and avian reovirus,
respectively. The strains’ pathogenicity was characterized
in vivo by inoculating SPF white leghorn chickens at dayold [9].
Animal trial
Embryonated SPF broiler eggs (Animal Health Service,
Deventer, The Netherlands) and SPF layer eggs (VALO,
Lohmann Tierzucht GmbH, Cuxhaven, Germany) were
incubated at our facilities. After hatch, the chicks were
individually tagged subcutaneously (Swiftack, Heartland Animal Health Inc., Fair Play, USA) and divided in
six groups: three groups of 27 SPF broiler chicks (groups
B0–2) and three groups of 20 SPF layer chicks (groups
L0–2). The groups were housed separately in isolator units
(Montair Andersen bv, HM 1500, Sevenum, Netherlands)
under negative pressure, where feed and water were available ad libitum throughout the animal experiment. At first
day of life, the body weight of all birds was measured and
birds from groups L1 and B1, and from groups L2 and B2
were orally inoculated with 0.5 mL of the 13/18 153 and
the 08/18 926 strains, respectively, while birds from groups
L0 and B0 were left uninoculated (Table 1). All birds were
daily monitored and an individual score was given based
on clinical signs: 0—active with no adverse clinical signs;
1—slightly weak with dropped wings; 2—depressed with
swollen crops; 3—weak, apathetic, with ruffled feathers and reluctant to move; 4—apathetic, unable to move
or stand, breathing intensely with eyes closed. Euthanasia was applied to birds clinically rated with the highest
score. The body weight of all birds was measured at 4, 7,
10, 14 and 21 days post-infection (dpi). Furthermore, at 4
dpi four randomly selected birds of each group were blood
sampled, euthanized and necropsied (Table 1). The same
procedure was performed at 7, 10, 14 and 21 dpi in groups
Page 2 of 10
L1, L2 and L0, whereas in groups B1 and B2 blood was
collected from five birds in poor condition, prior euthanasia and subsequent necropsy, between 5 and 7 dpi. In
group B0 five and eight randomly selected birds were sampled, euthanized and necropsied at 7, 10 and 14 or 21 dpi,
respectively.
The animal trial was discussed and approved by the
institutional ethics committee and the national authority according to §26 of the Law for Animal Experiments,
Tierversuchsgesetz 2012—TVG 2012, license number:
bmwf GZ 68.205/0041-WF/II/3b/2014.
Clinical chemistry
Preceding euthanasia, blood was collected from the jugular vein of the birds into heparin tubes (VACUETTE®,
Greiner Bio-One, Kremsmünster, Austria) and centrifuged at 1780 rcf for 12 min. Plasma was then separated
and the values of the following clinical chemistry analytes
were investigated by a fully selective clinical chemistry
analyzer (Cobas 501c®, Roche Diagnostics, Vienna, Austria): total protein, albumin, aspartate aminotransferase
(AST), glutamate dehydrogenase (GLDH), bile acids, uric
acid, lipase and glucose. All assays were applied according to manufacturer’s recommendations (Additional
file 1). The quality control was performed by analysing
two levels of control material before each run.
Post‑mortem examination
All euthanized and dead birds throughout the experiment
were examined by necropsy and gross lesions in liver,
pancreas, bursa of Fabricius and kidneys were recorded.
Specimens of these organs were further collected for
histopathological investigations. In addition, samples of
liver and pancreas were collected to determine the viral
load by real-time PCR.
Histopathology
Samples of liver, pancreas, bursa of Fabricius and kidney
were fixed in 4% neutral buffered formalin and embedded in paraffin blocks. Tissue sections with 4 μm of thickness were prepared using a microtome (Microm HM
360; Microm Laborgeräte GmbH, Walldorf, Germany),
mounted on glass slides and stained with haematoxylin
and eosin.
