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Pathogenicity of fowl adenovirus serotype 4 (FAdV-4) in chickens
Journal Pre-proof Pathogenicity of fowl adenovirus serotype 4 (FAdV-4) in chickens
Jie Sun, Yuanyuan Zhang, Shuo Gao, Jing Yang, Yi Tang, Youxiang Diao PII:
S1567-1348(19)30244-8
DOI:
https://doi.org/10.1016/j.meegid.2019.104017
Reference:
MEEGID 104017
To appear in:
Infection, Genetics and Evolution
Received date:
5 May 2019
Revised date:
19 August 2019
Accepted date:
22 August 2019
Please cite this article as: J. Sun, Y. Zhang, S. Gao, et al., Pathogenicity of fowl adenovirus serotype 4 (FAdV-4) in chickens, Infection, Genetics and Evolution(2019), https://doi.org/ 10.1016/j.meegid.2019.104017
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© 2019 Published by Elsevier.
Journal Pre-proof
Pathogenicity of Fowl Adenovirus Serotype 4 (FAdV-4) in
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Chickens
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Jie Sun1,2,3 , Yuanyuan Zhang1,2,3 , Shuo Gao1,2,3 , Jing Yang1,2,3 , Yi Tang1,2,3* , Youxiang
4
Diao1,2,3*
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1.College of Animal Science and Technology, Shandong Agricultural University,
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Tai’an, Shandong Province, China
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2.Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control
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and Prevention, Tai’an, Shandong, China
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3.Shandong Provincial Engineering Technology Research Center of Animal Disease
f
1
Control and Prevention, Tai’an, Shandong, China
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*Correspondence: Yi Tang and Youxiang Diao, College of Animal Science and
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Technology, Shandong Agricultural University, Tai’an, Shandong Province 271018,
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China.
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Emails: [email protected]; [email protected]
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Highlights
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province has strong pathogenicity to SPF chickens.
19 20 21
The SDSG strain isolated from the 10-day-old broiler flocks in Shandong
The pathogenicity of the SDSG strain to 10-day-old SPF chickens is stronger than that of 20-day-old chickens.
FAdV-4 isolate SDSG can replicate in many tissues of the infected chickens, with the highest viral load in livers.
Abstract
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Hepatitis-hydropericardium syndrome (HHS) is an acute infectious disease caused by
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fowl adenovirus serotype 4 (FAdV-4), which mainly infects broilers aged 3–5 weeks.
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In March 2018, a pathogenic disease, which was characterized by symptoms similar
26
to HHS, broke out in 10-day-old broiler flocks in Shandong province. In this study, a
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strain of FAdV-4 (SDSG) was isolated from naturally infected broilers. To assess its
28
pathogenicity, 10-day-old and 20-day-old specific pathogen- free (SPF) chickens were
29
inoculated separately with the FAdV-4 virus fluid via oral and intramuscular injection
30
routes.
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The results show that typical hydropericardium and hepatitis were observed in
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experimental chickens. The titer levels of the virus antibody and the levels of
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inflammatory cytokines were upregulated, which may be caused by the infection and
34
innate immune response. The detection of viral load showed that the presence of virus
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was detected in multiple organs, in which the liver contained the highest
36
concentration of viral DNA, and the virus content in the intramuscular injection group
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was higher than that of the oral injection group.
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In summary, these findings increase our understanding of the pathogenicity of FAdV-4
39
(SDSG) in chickens. The established model will be valuable for anti- viral drug testing
40
and vaccine evaluation, which can prevent and reduce the spread of HHS in the
41
poultry industry.
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Keywords: Hepatitis-hydropericardium syndrome; fowl adenovirus serotype 4; SPF
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chicken; day-old; experimental infection; pathogenicity
44
Abbreviations
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HHS, Hepatitis- hydropericardium syndrome; FAdV-4, fowl adenovirus serotype 4;
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SPF, specific pathogen- free; IBH, inclusion body hepatitis; CAM, chorioallantoic
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membrane; EID 50, median embryo infectious dose; dpi, day post- injection; HE,
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hematoxylin and eosin; qPCR, quantitative real time polymerase chain reaction; ALT,
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alanine
50
dehydrogenase; ELISA, enzyme- linked immunosorbent assay; OD, optical density; T
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cells, thymus-dependent lymphocytes; NK cells, natural killer cells.
aminotransferase;
AST,
aspartate
aminotransferase;
LDH,
lactate
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1. Introduction
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Fowl adenoviruses (FAdVs), belonging to the Aviadenovirus genus of the family
55
Adenoviridae, are non-enveloped double stranded DNA-viruses, which can be
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grouped into 5 species (FAdV-A to FAdV-E) with 12 serotypes (FAdV-1 to 12) (Hess,
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2000; McFerran and Smyth, 2000). FAdV-4 is classified as a species of FAdV-C
58
together with FAdV-10 based on the Ninth Report of the International Committee on
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Taxonomy of Viruses (Harrach and Kaján, 2011). FAdVs can cause acute avian
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infectious diseases, such as hydropericardium hepatitis syndrome (HHS), inclusion
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body hepatitis (IBH), and gizzard erosion in broilers and layers (Asthana et al., 2013;
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Domanska-Blicharz et al., 2011; Mase et al., 2010). FAdV-4 has been isolated from
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chickens with HHS, with each case showing similar clinical signs, including
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accumulation of slightly yellow transparent fluid in the pericardial sac, characteristic
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intranuclear inclusion bodies in hepatocytes and a high mortality of 20–80% (Ganesh
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et al., 2001; Toro et al., 2000; Vera-Hernandez et al., 2016). HHS was first reported in
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Angara Goth, Pakistan in 1987 and subsequently broke out in North America, Mexico,
68
Eastern Europe, India, South Asia, China, Japan, and South Korea, causing economic
69
losses mainly to the broiler industry worldwide (Choi et al., 2012; Mase et al., 2010;
70
Mittal et al., 2014; Schachner et al., 2018).
71
Since mid-2015, sporadic outbreaks of HHS have been increasingly observed in
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commercial chicken farms in China, which causes high mortality, resulting in
73
tremendous economic losses to poultry farmers. Therefore, strengthening the study of
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the disease is of great significance for protecting the development of the poultry
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industry in China. To determine different infection routes of FAdV-4 and the
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susceptibility of smaller-age-chickens, this study investigated the pathogenicity of 10
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and 20-day-old SPF chickens to provide a theoretical basis for preventing and
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controlling this disease.
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2. Materials and Methods
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2.1 Ethics Statement
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All the animal infection experiments were carried out in accordance with international,
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national, and institutional guidelines. The animal procedure was approved by the
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Committee on the Ethics of Animals of Shandong (permit number: 2017360331). The
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chickens used in the experiment at Shandong Agricultural University were euthanized
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using rapid cervical dislocation.
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2.2 Animals
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All SPF White Leghorn line chickens were purchased from Poultry Science and
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Technology Co., Ltd., Jinan Spirax Ferrer. The chickens were maintained in specific
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pathogen-free (SPF) chicken isolators with ad libitum feeding. Serum and swab
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samples of experimental chickens were collected before inoculation, using serum
91
neutralization and polymerase chain reaction (PCR) tests to confirm that all chickens
92
used in this study were serologically negative for FAdV-4.
