Transboundary and Emerging Diseases

ORIGINAL ARTICLE

Hydropericardium Hepatitis Syndrome Emerged in Cherry

Valley Ducks in China
H. Chen, Y. Dou, X. Zheng, Y. Tang, M. Zhang, Y. Zhang, Z. Wang and Y. Diao
College of Animal Science and Technology, Shandong Agricultural University, Tai’an, Shandong, China

Keywords: Summary
hydropericardium hepatitis syndrome; cherry
valley duck; fowl adenovirus C Since June 2015, a highly pathogenic disease occurred in duck flocks in China,
causing pericardial effusion, enlarged discoloured liver, renal enlargement and
Correspondences: haemorrhagic lung with a mortality ranging from 5% to 20%. Previous study
Z. Wang and Y. Diao. College of Animal confirmed that Fowl adenovirus group C (FAdV-C) and some field FAdVs iso-
Science and Technology, Shandong
lates had been identified as causative agents of hydropericardium hepatitis syn-
Agricultural University, Daizong Road 61,
drome (HHS) in chickens and geese world widely. In this study, we firstly report
Tai’an 271018, Shandong, China.
Tel.: +86 0538 8249851-8210;
the isolation of FAdV-C from ducks with HHS. The two isolates, designated as
fax: +86 0538 8241419; SDSX and SDJX, were separated from liver samples using 9-day-old SPF chicken
E-mail: [email protected] embryos and could cause severe cytopathic effects in duck and chicken embryonic
kidney cells. The entire ORF sequences of hexon gene of the two isolates were
Received for publication December 6, 2015 amplified, sequenced and analysed by restriction fragment length polymorphism.
Phylogenetic analysis of loop 1 sequences of hexon gene of FAdVs revealed that
doi:10.1111/tbed.12500
the two isolates were closely related to FAdV-C isolates, which could cause HHS
in chickens. Experimental infection indicated that the isolate was high
pathogenicity to 20-day-old ducks. Our study shows that the recently emerged
HHS in ducks was caused by FAdV-C and may possess a potential risk to other
poultry flocks.

Goth, Pakistan, which was designated as Angara disease,

Introduction
and has subsequently occurred in broiler flocks in South
Fowl adenoviruses (FAdVs), belonging to the Aviadenovirus Asia, the Arabian Peninsula, North and South America,
genus of the family Adenoviridae, are associated with inclu- Eastern Europe, Japan and South Korea (Khawaja et al.,
sion body hepatitis (IBH) and hydropericardium hepatitis 1988; Al-Sadi et al., 2000; Hess et al., 1999; Mendelson
syndrome (HHS) both in broilers and layers(Niczyporuk et al., 1995; Jantosovic et al., 1991; Mase et al., 2009; Kim
et al., 2010; McFerran and Connor, 1977). All 12 serotypes et al., 2008). Cross-neutralization test and restriction frag-
of FAdVs (FAdV 1-12) have been isolated from chickens ment length polymorphism (RFLP) analysis have been
and are grouped into five species (FAdV A-E) (Hess, 2000). established to distinguish FAdV serotypes (McFerran et al.,
Outbreaks of HHS caused by FAdV-C have occurred in 1972; Okuda et al., 2006; Takeuchi et al., 1999; Tang et al.,
many countries all over the world (Hafez, 2011). FAdV-C 2009). Recently, FAdV-C and FAdV-D were isolated in dis-
has been isolated from chickens and geese with HHS, with eased partridge chicken and hyline brown layers flock char-
each case showing similar clinical signs, including sudden acterized with IBH in China and the experimental infection
increase in mortality, hydropericardium, hepatitis and with the isolates caused HHS in 3-week-old SPF chicken.
nephritis (Ivanics et al., 2010; Glavits et al., 2005; Toro Previous study indicated the some strains of the FAdV-C
et al., 2000). The disease principally affects three- to 6- (FAdV serotypes of 4, 9, 10 and 11) and FAdV-D isolates
week-old broilers with a mortality of 10–40%, even up to can cause HHS and significant mortality in broiler chicken
80%. Egg production is decreased in diseased breeder flocks and goose. When virus infected 3- to 6-week-old broiler
(Nakamura et al., 1999). HHS was first reported in Angara chicken, the mortality usually varies from 2 to 40 per cent,

1262 © 2016 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 64 (2017) 1262–1267
 18651682, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.12500 by Cochrane Poland, Wiley Online Library on [28/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Chen et al. Hydropericardium Hepatitis Syndrome in Ducks