DNA extraction and determination of the viral load
DNA was extracted from 25 mg of liver and pancreas tissue from four birds of groups L1 and L2 at 4, 7 and 10
dpi, and from four and five birds at 4 and 5–7 dpi, respectively, of groups B1 and B2. For this, the DNeasy Blood
and Tissue Kit (Qiagen, Vienna, Austria) was used following the manufacturer’s instructions. The extracted
DNA was stored at −20 °C until use. A SYBR Green
Matos et al. Vet Res (2016) 47:69
Page 3 of 10
Table 1 Experimental design and mortality of birds after oral inoculation with FAdV isolates
Group
L1
B1
L2
B2
L0
B0
FAdV strain (species)
13/18 153 (FAdV-E)
13/18 153 (FAdV-E)
08/18 926 (FAdV-D)
08/18 926 (FAdV-D)
_c
_c
Chicken
type
Sampling scheme and mortality on the following days after inoculation
Layer
Broiler
Layer
Broiler
Layer
Broiler
4
5
6
7
8
9
10
14
21
Killed birds
4
_
_
3
_
_
4
4
2
Dead birdsa
_b
_
1
1
1
_
_
_
_
Killed birds
4
_
_
_
_
_
_
_
_
Dead birds
_
13
10
_
_
_
_
_
_
Killed birds
4
_
_
4
_
_
4
4
3
Dead birds
_
_
_
_
_
1
_
_
_
Killed birds
4
_
_
1
_
_
_
_
_
Dead birds
_
4
16
2
_
_
_
_
_
Killed birds
4
_
_
4
_
_
4
4
4
Dead birds
_
_
_
_
_
_
_
_
_
Killed birds
4
_
_
5
_
5
_
5
8
Dead birds
_
_
_
_
_
_
_
_
_
No.
of birds
20
27
20
27
20
27
Two groups of SPF broilers (B1–2) and two groups of SPF layers (L1–2) were inoculated orally at day-old with either a FAdV-D or -E strain, whereas one group SPF
broilers (B0) and one group of SPF layers (L0) were kept uninfected. Birds were routinely euthanized and sampled at 4, 7, 10, 14 and 21 dpi. In groups B1 and B2, five
birds with severe clinical signs were sampled between 5 and 7 dpi.
a
Birds found dead or had to be euthanized due to poor condition.
b
Not applicable.
c
Control group.
based real-time PCR with primers annealing within the
highly conserved 52 K region was performed to determine the viral load, as described by Günes et al. [12]. The
real-time PCR was performed on a Rotor-Gene Q thermal cycler (Qiagen, Hilden, Germany), using the doublestranded DNA-binding dye method with a Rotor-Gene
SYBR Green PCR kit (Qiagen). During the annealing/
extension step data were collected being further analysed in the Rotor-Gene Q software 1.7 (Qiagen). Standard curves were obtained by preparing 10-fold serial
dilutions of a linearized plasmid containing the partial
52 K gene of a FAdV-D strain (SR49) and were run two
times in duplicate. During sample preparation and realtime PCR run, negative extraction control and no template control (NTC) were included to monitor possible
contaminations. The number of viral genome copies per
reaction was calculated by comparing threshold cycle
(CT) values of the investigated samples with the standard
curves. An assessment of the specificity of the real-time
PCR products was accomplished by analysing the melting curve together with the separation of the amplification products by electrophoresis.
Statistical analysis
A Shapiro–Wilk test was performed together with a visual inspection of histograms, normal Q–Q plots and an
assessment of skewness and kurtosis z-values to confirm
the normal distribution assumptions of the data within
each group. Viral load data were log transformed to meet
the normality assumptions. Survival curves were estimated by the Kaplan–Meier method, in which routinely
killed birds were censored. A pairwise comparison by the
log-rank test was performed to investigate the significance of differences in survival rates. Survival rate data
were presented in terms of cumulative mortality (1 minus
the survival rate). An unpaired t test was used to compare the body weight and the clinical chemistry results
from each infected group with their respective control
group at each time point. Statistical differences regarding the viral load in liver and pancreas between broilers and layers infected with the same strain at each time
point were investigated by a one-way ANOVA succeeded
by pairwise comparisons using the Gabriel post hoc test.
In all cases, significant differences were assumed when
P < 0.05. Data were analysed with the statistical software
package SPSS Version 22 (IBM SPSS Statistics; IBM Corporation, Armonk, New York, USA).