93
2.3 Samples Collection
94
In March 2018, a pathogenic disease, which was characterized by symptoms similar
95
to HHS, broke out in broiler flocks in several farms (located in Shenxian, Gaotang
96
and Juxian) in Shandong province. The age of the diseased broilers was primarily
97
around 10 days, and it is known that broilers are not vaccinated with FAdV. We
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collected 87 liver samples from the sick and dead chickens for FAdV detection. The
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collected samples were kept at -4°C until being detected.
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2.4 DNA Sequencing
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DNA was extracted by phenol-chloroform method from liver samples. To detect the
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virus,
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GCCACTACCAACTTCTACTTCG
104
AGAGGAACTTCTTGTAGCTGAGG -3ʹ. The entire hexon open reading frame of
105
isolate was amplified, sequenced, and analyzed to identify the serotypes according to
106
previous study (Tang et al., 2009). PCR was carried out in a total volume of 20 μl
107
containing 1 μl (10 pmol) of each primer, 10 μl of Taq HS Perfect Mix (TaKaRa,
108
Beijing, China), 2 μl of DNA and 6 μl of nuclease-free water. Reactions were
109
performed according to the following protocol: 94°C for 3 min, followed by 32 cycles
PCR
was
performed
using
the -3ʹ;
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primer
pair
forward:
reverse:
5ʹ5ʹ-
Journal Pre-proof of 94°C for 30 s, 53°C for 30 s, 72°C for 1 min 30 s, and a final elongation step of
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3 min at 72 °C. All PCR products were cloned into pMD18 ‐ T vector and
112
transformed into DH5α E. coli-competent cells, and the positive clone was sequenced
113
(BGI Company Ltd., Beijing, China).
114
Phylogenetic analysis based on the nucleotide sequence s of hexon genes of the
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positive samples and other FAdV was constructed using MEGA7 software with
116
neighbor-joining method.
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2.5 Virus Isolation
118
Samples that were positive by PCR detection were used for virus isolation. In brief,
119
the homogenates were centrifuged at 8,000 × g for 10 min, and the supernatants were
120
filtered through 0.22-um filters. The isolates were passaged 5 times in SPF chicken
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embryos via the chorioallantoic membrane (CAM) route. The isolated strain was
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designated as SDSG and the median embryo infectious dose (EID 50) was determined
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to be 10-7.56 /0.2 mL by the Reed and Muench method (REED and MUENCH, 1938).
124
Allantoic fluids were harvested as the challenge virus in the present study and stored
125
at -80°C.
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2.6 Experimental Design
127
Animal regression experiment was performed with SDSG strain virus. Ninety
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10-day-old SPF chickens were randomly divided into 3 groups: oral injection group
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(30), muscle injection group (30), and control group (30). The chickens in the
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experimental group were inoculated with 1 mL of FAdV-4 (SDSG strain; EID 50,
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10−7.56/0.2 ml) either orally or by intramuscular route. The control group was
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inoculated with equal doses of physiological saline at the same injection site. The 3
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groups were separately reared in different SPF chicken isolators. Water and food were
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autoclaved before feeding and automatically refilled. The temperature of each house
135
was maintained using radiators at 20–24°C. The chicken feces was manually cleaned
136
every day. The same inoculation dose and test method were adopted in 20-day-old
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chickens. The chickens were observed daily for clinical signs.
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2.7 Body Weight and Histopathology
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Five chicks from the infected and control groups were respectively and randomly
140
drawn for weight detection and necropsy in each of 4th, 8th, 12th, 16th , and 20th days
141
post-injection (dpi) (chicks were euthanized). Chicks were examined for the presence
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Journal Pre-proof of gross lesions of the heart, liver, spleen, lung, kidney, thymus, pancreas, and bursa
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of Fabricius. Each organ was sectioned into 3 portions: one was placed into 10%
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formalin, routinely processed, and embedded in paraffin- wax, sections were cut
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approximately 4-μm-thick and stained with hematoxylin and eosin (HE), and all
146
stained sections were examined by light microscopy; the second was stored at −80°C
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for virology; and the third was stored at −80°C for quantitative real time PCR.
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2.8 Viral DNA Extraction
149
The viral DNA of tissue samples was extracted from the previously stored organs
150
using the Universal Genomic DNA Kit (Beijing ComWin Biotech Co., Ltd.)
151
according to the manufacturer’s instructions. The concentration of high-quality DNA
152
samples (i.e., A260/280 was between 1.8–2.2, A26/230 ≧2.0) was normalized to 50
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ng/ul, and used in subsequent experiments.
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2.9 QPCR Analysis
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To determine the viral load in tissues, real-time PCR was performed using the method
156
previously established in our laboratory for the detection of FAdV-4. The primers
157
used in this study are as follows: forward: 5ʹ -CGTCAACTTCAAGTACTC-3ʹ and
158
reverse: 5ʹ -AGAGGATGCTCATGTTAC-3ʹ. The relative TaqMan probe was a 24 bp
159
oligonucleotide,5′ FAM-CCTACTCAGATGGAGGCTTCTACC-3′ TAMRA, which
160
was labeled with FAM at the 5 end and TAMRA at the 3 end (Yu et al., 2018).
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QRT-PCR analysis was performed with Premix Ex Taq™ (Probe qPCR) (TaKaRa,
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China) following the manufacturer’s instructions, which contained 10 μL of Premix
163
Ex Taq, 0.4 μL of forward primer, 0.4 μL of reverse primer, 0.2 μL of ROX Reference
164
Dye II, 6.2 μL of ddH2O, 0.8 μL of probe, and 2.0 μL of DNA template.
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Amplification and detection were performed with an Applied Biosystems® 7500
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FAST Real- Time PCR System under the following conditions: 95°C for 30 s,
167
followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 62°C for 32 s.
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The fluorescent signals were measured during the annealing step. Each DNA sample
169
was processed with 3 replicates to guarantee the reproducibility of the amplification.
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2.10 Determination of Biochemical Indexes, Cytokines, and
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Antibodies
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On the 4th, 8th, 12th, 16th, and 20th days after the injection, 5 chickens were selected
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randomly, bled, and euthanatized. Serum samples were harvested for detecting and
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recording the changes of blood biochemical indexes, cytokines, and antibodies.
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Biochemical
176
aminotransferase (AST), and lactate dehydrogenase (LDH), were detected through the
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testing organization ADICON CLINICAL LABORATORIES, INC (Jinan, China).
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Cytokines were detected using the double-antibody sandwich enzyme-linked
179
immunosorbent assay (ELISA) kit of chicken interleukin 6 (IL-6) and interferon-γ
180
(IFN-γ) (Lengton, Shanghai, China) according to the manufacturer’s directions.
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The FAdV-4-specific antibodies were determined by the indirect ELISA which
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developed in previous work in our laboratory (Niu et al., 2017). In brief, 47 μg/mL of
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recombinant hexon protein in carbonate buffer solution was incubated in black
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Maxisorb 96-well plates (Greiner Bio-One, Germany) overnight at 4°C, and the plates
185
were blocked by 5% non- fat dry milk (Solarbio, Beijing, China, w/v) for 2 h at 37°C.
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Then, serum (1:10) was added at 200 μL/well and incubated at 37°C for 1 h.
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Afterward, the plates were incubated with rabbit anti-chicken immunoglobulin G (IgG,
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1: 800) (Solarbio,
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(Sigma-Aldrich) at 100 μL/well. The plates were incubated at 37°C for 1 h, and 3′, 3′,
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5′, 5′-tetramethylbenzidine substrate solution (TransGen, Beijing, China) was added
191
to each well (100 μL/well). Fifty microliters of 3 M H2SO4 (Wuxi JINGKE Chemical
192
CO., LTD.) was added to stop the reaction, and the optical density (OD) values were
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observed at 450 nm using an automated ELISA plate reader (Thermo, Waltham, MA,
194
USA).