even up to 60%. In 2015, an infectious disease outbreak in China), and the PCR condition was used at 95°C for
commercial duck flocks in Shandong province character- 5 min, followed by 35 cycles of 95°C for 45 s, 55°C for
ized similar symptoms to HHS in chicken. The age of dis- 1 min and 72°C for 1 min 30 s, and then 72°C for 10 min
eased ducks mainly ranged from 25 to 40 days old. In this to generate PCR products of 1219 bp and 1350 bp in
study, we attempt to confirm FAdV-C in suspected out- length, respectively. All PCR products were cloned into
breaks of HHS in ducks by virus isolation, PCR and pMD18-T vector and transformed into DH5a E. coli com-
sequencing for phylogenetic analysis of the hexon genes. petent cells, and the positive clone was sequenced (BGI
Furthermore, epidemiological survey was also carried out Company Ltd, Beijing, China). The PCR products were
in healthy duck flocks. analysed by restriction fragment length polymorphism
(Hae II) using NEB cutter software. Finally, samples
detected FAdV-positive without other agents were used in
Materials and Methods
further study.
Ethics statement
This study was approved by the Animal Care and Use
Virus isolation
Committee of Shandong Agricultural University (permit
number: 20150901) and performed in accordance with the Virus isolation from the tissue homogenates was performed
‘Guidelines for Experimental Animals’ of the Ministry of in chicken embryos, chicken embryonic kidney (CEK),
Science and Technology (Beijing, China). All the ducks fibroblasts (CEF) and liver (CEL) cells and duck embryonic
were cared for in accordance with the humane procedures. kidney (DEK) cells, respectively. The liver homogenates
were freeze-thawed three times and centrifuged at 8000 g
for 15 min. The supernatant was passed through a 0.22-lm
Samples and clinical signs
filter and then inoculated into the chorioallantoic mem-
After HHS was identified among Cherry Valley ducks from branes of 9-day-old SPF chicken embryos. After three pas-
four farms (located in Gaotang, Shenxian, Juxian and Fei- sages in embryonated eggs, embryo death occurred on
cheng counties) in Shandong province, China, 41 ducks passage days 3–5. Four avian embryonic cells were inocu-
were conveyed to the Poultry Diseases Lab of Shandong lated with virus allantoic fluids and monitored daily for
Agricultural University for identification of the causative cytopathic effects (CPE). The culture supernatants and
factors from six duck flocks. Of these, 30 ducks died during allantoic fluids were harvested as viral stocks and stored at
the transfer process and the 11 surviving ducks were eutha- 70°C.
nized. Sera samples were separated by centrifugation at
5000 g for 15 min. Viscera (e.g. liver, kidney, heart, spleen,
Phylogenetic analysis
glandular stomach, etc.) from diseased ducks were collected
and fixed in 10% neutral buffered formalin. Sections The amplified fragments of 1219 bp from 41 samples were
were stained with haematoxylin and eosin for microscopic aligned using the ClustalW methods in Megalin program
examination. included in Lasergene 7.0 software (DNASTAR Inc., Madi-
son WI, USA). The phylogenetic tree of hexon loop 1 (two
isolates from this study and 19 from the GenBank database)
Nucleic acid extraction and PCR assay
was constructed using the neighbour-joining method of the
Total DNA was extracted from tissue samples by phenol– MEGA program (version 5.0) with absolute distances fol-
chloroform method while the total RNA was acquired lowing 1000-bp replicates (Tamura et al., 2011).
using Trizol reagent. The RNA was reverse transcribed into
cDNA, which was used as template for PCR amplification.
Pathogenicity in ducks
To detect FAdV from liver samples of ducks, a PCR assay
was performed using a pair of specific primers (Fav-1F: To evaluate the pathogenicity in ducks, twenty 20-day-old
50 -TGGACATGGGGGCGACCTA-30 and Fav-1R: 50 -AAGG SPF ducks were inoculated with 103.0 ELD50 doses of SDJX
GATTGACGTTGTCCA-30 ), which was designed, based on strain by intracerebral injection, while ten ducks were
the conserved sequence of the hexon gene. In addition, the served as negative control. Two groups of ducks were sepa-
entire hexon open reading frame of two isolates was ampli- rately kept in different SPF chicken isolators and were
fied, sequenced and analysed with PCR-RFLP to identify monitored daily for 10 days. The appearance of clinical
the serotypes according to previous study (Tang et al., signs of disease and death was recorded. All ducks were
2009). The two PCRs were carried out in a total volume of euthanized with intravenous pentobarbital sodium (New
25 ll containing 2 ll viral DNA, 125 pmol each primer Asia pharmaceutical, Shanghai, China) at 10 days post-
and 2.5 U Ex-Taq DNA polymerase (Takara, Dalian, infection (dpi). Heart, liver, small intestine and kidney were

© 2016 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 64 (2017) 1262–1267 1263
 18651682, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.12500 by Cochrane Poland, Wiley Online Library on [28/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Hydropericardium Hepatitis Syndrome in Ducks H. Chen et al.