Results
Clinical signs, mortality and body weight
Specific pathogen-free broiler chickens from groups B1
and B2 showed severe clinical signs starting at 4 dpi, with
high clinical scores reached at 6 dpi (Figure 1A). The condition of the SPF broilers downgraded very quickly and
significant mortalities of 100 and 96% were recorded
between 5 and 6 dpi in group B1 and 5–7 dpi in group B2,
respectively (Figure 1B). Moreover, the body weight of
the inoculated SPF broilers was found to be significantly
Matos et al. Vet Res (2016) 47:69
Page 4 of 10
lower at 5–7 dpi compared with the body weight of SPF
broilers from the control group (B0) (Figure 1C).
Specific pathogen-free layer chickens showed milder
clinical signs in comparison to the broilers, from 6 to 9
dpi, and reached a peak at 7 dpi in both groups L1 and
L2 (Figure 1A). In these groups, mortalities of 20 and 8%
were recorded between 6 and 8 or at 9 dpi, respectively,
being significantly lower when compared to both groups
B1 and B2 (Figure 1B). Furthermore, a significantly lower
body weight was already observed in birds from group
L1 at 7 dpi and in birds from both groups L1 and L2 at
10 and 14 dpi when compared to the control group (L0)
(Figure 1C).
No clinical signs and mortality were recorded in the
control groups (L0 and B0).
Gross pathology
Figure 1 Mean clinical score, cumulative mortality and mean
body weight difference. A Infected birds belonging to groups L1,
B1, L2 and B2 were individually scored based on the following clinical
signs: 0—active with no clinical signs; 1—slightly weak with dropped
wings; 2—depressed with swollen crops; 3—weak, apathetic, with
ruffled feathers and reluctant to move; 4—apathetic, unable to
move or stand, breathing intensely with eyes closed. An average
of each group’s clinical score was calculated at each time point. All
clinical signs were observed between 4 and 9 dpi. No clinical signs
were observed in the control birds. B Mortality rates (%), recorded in
groups L1, B1, L2, B2, L0 and B0 throughout the animal experiment.
Mortality curves with different lowercase letters are significantly different (P < 0.05). C Mean differences in body weight (%) of infected
birds belonging to groups L1, B1, L2 and B2 in comparison to the
respective control group (L0 or B0), at 1, 4, 7, 10, 14 and 21 dpi. Values
of groups B1 and B2 at 7 dpi correspond to pooled birds that were
euthanized and sampled between 5 and 7 dpi due to poor condition.
Asterisks indicate statistical significant difference (P < 0.05). From 7
dpi onwards there were no SPF broilers alive in groups B1 and B2.
In all SPF broiler chickens from groups B1 and B2 killed
at 4 dpi, swollen livers were the most prominent finding
during necropsy. Additionally, small necrotic foci were
present in the liver of one bird from group B2. At this
time point, no other lesions were recorded. Furthermore,
no pathomorphological lesions were observed in SPF layers. However, all dead and killed birds of the inoculated
groups between 5 and 9 dpi, regardless of host and virus
strain, presented swollen marble-like livers with a colour
ranging from yellow to brown. Moreover, swollen kidneys were observed during the necropsy of two SPF layer
chickens (one found dead and one killed) from group L1
at 7 dpi, whereas in SPF broilers no lesions were found in
kidneys.
No gross lesions were observed in other organs. Furthermore, no macroscopical changes were present in
organs from birds of the control groups (L0 and B0).
Histopathology
Although microscopical lesions were already observed at
4 dpi, most severe histological changes in infected birds
were recorded at 5–7 dpi, when large basophilic intranuclear inclusion bodies were observed in the hepatocytes and acinar cells of liver and pancreas, respectively,
together with large areas of cellular degeneration and
necrosis (Additional file 2). In coincidence with this,
areas of lymphocyte infiltration were found in liver and
pancreas of birds from groups L1 and L2. Lymphocyte
depletion in bursa of Fabricius was observed in all birds
from group B2 and in 1 and 2 birds from groups L1 and
B1, respectively, whereas signs of atrophy of the bursa of
Fabricius was observed in 1 bird each from groups B1, L2
and B2 at 5–7 dpi.