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2.11 Statistical Analysis
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All results are presented at means ± standard deviations and analyzed using the
197
one-way analysis of variance (ANOVA) procedure of GraphPad Prism 6.0 (GraphPad
198
Software Inc., San Diego, CA, USA). P values of <0.05 or <0.01 were used to define
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statistical significance.
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3. Results
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3.1 Clinical Investigation
including
alanine
aminotransferase
(ALT),
aspartate
China) conjugated
to
horseradish peroxidase
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Journal Pre-proof The PCR results showed that 79 of the 87 samples were positive for FAdV.
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Postmortem examination of the 79 chickens showed severe lesions, including
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hydropericardium in the heart, hemorrhaging and enlargement of livers, edema with
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congestion in lungs and kidneys, and swelling and bleeding in the thymus (Fig 1
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(A)-(D)). The remaining 8 had no obvious lesions.
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3.2 Sequence Analysis of Hexon Gene
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The isolate was designated as SDSG (GenBank accession: MK424834). Multiple
209
alignments showed that the hexon genome of the isolate shared 60.7–99.7% of the
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nucleotides and 66.4–99.9% of the deducted amino acid sequences with those of
211
representative strains from different lineages. As shown in Figure 2, the phylogenetic
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tree for hexon genes was constructed based on the sequences obtained from the
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GenBank database, and it revealed that the isolate SDSG was clustered together with
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chicken FAdV-4 isolates (PK-01 and KR5) (Steer et al., 2009).
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Notably, the SDSG strain shared 99.7% of the nucleotide and amino acid sequences
216
with the India- isolated strain (EU931693, PK-01), which indicated that this strain
217
might have originated from early India isolates.
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3.3 Clinical Symptoms
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After infection with FAdV-4, the injection group of 10-day-old and 20-day-old SPF
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chickens showed signs of depression, lack of appetite, and stunted growth. The
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chickens of the control group did not demonstrate any clinical symptoms throughout
222
the experiment. The mortality rate of the 10-day-old muscle injection group reached
223
50% (Table 1). Chickens in the injection group grew slowly, and the body weights
224
were lower than those of the control group (Fig 3).
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Table 1. Grouping and death status
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Grouping
10-day-old group
20-day-old group
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Experiment
Quantity
dead number
Mortality
Oral injection
30
7
23.333%
Intramuscular injection
30
15
50%
Control
30
0
0
Oral injection
30
5
16.667%
Intramuscular injection
30
5
16.667%
Control
30
0
0
3.4 Pathologic Change
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Journal Pre-proof Dissecting the chickens showed that hearts have a severe pericardial effusion; livers
228
are swollen with bleeding spots. In addition to the above typical clinical symptoms,
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the infected chickens also presented significant lesions characterized by swelling with
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hemorrhage in spleens, edema with varying degrees of congestion in lungs and
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kidneys, and swelling with different extents of bleeding in the thymus. The chickens
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of the control group had no obvious necrotic lesions (Figs 4 and 5).
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3.5 Histopathologic Analysis
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Histopathological analysis showed that chickens from both experimental groups had
235
obvious histopathological changes (Fig 6). The heart showed interstitial edema
236
accompanied with lymphocytic infiltration. Also, the 10-day-old muscle injection
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group (Fig 6(B)) exhibited myocardial fiber granular degeneration, necrosis, and
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hemorrhage. The livers exhibited hepatocyte steatosis, necrosis, and hemorrhage, and
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the 10-day-old muscle injection group (Fig 6(D)) also exhibited hepatocellular nuclear
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concentration and dissolution necrosis. Lesions in the spleens of 10-day-old injection
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group (Fig 6(E), (F)) were lymphocyte of white pulp decreased and necrosis, but in
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the 20-day-old injection group (Fig 6(e), (f)) were hemorrhage in the white pulp and
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increase in the number of lymphocytes; In the lungs, there were exudates in the
244
bronchi, as well as capillary congestion. Lesions in the kidneys were characterized by
245
renal tubular epithelial cell degeneration and renal interstitial hemorrhage. In the
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thymus, a large amount of necrosis of lymphocytes in the medulla was observed.
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lesions in bursas were characterized by reduction and necrosis of lymphocytes; in
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particular, the muscle injection group was more serious than the oral injection group.
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3.6 Real-time PCR Analysis
250
After injection, the presence of virus was detected in the heart, liver, spleen, lung,
251
kidney, thymus, pancreas and bursae of both the oral and intramuscular groups (Fig 7).
252
Notably, more FAdV-4 copies were found in the liver than in other tissues. Moreover,
253
the overall number of virus copies in the tested tissues was higher in the intramuscular
254
group than in the oral group.
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3.7 Detection of Biochemical Indices
256
The results of serum biochemical indices is presented in Figure 8. In the 10-day-old
257
group, the AST, ALT, and LDH of experimental groups were higher than those of the
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control group. Briefly, ALT reached the peak at 8 dpi (p < 0.01), AST at 4 dpi (p <
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Journal Pre-proof 0.01), and LDH at 8 dpi (p < 0.01). After reaching peak levels, the above biochemical
260
indices declined with time. The results of serum biochemical indicators in the
261
20-day-old group were similar to those in the 10-day-old group, except the LDH
262
reached the peak at 4 dpi (p < 0.01).
263
3.8 Detection of Cytokines in Serum
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As Figure 9 shows, in the 10-day-old group, the levels of IL-6 in the serum reached
265
the peak at 16 dpi and then tended to decline. The level of the IFN-γ in the
266
intramuscular group was higher than that in the oral group, which showed a slowly
267
upward trend, but decreased rapidly after reaching the peak, and was finally lower
268
than the oral group. In the 20-day-old group, IL-6 levels rose slowly and peaked at 16
269
dpi. The IFN-γ levels were significantly higher than in the control group (P < 0.01) at
270
8 and 16 dpi, but the IFN-γ level in the intramuscular injection group was
271
downregulated rapidly after 16 dpi.
272
3.9 Detection of Antibodies in Serum
273
FAdV-4 antibody levels in the serum were detected throughout the experiment. As
274
Figure 10 shows, the positive serum could be detected earlier in the intramuscular
275
injection group at 4 dpi, compared with the oral group. The antibody level in the
276
experimental group continued to increase throughout the experiment; however, the
277
antibody level in the 10-day-old intramuscular injection group showed a downward
278
trend at 8 dpi and 16 dpi.
279
4. Discussion
280
HHS caused by FAdV-4 is a highly pathogenic communicable disease characterized
281
by pericardial effusion and IBH (Mase et al., 2010; Schachner et al., 2018). HHS
282
mainly infects broilers aged 3–5 weeks with a high mortality of 20–80%. Currently,
283
this disease can also occur in hybrid chickens, breeders, and layers (Ye et al., 2016),
284
resulting in considerable economic losses in China.