Matos et al. Vet Res (2016) 47:69
Page 5 of 10
Table 2 Clinical chemistry analytes. Means and standard deviations of total protein, albumin, AST, GLDH, bile acids, uric acid
and lipase measured in the plasma of orally inoculated SPF layers and SPF broilers from groups L1, B1, L2, B2, L0 and B0 at 4, 7, 10,
14 and 21 days post infection (dpi).
Dpia
4
7
Group
Total protein
(g/dL)
Albumin
(g/dL)
L1
3.05 ± 0.16
1.41 ± 0.13
182.25 ± 17.10
9.27 ± 4.38
48.00 ± 15.25
B1
2.48 ± 0.27
1.09 ± 0.09
226.50 ± 18.98
8.85 ± 0.76
53.75 ± 4.57
L2
2.84 ± 0.22
1.35 ± 0.13
164.50 ± 3.32c
8.47 ± 2.44
41.75 ± 12.69
B2
1.93 ± 0.12
0.84 ± 0.06
256.75 ± 82.43
21.01 ± 21.18
51.00 ± 17.80
13.65 ± 5.57
6.00 ± 0.71
L0
3.04 ± 0.16
1.28 ± 0.31
228.25 ± 35.72
8.51 ± 2.50
54.00 ± 6.98
12.48 ± 3.34
6.75 ± 1.78
B0
2.16 ± 0.43
1.01 ± 0.20
211.67 ± 56.54
13.44 ± 19.43
49.67 ± 26.10
12.33 ± 3.84
9.75 ± 7.66
L1
2.91 ± 0.44
0.99 ± 0.08c
1304.00 ± 1648.35
92.34 ± 148.87
49.75 ± 45.98
8.38 ± 4.00
78.50 ± 102.60
B1b
1.96 ± 0.29c
0.64 ± 0.04c
2382.40 ± 817.10c
35.88 ± 8.43c
272.40 ± 129.75c
8.34 ± 3.42
36.25 ± 11.50c
c
c
5.45 ± 1.06
35.33 ± 12.66c
10.38 ± 2.55
271.40 ± 132.48c
L2
3.14 ± 0.11
b
B2
10
14
21
2.53 ± 0.37
AST (U/L)
1.13 ± 0.12
c
GLDH (U/L)
425.25 ± 153.27
6.21 ± 2.34
c
0.84 ± 0.25
4319.67 ± 2970.77
166.50 ± 19.49
Bile acids
(µmol/L)
Uric acid (mg/
dL)
6.73 ± 0.88c
15.48 ± 8.24
7.95 ± 0.95c
c
167.75 ± 68.27
c
268.59 ± 274.16 197.00 ± 158.42
Lipase (U/L)
5.75 ± 0.83
7.25 ± 0.43
5.75 ± 1.09
L0
3.23 ± 0.18
1.54 ± 0.1
4.39 ± 2.07
24.00 ± 6.98
6.58 ± 0.77
5.75 ± 1.09
B0
2.34 ± 0.22
1.25 ± 0.13
203 ± 8.09
13.19 ± 4.60
29.80 ± 7.53
8.60 ± 1.72
7.20 ± 0.98
L1
3.15 ± 0.22
1.29 ± 0.12
237.00 ± 18.13c
30.60 ± 22.86
40.25 ± 12.50c
7.68 ± 2.31
7.00 ± 0.82c
c
L2
3.07 ± 0.36
1.29 ± 0.27
278.75 ± 129.96
9.80 ± 6.75
42.50 ± 8.39
6.55 ± 1.16
20.25 ± 19.14
L0
2.52 ± 0.42
0.70 ± 0.49
149.08 ± 26.61
5.52 ± 2.63
23.50 ± 9.20
6.87 ± 1.28
5.50 ± 0.50
B0
2.32 ± 0.31
0.62 ± 0.44
182.17 ± 24.60
7.78 ± 4.50
30.83 ± 10.17
9.59 ± 2.25
8.80 ± 3.82
L1
3.18 ± 0.20
1.33 ± 0.07
166.75 ± 6.99
4.17 ± 2.37
26.00 ± 9.83
6.53 ± 3.20
7.75 ± 3.03
L2
3.35 ± 0.21c
1.47 ± 0.04
225.00 ± 31.55c
66.35 ± 94.58
32.25 ± 2.63c
5.55 ± 1.50
5.33 ± 0.47
L0
2.60 ± 0.42
0.84 ± 0.53
158.25 ± 29.93
5.10 ± 1.82
19.88 ± 5.96
6.78 ± 1.88
7.50 ± 1.12
B0
2.46 ± 0.41
0.74 ± 0.47
162.31 ± 28.62
6.05 ± 4.54
21.92 ± 8.14
9.57 ± 1.42
5.80 ± 0.40
L1
3.07 ± 0.30
1.27 ± 0.06
164.50 ± 4.95
4.61 ± 0.14
21.00 ± 4.24
5.10 ± 0.00
6.00 ± 0.00
L2
3.06 ± 0.28
1.43 ± 0.09
168.00 ± 21.63
3.12 ± 1.88
29.33 ± 7.57
5.47 ± 1.86
5.50 ± 0.50
L0
3.22 ± 0.64
1.44 ± 0.40
182.00 ± 24.54
4.11 ± 1.46
26.25 ± 7.80
7.53 ± 1.69
6.50 ± 0.50
B0
2.75 ± 0.17
1.23 ± 0.08
199.50 ± 18.61
10.29 ± 9.45
16.25 ± 11.35
7.81 ± 0.85
5.75 ± 0.97
From 10 dpi onwards there were no SPF broilers alive in groups B1 and B2. All dead and killed birds at each investigated time point of each group (Table 1) were