285
At present, a few studies have been reported on the pathogenicity of FAdV-4 in
286
chickens such as one on oral infection of 3-week-old SPF chickens (Zhao et al., 2016),
287
and another on both oral and intramuscular infection of 40-day-old chickens (Li et al.,
288
2016). However, these studies were all conducted on chickens older than 3 weeks of
289
age. There is no article about the pathogenicity of smaller-day-old SPF chickens via
290
different injection routes. Some studies included a small portion of data on the
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Journal Pre-proof pathogenicity of FAdV-4 to chickens (Li et al., 2018a; Li et al., 2018b; Ren et al.,
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2019), but they did not systematically and comprehensively test cytokines,
293
biochemical indices, and antibody levels in infected chickens. Existing articles lack
294
this part of the data. The pathogenesis of the virus in chickens is not comprehensively
295
understood, and it is necessary to better understand the pathogenic mechanisms of
296
FAdV-4 to effectively control a viral epidemic.
297
Because the pathogenicity of FAdV-4 isolated from small-day-old chickens and the
298
pathogenic difference of this strain to small-day-old and large-day-old chickens
299
remains unclear, this study established animal regression experiments. Ten-day-old
300
and 20-day-old SPF chickens were injected separately with the same doses of the
301
FAdV-4 serum via oral and intramuscular injection routes.
302
From the gross lesions, body weight, mortality percent, and viral load we observed
303
that the pathogenicity of FAdV-4 in SPF chickens is related to the age and the route of
304
injection. Ten-day-old chickens are more susceptible than 20-day-old chickens, and
305
the muscular injection group was more serious than the oral injection group.
306
Most chickens in the 2 experimental groups showed obvious clinical symptoms,
307
including hydropericardium and hepatitis manifestation, bleeding ,and swelling of the
308
spleen, lungs, and thymus. The inoculated chickens in the 10-day-old and 20-day-old
309
groups started to die on the 4th and 7th days after injection, respectively. Mortality
310
rates among the 10-day-old chickens inoculated intramuscularly were 50%.
311
Histopathological analysis also
312
including the interstitial edema around cardiocytes, visible inflammatory cell
313
infiltration in the liver and spleen, and lymphocyte necrosis in the thymus and bursae.
314
The results demonstrate that FAdV-4 can invade multiple organs and lead to the
315
pathogenesis of chickens. The clinical manifestations and pathological changes after
316
infection were highly consistent with those of other scholars, such as Abdul-Aziz
317
(Abdul-Aziz and Hasan, 1995).
318
Real-time PCR results show that the virus is widely distributed in the body, and virus
319
DNA can be detected in various organs, such as the heart, liver, spleen, lung, kidney,
320
thymus, and bursae, which is consistent with the necropsy and histopathological
321
changes of the infected chickens. The quantity of virus in the liver was the highest, so
322
the liver is the primary choice for clinical detection of adenovirus. Notably, the
323
quantity of virus in the intramuscular injection group was higher than that in the oral
indicated obvious histopathological changes,
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Journal Pre-proof injection group, indicating that the pathogenicity of the body was stronger by
325
intramuscular injection of the virus. In the natural infection model, there is no
326
intramuscular injection route. However, the established 10-day-old muscle infection
327
model is more infectious and pathogenic to the body, so this model can be valuable
328
for anti-viral drug testing and vaccine evaluation.
329
AST mainly exists in cardiomyocytes, but also in mitochondria of hepatocytes (Fan et
330
al., 2014). If hepatic cells or myocardial cells are destroyed, the enzymes will leak
331
into the blood and levels of it will increase rapidly. As the cells are repaired, its
332
content gradually decreases. ALT is mainly distributed in the body's hepatic
333
cytoplasm. The activity of this enzyme is approximately 100 times higher in the liver
334
than in serum. Therefore, 1% hepatocyte necrosis results in a 1- fold increase in serum
335
ALT (Kew, 2000; Rosenthal, 1997). LDH exists in the cytoplasm of all histiocytes in
336
the body, and its content is highest in the kidney. Therefore, LDH levels can reflect
337
tissue damage, especially kidney damage. The detection results of this study showed
338
that almost all blood biochemical indices of experimental groups were higher than
339
those of the control group and then decreased with time. As the cells self- repair, the
340
levels of enzymes in the plasma are reduced, which may be due to the damage of the
341
heart, liver, and kidney after injection. These were highly consistent with Asrani’s
342
report (Asrani et al., 1997).
343
histopathological damage.
344
IL-6 was produced by a variety of lymphocytes and non- lymphocytes, which can
345
promote the proliferation of B cells, enhance their ability to produce antibodies, and
346
promote humoral immunity. The level of secretion can reflect the body's humoral
347
immune function (Thacker et al., 2009). IFN-γ is produced by thymus-dependent
348
lymphocytes (T cells) and natural killer cells (NK cells). It is a pleiotropic factor for
349
immune regulation that can participate in the body's immune regulation and improve
350
antigen presentation ability. In this study, the levels of IL-6 in the 2 injection groups
351
first rose and then decreased. This shows that the body is in a normal state of
352
immunity at the initial stage after injecting the virus, but at the late stage,
353
immunosuppression appears (Schonewille et al., 2008). The IFN-γ in the
354
intramuscular group was significantly higher than that in the control group and was
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maintained in the first 16 dpi, which reflected that many of these cytokines with high
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concentrations were produced by T cells and NK cells under the stimulation of
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the immune organs of the infected chickens were severely damaged, resulting in a
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decrease in IFN-γ levels. After the injection, the antibody levels also showed an
360
increasing trend, indicating that humoral immunity plays an important role in host
361
antiviral properties.
362
In conclusion, this experiment determined the accurate value of viral load, cytokines,
363
biochemical indicators and antibodies of FAdV-4 (SDSG)- induced chickens at 10 and
364
20 days old. Filling in the gaps in this aspect of data may provide references for future
365
research.
366
Acknowledgments
367
This work was supported by Tai’shan Industry Leader (LJNY201610), the National
368
Key Research and Development Program of China (2018YFD0501506), the China
369
Agriculture Research System (CARS-42-19), and Funds of Shandong "Double Top"
370
Program.
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Conflict of Interest Statement
372
The authors declare that the research was conducted in the absence of any commercial
373
or financial relationship that could be construed as a potential conflict of interest.
374
Fig 1: Clinical investigation results. (A) Severe hydropericardium and enlarged
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livers with blood spots; (B, C) edema with congestion in lungs and kidney; (D)
376
swelling and bleeding in thymus.
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Fig 2: Phylogenetic tree of hexon gene sequences of the isolate SDSG (marked by
378
a red dot) and other avian strains. The phylogenetic tree was built using
379
neighbor-joining analysis and MEGA 7.0, and the bootstrap confidence values were
380
determined using 1,000-bp replicates.
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Fig 3: Weight changes after injection. (A) 10-day-old group; (B) 20-day-old group.
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Fig 4: Gross lesions of 10-day-old group chickens inoculated with FAdV-4.
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Fig 5: Gross lesions of 20-day-old group chickens inoculated with FAdV-4.
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Fig 6: Histopathologic changes of FAdV-4-infected chickens. (A-H) (K) and (L)
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Magnification, ×400; other magnification, ×200.
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oral injection group; (B) 10-day-old intramuscular injection group; (C) 20-day-old
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oral injection group; (D) 20-day-old intramuscular injection group.
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Fig 8: Dynamics of ALT, AST, and LDH in serum of the chickens post artificially
390
infected with FAdV-4. (A-C) 10-day-old group; (D-F) 20-day-old group.
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Fig 9: Dynamics of IL-6 and IFN-γ in serum of the chickens post artificially
392
infected with FAdV-4. (A-B) 10-day-old group; (C-D) 20-day-old group.