included.
a
Day post infection.
b
Data from pooled birds, which were euthanized between 5 and 7 dpi due to poor condition.
c
Statistical significant difference—P < 0.05 when compared to the respective control group (L1 and L2 compared to L0, and B1 and B2 compared to B0).
At 10 dpi, areas of lymphocyte infiltration in liver and
pancreas were seen in all birds from groups L1 and L2,
with very few birds presenting small areas of necrosis in
liver and pancreas.
No histological changes were observed in the kidney.
Furthermore, no microscopical lesions were present in
organs from birds of the control groups (L0 and B0).
Clinical chemistry
The clinical chemistry results are presented in Table 2
and Figure 2. Total plasmatic protein was significantly
changed in group B1 at 5–7 dpi and in group L2 at 14
dpi, being lower at the former and higher at the latter sampling point, when compared to their respective
control group. The levels of plasmatic albumin were
significantly lower at 5–7 dpi in all inoculated groups
when compared to the levels observed in the respective
control group.
Two liver enzymes were measured in the plasma of the
birds—AST and GLDH—and AST activity was significantly higher at 5–7 dpi in groups B1, L2, B2, in comparison to the respective control. At 7 dpi, high values of AST
were also observed in group L1, however, a significant
difference could not be found. At 10 and 14 dpi significantly higher values were recorded in groups L1 and L2,
respectively, compared to the control group (L0). GLDH
values were found to be very high at 5–7 dpi in groups L1,
B1 and B2, at 10 dpi in group L1 and at 14 dpi in group
L2. However, significant differences with the respective
control were only found at 5–7 dpi in group B1.
Matos et al. Vet Res (2016) 47:69
Page 6 of 10
birds from group L1 were higher in comparison to group
L2 at 4 and 7 dpi.
Figure 2 Blood glucose concentration. Means and standard
deviations of blood glucose concentration values (mg/dL) of SPF
layer and SPF broiler chickens from groups L1, B1, L2, B2, L0 and B0 at
4, 7, 10, 14 and 21 dpi. Values of groups B1 and B2 at 7 dpi correspond to pooled birds that were euthanized and sampled between
5 and 7 dpi due to poor condition. From 7 dpi onwards there were
no SPF broilers alive in groups B1 and B2. All dead and killed birds at
each investigated time point of each group (Table 1) were included.
Asterisks indicate statistical significant difference when compared to
the respective control group (L0 or B0) (P < 0.05).
Significant changes were observed in the levels of
metabolites measured in the plasma. Bile acids plasmatic
concentration was significantly increased at 5–7, 10 and
14 dpi in groups B1, L2 and B2, groups L1 and L2, and
group L2, respectively. Plasmatic uric acid levels were
significantly lower at 4 dpi in groups L1 and L2, in comparison to the control group L0.