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Fig 10: Dynamics of antibodies against FAdV-4 in serum of the chickens post
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artificially infected with FAdV-4. (A) 10-day-old group; (B) 20-day-old group.
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Jie Sun, Yuanyuan Zhang, Shuo Gao, Jing Yang, Yi Tang, Youxiang Diao PII:
S1567-1348(19)30244-8
DOI:
https://doi.org/10.1016/j.meegid.2019.104017
Reference:
MEEGID 104017
To appear in:
Infection, Genetics and Evolution
Received date:
5 May 2019
Revised date:
19 August 2019
Accepted date:
22 August 2019
Please cite this article as: J. Sun, Y. Zhang, S. Gao, et al., Pathogenicity of fowl adenovirus serotype 4 (FAdV-4) in chickens, Infection, Genetics and Evolution(2019), https://doi.org/ 10.1016/j.meegid.2019.104017
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
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Pathogenicity of Fowl Adenovirus Serotype 4 (FAdV-4) in
2
Chickens
3
Jie Sun1,2,3 , Yuanyuan Zhang1,2,3 , Shuo Gao1,2,3 , Jing Yang1,2,3 , Yi Tang1,2,3* , Youxiang
4
Diao1,2,3*
5
1.College of Animal Science and Technology, Shandong Agricultural University,
6
Tai’an, Shandong Province, China
7
2.Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control
8
and Prevention, Tai’an, Shandong, China
9
3.Shandong Provincial Engineering Technology Research Center of Animal Disease
f
1
Control and Prevention, Tai’an, Shandong, China
11
*Correspondence: Yi Tang and Youxiang Diao, College of Animal Science and
12
Technology, Shandong Agricultural University, Tai’an, Shandong Province 271018,
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China.
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Emails: [email protected]; [email protected]
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Highlights
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17 18
province has strong pathogenicity to SPF chickens.
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The SDSG strain isolated from the 10-day-old broiler flocks in Shandong
The pathogenicity of the SDSG strain to 10-day-old SPF chickens is stronger than that of 20-day-old chickens.
FAdV-4 isolate SDSG can replicate in many tissues of the infected chickens, with the highest viral load in livers.
Abstract
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Hepatitis-hydropericardium syndrome (HHS) is an acute infectious disease caused by
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fowl adenovirus serotype 4 (FAdV-4), which mainly infects broilers aged 3–5 weeks.
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In March 2018, a pathogenic disease, which was characterized by symptoms similar
26
to HHS, broke out in 10-day-old broiler flocks in Shandong province. In this study, a
27
strain of FAdV-4 (SDSG) was isolated from naturally infected broilers. To assess its
28
pathogenicity, 10-day-old and 20-day-old specific pathogen- free (SPF) chickens were
29
inoculated separately with the FAdV-4 virus fluid via oral and intramuscular injection
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routes.
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The results show that typical hydropericardium and hepatitis were observed in
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experimental chickens. The titer levels of the virus antibody and the levels of
33
inflammatory cytokines were upregulated, which may be caused by the infection and
34
innate immune response. The detection of viral load showed that the presence of virus
35
was detected in multiple organs, in which the liver contained the highest
36
concentration of viral DNA, and the virus content in the intramuscular injection group
37
was higher than that of the oral injection group.
38
In summary, these findings increase our understanding of the pathogenicity of FAdV-4
39
(SDSG) in chickens. The established model will be valuable for anti- viral drug testing
40
and vaccine evaluation, which can prevent and reduce the spread of HHS in the
41
poultry industry.
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Keywords: Hepatitis-hydropericardium syndrome; fowl adenovirus serotype 4; SPF
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chicken; day-old; experimental infection; pathogenicity
44
Abbreviations
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HHS, Hepatitis- hydropericardium syndrome; FAdV-4, fowl adenovirus serotype 4;
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SPF, specific pathogen- free; IBH, inclusion body hepatitis; CAM, chorioallantoic
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membrane; EID 50, median embryo infectious dose; dpi, day post- injection; HE,
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hematoxylin and eosin; qPCR, quantitative real time polymerase chain reaction; ALT,
49
alanine
50
dehydrogenase; ELISA, enzyme- linked immunosorbent assay; OD, optical density; T
51
cells, thymus-dependent lymphocytes; NK cells, natural killer cells.
aminotransferase;
AST,
aspartate
aminotransferase;
LDH,
lactate
52
1. Introduction
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Fowl adenoviruses (FAdVs), belonging to the Aviadenovirus genus of the family
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Adenoviridae, are non-enveloped double stranded DNA-viruses, which can be
56
grouped into 5 species (FAdV-A to FAdV-E) with 12 serotypes (FAdV-1 to 12) (Hess,
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2000; McFerran and Smyth, 2000). FAdV-4 is classified as a species of FAdV-C
58
together with FAdV-10 based on the Ninth Report of the International Committee on
59
Taxonomy of Viruses (Harrach and Kaján, 2011). FAdVs can cause acute avian
60
infectious diseases, such as hydropericardium hepatitis syndrome (HHS), inclusion
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body hepatitis (IBH), and gizzard erosion in broilers and layers (Asthana et al., 2013;
62
Domanska-Blicharz et al., 2011; Mase et al., 2010). FAdV-4 has been isolated from
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chickens with HHS, with each case showing similar clinical signs, including
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accumulation of slightly yellow transparent fluid in the pericardial sac, characteristic
65
intranuclear inclusion bodies in hepatocytes and a high mortality of 20–80% (Ganesh
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et al., 2001; Toro et al., 2000; Vera-Hernandez et al., 2016). HHS was first reported in
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Angara Goth, Pakistan in 1987 and subsequently broke out in North America, Mexico,
68
Eastern Europe, India, South Asia, China, Japan, and South Korea, causing economic
69
losses mainly to the broiler industry worldwide (Choi et al., 2012; Mase et al., 2010;
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Mittal et al., 2014; Schachner et al., 2018).
71
Since mid-2015, sporadic outbreaks of HHS have been increasingly observed in
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commercial chicken farms in China, which causes high mortality, resulting in
73
tremendous economic losses to poultry farmers. Therefore, strengthening the study of
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the disease is of great significance for protecting the development of the poultry
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industry in China. To determine different infection routes of FAdV-4 and the
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susceptibility of smaller-age-chickens, this study investigated the pathogenicity of 10
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and 20-day-old SPF chickens to provide a theoretical basis for preventing and
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controlling this disease.
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2. Materials and Methods
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2.1 Ethics Statement
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All the animal infection experiments were carried out in accordance with international,
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national, and institutional guidelines. The animal procedure was approved by the
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Committee on the Ethics of Animals of Shandong (permit number: 2017360331). The
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chickens used in the experiment at Shandong Agricultural University were euthanized
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using rapid cervical dislocation.
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2.2 Animals
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All SPF White Leghorn line chickens were purchased from Poultry Science and
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Technology Co., Ltd., Jinan Spirax Ferrer. The chickens were maintained in specific
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pathogen-free (SPF) chicken isolators with ad libitum feeding. Serum and swab
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samples of experimental chickens were collected before inoculation, using serum
91
neutralization and polymerase chain reaction (PCR) tests to confirm that all chickens
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used in this study were serologically negative for FAdV-4.
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2.3 Samples Collection
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In March 2018, a pathogenic disease, which was characterized by symptoms similar
95
to HHS, broke out in broiler flocks in several farms (located in Shenxian, Gaotang
96
and Juxian) in Shandong province. The age of the diseased broilers was primarily
97
around 10 days, and it is known that broilers are not vaccinated with FAdV. We
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collected 87 liver samples from the sick and dead chickens for FAdV detection. The
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collected samples were kept at -4°C until being detected.
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2.4 DNA Sequencing
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DNA was extracted by phenol-chloroform method from liver samples. To detect the
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virus,
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GCCACTACCAACTTCTACTTCG
104
AGAGGAACTTCTTGTAGCTGAGG -3ʹ. The entire hexon open reading frame of
105
isolate was amplified, sequenced, and analyzed to identify the serotypes according to
106
previous study (Tang et al., 2009). PCR was carried out in a total volume of 20 μl
107
containing 1 μl (10 pmol) of each primer, 10 μl of Taq HS Perfect Mix (TaKaRa,
108
Beijing, China), 2 μl of DNA and 6 μl of nuclease-free water. Reactions were
109
performed according to the following protocol: 94°C for 3 min, followed by 32 cycles
PCR
was
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primer
pair
forward:
reverse:
5ʹ5ʹ-
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111
3 min at 72 °C. All PCR products were cloned into pMD18 ‐ T vector and
112
transformed into DH5α E. coli-competent cells, and the positive clone was sequenced
113
(BGI Company Ltd., Beijing, China).
114
Phylogenetic analysis based on the nucleotide sequence s of hexon genes of the
115
positive samples and other FAdV was constructed using MEGA7 software with
116
neighbor-joining method.
117
2.5 Virus Isolation
118
Samples that were positive by PCR detection were used for virus isolation. In brief,
119
the homogenates were centrifuged at 8,000 × g for 10 min, and the supernatants were
120
filtered through 0.22-um filters. The isolates were passaged 5 times in SPF chicken
121
embryos via the chorioallantoic membrane (CAM) route. The isolated strain was
122
designated as SDSG and the median embryo infectious dose (EID 50) was determined
123
to be 10-7.56 /0.2 mL by the Reed and Muench method (REED and MUENCH, 1938).
124
Allantoic fluids were harvested as the challenge virus in the present study and stored
125
at -80°C.
126
2.6 Experimental Design
127
Animal regression experiment was performed with SDSG strain virus. Ninety
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10-day-old SPF chickens were randomly divided into 3 groups: oral injection group
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(30), muscle injection group (30), and control group (30). The chickens in the
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experimental group were inoculated with 1 mL of FAdV-4 (SDSG strain; EID 50,
131
10−7.56/0.2 ml) either orally or by intramuscular route. The control group was
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inoculated with equal doses of physiological saline at the same injection site. The 3
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groups were separately reared in different SPF chicken isolators. Water and food were
134
autoclaved before feeding and automatically refilled. The temperature of each house
135
was maintained using radiators at 20–24°C. The chicken feces was manually cleaned
136
every day. The same inoculation dose and test method were adopted in 20-day-old
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chickens. The chickens were observed daily for clinical signs.
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2.7 Body Weight and Histopathology
139
Five chicks from the infected and control groups were respectively and randomly
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drawn for weight detection and necropsy in each of 4th, 8th, 12th, 16th , and 20th days
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post-injection (dpi) (chicks were euthanized). Chicks were examined for the presence
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of Fabricius. Each organ was sectioned into 3 portions: one was placed into 10%
144
formalin, routinely processed, and embedded in paraffin- wax, sections were cut
145
approximately 4-μm-thick and stained with hematoxylin and eosin (HE), and all
146
stained sections were examined by light microscopy; the second was stored at −80°C
147
for virology; and the third was stored at −80°C for quantitative real time PCR.
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2.8 Viral DNA Extraction
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The viral DNA of tissue samples was extracted from the previously stored organs
150
using the Universal Genomic DNA Kit (Beijing ComWin Biotech Co., Ltd.)
151
according to the manufacturer’s instructions. The concentration of high-quality DNA
152
samples (i.e., A260/280 was between 1.8–2.2, A26/230 ≧2.0) was normalized to 50
153
ng/ul, and used in subsequent experiments.
154
2.9 QPCR Analysis
155
To determine the viral load in tissues, real-time PCR was performed using the method
156
previously established in our laboratory for the detection of FAdV-4. The primers
157
used in this study are as follows: forward: 5ʹ -CGTCAACTTCAAGTACTC-3ʹ and
158
reverse: 5ʹ -AGAGGATGCTCATGTTAC-3ʹ. The relative TaqMan probe was a 24 bp
159
oligonucleotide,5′ FAM-CCTACTCAGATGGAGGCTTCTACC-3′ TAMRA, which
160
was labeled with FAM at the 5 end and TAMRA at the 3 end (Yu et al., 2018).
161
QRT-PCR analysis was performed with Premix Ex Taq™ (Probe qPCR) (TaKaRa,
162
China) following the manufacturer’s instructions, which contained 10 μL of Premix
163
Ex Taq, 0.4 μL of forward primer, 0.4 μL of reverse primer, 0.2 μL of ROX Reference
164
Dye II, 6.2 μL of ddH2O, 0.8 μL of probe, and 2.0 μL of DNA template.
165
Amplification and detection were performed with an Applied Biosystems® 7500
166
FAST Real- Time PCR System under the following conditions: 95°C for 30 s,
167
followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 62°C for 32 s.
168
The fluorescent signals were measured during the annealing step. Each DNA sample
169
was processed with 3 replicates to guarantee the reproducibility of the amplification.
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2.10 Determination of Biochemical Indexes, Cytokines, and
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Antibodies
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On the 4th, 8th, 12th, 16th, and 20th days after the injection, 5 chickens were selected
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randomly, bled, and euthanatized. Serum samples were harvested for detecting and
174
recording the changes of blood biochemical indexes, cytokines, and antibodies.
175
Biochemical
176
aminotransferase (AST), and lactate dehydrogenase (LDH), were detected through the
177
testing organization ADICON CLINICAL LABORATORIES, INC (Jinan, China).
178
Cytokines were detected using the double-antibody sandwich enzyme-linked
179
immunosorbent assay (ELISA) kit of chicken interleukin 6 (IL-6) and interferon-γ
180
(IFN-γ) (Lengton, Shanghai, China) according to the manufacturer’s directions.
181
The FAdV-4-specific antibodies were determined by the indirect ELISA which
182
developed in previous work in our laboratory (Niu et al., 2017). In brief, 47 μg/mL of
183
recombinant hexon protein in carbonate buffer solution was incubated in black
184
Maxisorb 96-well plates (Greiner Bio-One, Germany) overnight at 4°C, and the plates
185
were blocked by 5% non- fat dry milk (Solarbio, Beijing, China, w/v) for 2 h at 37°C.
186
Then, serum (1:10) was added at 200 μL/well and incubated at 37°C for 1 h.
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Afterward, the plates were incubated with rabbit anti-chicken immunoglobulin G (IgG,
188
1: 800) (Solarbio,
189
(Sigma-Aldrich) at 100 μL/well. The plates were incubated at 37°C for 1 h, and 3′, 3′,
190
5′, 5′-tetramethylbenzidine substrate solution (TransGen, Beijing, China) was added
191
to each well (100 μL/well). Fifty microliters of 3 M H2SO4 (Wuxi JINGKE Chemical
192
CO., LTD.) was added to stop the reaction, and the optical density (OD) values were
193
observed at 450 nm using an automated ELISA plate reader (Thermo, Waltham, MA,
194
USA).