Lipase activities were very high in all inoculated groups
at 5–7 dpi, being significantly increased in groups B1,
L2 and B2. At 10 dpi lipase activities were significantly
and non-significantly increased in groups L1 and L2,
respectively.
Levels of plasmatic glucose were non-significantly
increased at 4 dpi in group B2 and significantly decreased
at 5–7 dpi in groups B1 and B2, in comparison with the
control group B0 (Figure 2).
Viral load
At 4 dpi the viral load in the liver of birds from both
groups B1 and B2 was significantly higher in comparison
to group L2 and non-significantly higher than in group
L1 (Figure 3A). At 7 dpi, the viral load peaked in the liver
of birds from groups L1 and L2 and the viral genome copies per reaction in these groups were significantly higher
in comparison to groups B1 and B2 at 5–7 dpi.
In the pancreas, high loads of viral DNA were determined in both groups B1 and B2 at 4 and 5–7 dpi, being
significantly higher compared with groups L1 and L2
(Figure 3B). Furthermore, the viral load in pancreas of
Discussion
In a recent study we used five FAdV field strains belonging to species FAdV-D and E to orally inoculate separate
groups of day-old SPF white leghorn chickens and a panel
of biomarkers based on clinical chemistry was established, which correlated with the pathogenicity of FAdV
strains and the pathogenesis of IBH [9]. It was shown that
pathogenic FAdV strains are capable to interfere with
enzyme systems and metabolites concentration which are
related to liver and pancreas functions.
In continuity with the aforementioned investigations,
the aim of the present study was to compare and assess
the influence of the genetic background of the host on
the outcome of a FAdV infection considering the different metabolism and growth rate between meat-producing (SPF broilers) and egg-producing (SPF layers)
chickens. For this, two previously tested FAdV-D and E
field strains were chosen based on their pathogenicity in
SPF layers [9]. Independent of the virus used for infection in the present investigation, significant mortalities
approaching 100% together with severe clinical signs
were recorded in the inoculated SPF broilers, differing
substantially from the infection outcome in SPF layers.
However, the recorded mortality in SPF layers was considerably lower when compared with the results which
we previously reported, following the same inoculation
procedure [9]. In the previous study, the occurrence of
non-specific mortality in the first week of life together
with a lower body weight of the control birds throughout the animal experiment, in comparison to the control
SPF layers of the present study (data not shown), indicates a diminished quality and performance of the chicks,
which may have influenced the outcome of the infection.
Nonetheless, further investigations are needed to test this
hypothesis.
Previous pathogenicity studies were mostly performed in layer-type chickens and mortalities of 30–50%
were reported when birds were inoculated by natural
routes with a similar dose of a virulent IBH strain [5,
9, 13–15], hence considerably lower than the mortalities recorded in the inoculated SPF broilers’ groups of
the current study. Genetically different chickens were
used earlier to investigate IBH in experimental studies
[16–20]. However, in nearly all of these studies broilers
of commercial origin were used, whose serological status
regarding maternal antibodies against FAdVs was either
positive or unknown [16, 18–20]. Exceptional to this,
Cook [17] used different chicken breeds of SPF and nonSPF origin to study the influence of host, age and route
Matos et al. Vet Res (2016) 47:69
Page 7 of 10
Figure 3 Viral load in liver and pancreas. Means and standard deviations of the viral genome copies per reaction (log10) in (A) liver and (B)
pancreas of orally inoculated SPF layer and SPF broiler chickens from groups L1, B1, L2 and B2 at 4, 7 and 10 dpi quantified by real-time PCR. Values
of groups B1 and B2 at 7 dpi correspond to pooled birds that were euthanized and sampled between 5 and 7 dpi due to poor condition. All dead
and killed birds at each investigated time point from each group (Table 1) were included. Negative results are indicated with §. There were no SPF
broilers alive at 10 dpi (¥). Mean values with different lowercase letters at each time point are significantly different (P < 0.05).