195
2.11 Statistical Analysis
196
All results are presented at means ± standard deviations and analyzed using the
197
one-way analysis of variance (ANOVA) procedure of GraphPad Prism 6.0 (GraphPad
198
Software Inc., San Diego, CA, USA). P values of <0.05 or <0.01 were used to define
199
statistical significance.
200
3. Results
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3.1 Clinical Investigation
including
alanine
aminotransferase
(ALT),
aspartate
China) conjugated
to
horseradish peroxidase
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indexes,
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Journal Pre-proof The PCR results showed that 79 of the 87 samples were positive for FAdV.
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Postmortem examination of the 79 chickens showed severe lesions, including
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hydropericardium in the heart, hemorrhaging and enlargement of livers, edema with
205
congestion in lungs and kidneys, and swelling and bleeding in the thymus (Fig 1
206
(A)-(D)). The remaining 8 had no obvious lesions.
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3.2 Sequence Analysis of Hexon Gene
208
The isolate was designated as SDSG (GenBank accession: MK424834). Multiple
209
alignments showed that the hexon genome of the isolate shared 60.7–99.7% of the
210
nucleotides and 66.4–99.9% of the deducted amino acid sequences with those of
211
representative strains from different lineages. As shown in Figure 2, the phylogenetic
212
tree for hexon genes was constructed based on the sequences obtained from the
213
GenBank database, and it revealed that the isolate SDSG was clustered together with
214
chicken FAdV-4 isolates (PK-01 and KR5) (Steer et al., 2009).
215
Notably, the SDSG strain shared 99.7% of the nucleotide and amino acid sequences
216
with the India- isolated strain (EU931693, PK-01), which indicated that this strain
217
might have originated from early India isolates.
218
3.3 Clinical Symptoms
219
After infection with FAdV-4, the injection group of 10-day-old and 20-day-old SPF
220
chickens showed signs of depression, lack of appetite, and stunted growth. The
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chickens of the control group did not demonstrate any clinical symptoms throughout
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the experiment. The mortality rate of the 10-day-old muscle injection group reached
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50% (Table 1). Chickens in the injection group grew slowly, and the body weights
224
were lower than those of the control group (Fig 3).
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Table 1. Grouping and death status
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Grouping
10-day-old group
20-day-old group
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Experiment
Quantity
dead number
Mortality
Oral injection
30
7
23.333%
Intramuscular injection
30
15
50%
Control
30
0
0
Oral injection
30
5
16.667%
Intramuscular injection
30
5
16.667%
Control
30
0
0
3.4 Pathologic Change
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Journal Pre-proof Dissecting the chickens showed that hearts have a severe pericardial effusion; livers
228
are swollen with bleeding spots. In addition to the above typical clinical symptoms,
229
the infected chickens also presented significant lesions characterized by swelling with
230
hemorrhage in spleens, edema with varying degrees of congestion in lungs and
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kidneys, and swelling with different extents of bleeding in the thymus. The chickens
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of the control group had no obvious necrotic lesions (Figs 4 and 5).
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3.5 Histopathologic Analysis
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Histopathological analysis showed that chickens from both experimental groups had
235
obvious histopathological changes (Fig 6). The heart showed interstitial edema
236
accompanied with lymphocytic infiltration. Also, the 10-day-old muscle injection
237
group (Fig 6(B)) exhibited myocardial fiber granular degeneration, necrosis, and
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hemorrhage. The livers exhibited hepatocyte steatosis, necrosis, and hemorrhage, and
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the 10-day-old muscle injection group (Fig 6(D)) also exhibited hepatocellular nuclear
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concentration and dissolution necrosis. Lesions in the spleens of 10-day-old injection
241
group (Fig 6(E), (F)) were lymphocyte of white pulp decreased and necrosis, but in
242
the 20-day-old injection group (Fig 6(e), (f)) were hemorrhage in the white pulp and
243
increase in the number of lymphocytes; In the lungs, there were exudates in the
244
bronchi, as well as capillary congestion. Lesions in the kidneys were characterized by
245
renal tubular epithelial cell degeneration and renal interstitial hemorrhage. In the
246
thymus, a large amount of necrosis of lymphocytes in the medulla was observed.
247
lesions in bursas were characterized by reduction and necrosis of lymphocytes; in
248
particular, the muscle injection group was more serious than the oral injection group.
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3.6 Real-time PCR Analysis
250
After injection, the presence of virus was detected in the heart, liver, spleen, lung,
251
kidney, thymus, pancreas and bursae of both the oral and intramuscular groups (Fig 7).
252
Notably, more FAdV-4 copies were found in the liver than in other tissues. Moreover,
253
the overall number of virus copies in the tested tissues was higher in the intramuscular
254
group than in the oral group.
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3.7 Detection of Biochemical Indices
256
The results of serum biochemical indices is presented in Figure 8. In the 10-day-old
257
group, the AST, ALT, and LDH of experimental groups were higher than those of the
258
control group. Briefly, ALT reached the peak at 8 dpi (p < 0.01), AST at 4 dpi (p <
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Journal Pre-proof 0.01), and LDH at 8 dpi (p < 0.01). After reaching peak levels, the above biochemical
260
indices declined with time. The results of serum biochemical indicators in the
261
20-day-old group were similar to those in the 10-day-old group, except the LDH
262
reached the peak at 4 dpi (p < 0.01).
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3.8 Detection of Cytokines in Serum
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As Figure 9 shows, in the 10-day-old group, the levels of IL-6 in the serum reached
265
the peak at 16 dpi and then tended to decline. The level of the IFN-γ in the
266
intramuscular group was higher than that in the oral group, which showed a slowly
267
upward trend, but decreased rapidly after reaching the peak, and was finally lower
268
than the oral group. In the 20-day-old group, IL-6 levels rose slowly and peaked at 16
269
dpi. The IFN-γ levels were significantly higher than in the control group (P < 0.01) at
270
8 and 16 dpi, but the IFN-γ level in the intramuscular injection group was
271
downregulated rapidly after 16 dpi.
272
3.9 Detection of Antibodies in Serum
273
FAdV-4 antibody levels in the serum were detected throughout the experiment. As
274
Figure 10 shows, the positive serum could be detected earlier in the intramuscular
275
injection group at 4 dpi, compared with the oral group. The antibody level in the
276
experimental group continued to increase throughout the experiment; however, the
277
antibody level in the 10-day-old intramuscular injection group showed a downward
278
trend at 8 dpi and 16 dpi.
279
4. Discussion
280
HHS caused by FAdV-4 is a highly pathogenic communicable disease characterized
281
by pericardial effusion and IBH (Mase et al., 2010; Schachner et al., 2018). HHS
282
mainly infects broilers aged 3–5 weeks with a high mortality of 20–80%. Currently,
283
this disease can also occur in hybrid chickens, breeders, and layers (Ye et al., 2016),
284
resulting in considerable economic losses in China.