of inoculation on the outcome of a FAdV infection. As a
result, SPF Light Sussex chickens were highly susceptible
to the infection in comparison to SPF Rhode Island Red
chickens, with mortalities of 67 and 33% being recorded,
respectively, following intraperitoneal inoculation at dayold. Nonetheless, no broiler chickens were used in the
investigation. Different to this, Alvarado et al. [20] used
broilers and layers of SPF origin together with broilers
from vaccinated breeders to investigate the pathogenicity
of a FAdV-E strain, and different mortalities in SPF broilers (40%) and SPF layers (20%) were reported, following
subcutaneous infection at week-old. However, the study
did not focus to elucidate the mechanisms of these differences in susceptibility, which would have been hampered
Matos et al. Vet Res (2016) 47:69
by the fact that subcutaneous infection route was used.
Thus, this is the first study to report a significant susceptibility of SPF broiler chickens following an oral infection
with FAdV strains belonging to species FAdV-D and E, in
direct comparison to SPF layers, and to unravel the background of this phenomenon.
Therefore, in the actual study, further investigations were
carried out to elucidate the pathogenesis of IBH in general
and the influence of the birds’ metabolism. Liver and pancreas are important target organs for FAdVs [9] and, therefore, the blood glucose concentration was determined in
all birds throughout the study, as the glucose metabolism
is a good indicator for liver and pancreas functions [21]. A
recent investigation demonstrated that chickens from lines
bred for high juvenile body weight have an impaired glucose homeostasis and a different pancreas physiology in
comparison to chickens with a low juvenile body weight
[22]. In the current investigation significantly lower blood
glucose concentrations were recorded in both inoculated
SPF broilers’ groups during the peak of infection, in comparison to the control group, corresponding to a hypoglycaemic status according to previously established reference
intervals [21, 23]. It seems that IBH, to which broilers are
highly susceptible, evolves to a metabolic disorder. Furthermore, significant changes in clinical chemistry analytes measured in the plasma of infected birds confirmed
tissue damage and functional impairment of both liver
and pancreas, which were further validated by histopathological studies. In a field investigation, Goodwin et al. [24]
reported the presence of adenoviral inclusion bodies in
liver, pancreas and small intestine of hypoglycaemic broiler
chicks from a flock suffering from spiking mortality, to
which the outcome of the present experimental study parallels. Glucose together with sodium bicarbonate and calcium were once effective as a supportive treatment during
an IBH outbreak, in which broiler chickens were suffering
from hypoglycaemia, metabolic acidosis and hypocalcaemia [25], highlighting the potential of FAdV infections to
unfold in a metabolic impairment. It can be hypothesized
that the blood glucose of the infected birds would decrease
due to apathy and anorexia together with malabsorption
related to the ongoing pancreatitis caused by the infection.
However, during short-term fasting periods, the blood
glucose concentration in granivorous birds is maintained
by hepatic glycogenolysis, in which glucagon—a hormone
produced by pancreatic alpha-cells—plays an important
role as a regulator [21]. Therefore, severe lesions in liver
and/or pancreas can contribute towards disturbances in
the glucose homeostasis. Recently, it was demonstrated
that birds with extensive lesions in pancreas due to an
experimental infection with low-pathogenic avian influenza viruses (LPAIVs) experienced hyperglycaemia and
not hypoglycaemia as observed in our study [26, 27]. In
Page 8 of 10
comparison, FAdV induced IBH severely affects not only
the pancreas but also the liver of birds, preventing compensation mechanisms to occur. Nevertheless, further
studies would be of interest to fully address this issue.
In addition to hepatic and pancreatic changes, lesions
in kidneys have been described throughout the years
as a consequence of experimental FAdV infections [9,
15, 28–30]. Kidneys are mostly responsible for removing uric acid from the blood and, therefore, concentrations greater than 13 mg/dL are suggestive for impaired
renal function [21]. This was never the case in the present study and, in fact, plasmatic uric acid concentration was significantly lower only in the infected SPF
layers’ groups at 4 dpi when compared with the control
group. This can rather be interpreted as a consequence
of liver instead of kidney damage, since the liver is highly
responsible for uric acid production [21].Therefore, the
findings of the present study do not provide evidence
of kidney function impairment due to FAdV infection,
confirming our previously published data [9]. In agreement with this, no lesions were observed in the kidneys
during the histological investigation, despite of swollen
kidneys noticed during necropsy in two birds. Although
glomerulonephritis has been suggested as the underlying cause for the macroscopical changes in kidneys of
broilers suffering from IBH in the field [31], a direct connection between FAdVs and glomerulonephritis has not
been demonstrated, so far, in experimentally infected
birds. Assessing the glomerular size and cellularity in
kidneys of experimentally FAdV infected birds by histomorphometric studies would be of interest to test this
hypothesis.
In our previous studies we were aiming to establish a
link between viral load in target organs with histopathological and macroscopical lesions, clinical chemistry and
clinical signs, in chickens experimentally infected with
FAdVs, although different diseases were investigated and
not all parameters were investigated at the same time [9,
32]. In the present study, very high loads of viral DNA
were seen already at 4 dpi in both liver and pancreas of
infected SPF broilers, preceding the onset of severe clinical signs and high mortality together with severe changes
in clinical chemistry and histopathological lesions. Thus,
these findings provide evidence that FAdVs replicate
faster in both liver and pancreas of broilers in comparison to layers, harmonizing with the outcome of the infection. Furthermore, independent of the virus, much higher
viral loads were determined in the pancreas of broilers in
comparison to livers throughout the experiment, something not observed in layers. This highlights the importance of the pancreas as a target organ for FAdVs with
its crucial function in context of the noticed metabolic
derangement.
Matos et al. Vet Res (2016) 47:69
In conclusion, this is the first study to report a significant difference in susceptibility of SPF broiler chickens to
an oral infection with FAdV strains belonging to species
FAdV-D and E in direct comparison to SPF layers, underlining the importance of the genetic background of the host
on the outcome of a FAdV infection. Furthermore, during
the peak of infection SPF broilers suffered from hypoglycaemia, in which the severe lesions in liver and pancreas
seemed to play an important role. Therefore, we propose
that FAdV infections, to which broiler chickens are very
susceptible, can lead to metabolic disorders. Nevertheless,
further studies are needed to better understand the pathogenesis of IBH in broilers and the involvement of the liver
and pancreas in metabolic derangements by FAdVs.
Additional files
Additional file 1. Kits and methods used to investigate the clinical
chemistry analytes in the plasma of birds. Total protein, albumin,
aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH),
bile acids, uric acid and lipase were investigated in the plasma of birds at
different time points by a fully selective clinical chemistry analyzer (Cobas
501c®, Roche Diagnostics, Vienna, Austria).
Additional file 2. Histopathological findings in infected birds
at different time points. Histopathological lesions recorded in liver,
pancreas and bursa of Fabricius of infected birds from groups L1, B1, L2
and B2 at 4, 7 and 10 dpi. No histopathological changes were observed in
the kidney. Furthermore, no microscopical lesions were present in organs
from birds of the control groups.
Abbreviations
AGE: adenoviral gizzard erosion; AST: aspartate aminotransferase; CEL: chicken
embryo liver; CT: threshold cycle; FAdV: fowl aviadenovirus; GLDH: glutamate
dehydrogenase; HHS: hydropericardium hepatitis syndrome; IBH: inclusion
body hepatitis; NTC: negative template control; PCR: polymerase chain reaction; SPF: specific-pathogen-free; TCID50: median tissue culture infective dose.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MH, BG and MM participated in the design of the study. MM and BG performed the animal experiment. MM and DL carried out the histopathological
analysis. MM performed the viral load investigations by real-time PCR, statistical analysis and drafted the manuscript. All authors read and approved the
final manuscript.
Acknowledgements
The authors would like to thank Prof. Dr. Ilse Schwendenwein from the Clinical
Pathology Platform, Department of Pathobiology, University of Veterinary
Medicine Vienna, for the clinical chemistry analysis.
Author details
1
Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz
1, 1210 Vienna, Austria. 2 Christian Doppler Laboratory for Innovative Poultry
Vaccines (IPOV), University of Veterinary Medicine Vienna, Veterinaerplatz 1,
1210 Vienna, Austria.
Received: 15 March 2016 Accepted: 2 June 2016
Page 9 of 10
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