285
At present, a few studies have been reported on the pathogenicity of FAdV-4 in
286
chickens such as one on oral infection of 3-week-old SPF chickens (Zhao et al., 2016),
287
and another on both oral and intramuscular infection of 40-day-old chickens (Li et al.,
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2016). However, these studies were all conducted on chickens older than 3 weeks of
289
age. There is no article about the pathogenicity of smaller-day-old SPF chickens via
290
different injection routes. Some studies included a small portion of data on the
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2019), but they did not systematically and comprehensively test cytokines,
293
biochemical indices, and antibody levels in infected chickens. Existing articles lack
294
this part of the data. The pathogenesis of the virus in chickens is not comprehensively
295
understood, and it is necessary to better understand the pathogenic mechanisms of
296
FAdV-4 to effectively control a viral epidemic.
297
Because the pathogenicity of FAdV-4 isolated from small-day-old chickens and the
298
pathogenic difference of this strain to small-day-old and large-day-old chickens
299
remains unclear, this study established animal regression experiments. Ten-day-old
300
and 20-day-old SPF chickens were injected separately with the same doses of the
301
FAdV-4 serum via oral and intramuscular injection routes.
302
From the gross lesions, body weight, mortality percent, and viral load we observed
303
that the pathogenicity of FAdV-4 in SPF chickens is related to the age and the route of
304
injection. Ten-day-old chickens are more susceptible than 20-day-old chickens, and
305
the muscular injection group was more serious than the oral injection group.
306
Most chickens in the 2 experimental groups showed obvious clinical symptoms,
307
including hydropericardium and hepatitis manifestation, bleeding ,and swelling of the
308
spleen, lungs, and thymus. The inoculated chickens in the 10-day-old and 20-day-old
309
groups started to die on the 4th and 7th days after injection, respectively. Mortality
310
rates among the 10-day-old chickens inoculated intramuscularly were 50%.
311
Histopathological analysis also
312
including the interstitial edema around cardiocytes, visible inflammatory cell
313
infiltration in the liver and spleen, and lymphocyte necrosis in the thymus and bursae.
314
The results demonstrate that FAdV-4 can invade multiple organs and lead to the
315
pathogenesis of chickens. The clinical manifestations and pathological changes after
316
infection were highly consistent with those of other scholars, such as Abdul-Aziz
317
(Abdul-Aziz and Hasan, 1995).
318
Real-time PCR results show that the virus is widely distributed in the body, and virus
319
DNA can be detected in various organs, such as the heart, liver, spleen, lung, kidney,
320
thymus, and bursae, which is consistent with the necropsy and histopathological
321
changes of the infected chickens. The quantity of virus in the liver was the highest, so
322
the liver is the primary choice for clinical detection of adenovirus. Notably, the
323
quantity of virus in the intramuscular injection group was higher than that in the oral
indicated obvious histopathological changes,
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325
intramuscular injection of the virus. In the natural infection model, there is no
326
intramuscular injection route. However, the established 10-day-old muscle infection
327
model is more infectious and pathogenic to the body, so this model can be valuable
328
for anti-viral drug testing and vaccine evaluation.
329
AST mainly exists in cardiomyocytes, but also in mitochondria of hepatocytes (Fan et
330
al., 2014). If hepatic cells or myocardial cells are destroyed, the enzymes will leak
331
into the blood and levels of it will increase rapidly. As the cells are repaired, its
332
content gradually decreases. ALT is mainly distributed in the body's hepatic
333
cytoplasm. The activity of this enzyme is approximately 100 times higher in the liver
334
than in serum. Therefore, 1% hepatocyte necrosis results in a 1- fold increase in serum
335
ALT (Kew, 2000; Rosenthal, 1997). LDH exists in the cytoplasm of all histiocytes in
336
the body, and its content is highest in the kidney. Therefore, LDH levels can reflect
337
tissue damage, especially kidney damage. The detection results of this study showed
338
that almost all blood biochemical indices of experimental groups were higher than
339
those of the control group and then decreased with time. As the cells self- repair, the
340
levels of enzymes in the plasma are reduced, which may be due to the damage of the
341
heart, liver, and kidney after injection. These were highly consistent with Asrani’s
342
report (Asrani et al., 1997).
343
histopathological damage.
344
IL-6 was produced by a variety of lymphocytes and non- lymphocytes, which can
345
promote the proliferation of B cells, enhance their ability to produce antibodies, and
346
promote humoral immunity. The level of secretion can reflect the body's humoral
347
immune function (Thacker et al., 2009). IFN-γ is produced by thymus-dependent
348
lymphocytes (T cells) and natural killer cells (NK cells). It is a pleiotropic factor for
349
immune regulation that can participate in the body's immune regulation and improve
350
antigen presentation ability. In this study, the levels of IL-6 in the 2 injection groups
351
first rose and then decreased. This shows that the body is in a normal state of
352
immunity at the initial stage after injecting the virus, but at the late stage,
353
immunosuppression appears (Schonewille et al., 2008). The IFN-γ in the
354
intramuscular group was significantly higher than that in the control group and was
355
maintained in the first 16 dpi, which reflected that many of these cytokines with high
356
concentrations were produced by T cells and NK cells under the stimulation of
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358
the immune organs of the infected chickens were severely damaged, resulting in a
359
decrease in IFN-γ levels. After the injection, the antibody levels also showed an
360
increasing trend, indicating that humoral immunity plays an important role in host
361
antiviral properties.
362
In conclusion, this experiment determined the accurate value of viral load, cytokines,
363
biochemical indicators and antibodies of FAdV-4 (SDSG)- induced chickens at 10 and
364
20 days old. Filling in the gaps in this aspect of data may provide references for future
365
research.
366
Acknowledgments
367
This work was supported by Tai’shan Industry Leader (LJNY201610), the National
368
Key Research and Development Program of China (2018YFD0501506), the China
369
Agriculture Research System (CARS-42-19), and Funds of Shandong "Double Top"
370
Program.
371
Conflict of Interest Statement
372
The authors declare that the research was conducted in the absence of any commercial
373
or financial relationship that could be construed as a potential conflict of interest.
374
Fig 1: Clinical investigation results. (A) Severe hydropericardium and enlarged
375
livers with blood spots; (B, C) edema with congestion in lungs and kidney; (D)
376
swelling and bleeding in thymus.
377
Fig 2: Phylogenetic tree of hexon gene sequences of the isolate SDSG (marked by
378
a red dot) and other avian strains. The phylogenetic tree was built using
379
neighbor-joining analysis and MEGA 7.0, and the bootstrap confidence values were
380
determined using 1,000-bp replicates.
381
Fig 3: Weight changes after injection. (A) 10-day-old group; (B) 20-day-old group.
382
Fig 4: Gross lesions of 10-day-old group chickens inoculated with FAdV-4.
383
Fig 5: Gross lesions of 20-day-old group chickens inoculated with FAdV-4.
384
Fig 6: Histopathologic changes of FAdV-4-infected chickens. (A-H) (K) and (L)
385
Magnification, ×400; other magnification, ×200.
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387
oral injection group; (B) 10-day-old intramuscular injection group; (C) 20-day-old
388
oral injection group; (D) 20-day-old intramuscular injection group.
389
Fig 8: Dynamics of ALT, AST, and LDH in serum of the chickens post artificially
390
infected with FAdV-4. (A-C) 10-day-old group; (D-F) 20-day-old group.
391
Fig 9: Dynamics of IL-6 and IFN-γ in serum of the chickens post artificially
392
infected with FAdV-4. (A-B) 10-day-old group; (C-D) 20-day-old group.
393
Fig 10: Dynamics of antibodies against FAdV-4 in serum of the chickens post
394
artificially infected with FAdV-4. (A) 10-day-old group; (B) 20-day-old group.
395
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