collected for the histopathological observation and virus ducks and 6 (12%) of the 50 meat-type ducks were detected
detection. to be FAdV-C positive.
All 41 field samples were detected FAdV-positive, and of
which, 6 ducks were co-infected with FAdV and H9N2 ser-
Epidemiological survey
otype avian influenza virus (AIV). These isolates shared
To investigate the epidemiological characteristics of FAdV- 99.2–100% sequence identity of amplified 1219-bp frag-
C in duck flocks, cloacal swabs were collected from 50 bree- ments. Among these, two strains designated as SDSX and
der ducks and 50 commercial meat-type ducks from duck SDJX were isolated from liver samples, which were FAdV-
farms in Shenxian county, western Shandong province. positive and excluded many common duck-origin causa-
Viral DNA was detected from all specimens by PCR. tive agents (e.g. AIV, tembusu virus, novel goose par-
vovirus-related virus (N-GPV), duck plague virus (DPV),
etc.). The entire ORF of hexon gene was assembled to be
Results
2815 bp in length. Fragment A and B were analysed by
Post-mortem examination revealed severe gross lesions of NEB cutter software (Hae II at 983 bp in fragment A, and
diseased ducks with enlarged and yellowish livers with 357 bp, 488 bp in fragment B). RFLPs analysis demon-
necrotic foci, pericardial effusion, congestion, haemorrhage strated that the two isolates were classified into group
in the lungs, and haemorrhaging and enlargement of the FAdV-C (FAdV-4 serotype). Multiple alignments showed
kidneys (Fig. 1a–c). Post-mortem examination of the mod- that the two isolates shared hexon sequence identities of
erate lesions showed haemorrhagic enteritis in the intestine, 98.9–100% with other FAdV-4 isolates (JSJ13, PK-01 and
thelorrhagia of the glandular stomach, pancreatic oedema KR5). There were only three nucleic acid variations
and spotted bleeding. The livers and lungs were homoge- between JSJ13 and field isolates in this study. A phyloge-
nized for virus isolation and nucleic acid extraction. netic tree for loop 1 amino acid was constructed based on
Four ducks died without significant clinical signs during the sequences obtained from GenBank database, and it
5–7 dpi from which liver samples were collected and revealed that the two isolates were clustered together with
detected to be FAdV-positive and seventeen ducks demon- chicken FAdV-C isolates (JSJ13, KR5, PK-1 and C2B)
strated symptoms of HHS (severe in 11, moderate in four (Steer et al., 2009) (Fig. 2). The deducted amino acid
and mild in two) in experimental infection. Besides the sequences of hexon genes of isolates (SDJX and SDSX)
clinical HHS, other symptoms of FAdV-C-infected ducks shared 97.6–100% identities with those of reference isolates
included ascites, enteritis, haemorrhaging of the glandular included in the FAdV-C lineage.
stomach and light coloration of the bone marrow. The
main histopathological changes of field diseased and exper-
Discussion
imental infected ducks with HHS included cardiac myocyte
necrosis and disordered arrangement, moderate neu- Hydropericardium hepatitis syndrome has been subse-
trophils and monocytes infiltration in the heart, necrosis quently reported in chickens and geese and causes fluid
and fatty degeneration of hepatocytes, intranuclear inclu- accumulation in the pericardium and enlargement and dis-
sions in liver parenchymal cells and severe haemorrhage coloration of the liver with foci of haemorrhage and/or
accompanied by lymphocyte infiltration in the liver, and necrosis world widely. FAdV-4 isolates were the major cau-
degeneration and necrosis of renal tubular epithelial cells, a sative agents of HHS and highly virulent for broilers, com-
protein-like substance in the proximal convoluted tubules, pared with other serotype FAdVs (belonging to the genus
and extensive congestion in the renal interstitium in the Aviadenovirus) which caused birds with IBH. The disease
kidneys (Fig. 1g–i). No significant histologic lesions and was first isolated from broiler flocks in Jiangsu province in
mortality were present in the negative control ducks until China during 2012 to 2013 (Zhao et al., 2015). Currently,
all ducks were euthanized at 10 dpi. HHS is becoming one of the major infectious diseases that
The chicken embryos died at 3–5 dpi, characterized by lead to huge economic losses or risk to poultry industry in
stunted growth, severe diffuse haemorrhaging and hepator- China.
rhagia, compared with healthy embryos (Fig. 1d). Viral In the present study, we reported the detection and
titres of allantoic fluid were 104.3–104.5 median embryo identification of FAdV-C in Cherry Valley ducks, related
lethal dose (ELD50)/ml using the methods of Reed and with HHS, for the first time. As vast numbers of chickens
Muench (1938). Severe cytopathic effects were observed in and ducks are raised in Shandong (≥1.5 billion in total),
DEK and CEK cells for the primary infection (Fig. 1e and cross-host transmission of many causative agents can
f). Both of the two isolates did not have the ability to agglu- occur in adjacent poultry farms. These results indicated
tinate duck or chicken erythrocytes. PCR analysis showed that the possibility risk of cross-host transmission of
that cloacal swab samples from 12 (24%) of the 50 breeder FAdV-C might occur from chicken to ducks. High

1264 © 2016 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 64 (2017) 1262–1267
 18651682, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.12500 by Cochrane Poland, Wiley Online Library on [28/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Chen et al. Hydropericardium Hepatitis Syndrome in Ducks

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 1. Pathologic and histopathological changes in ducks, duck embryos and embryo kidney cells infected by FAdV-C. Diseased ducks showed (a)
pericardial effusion and enlarged and light coloured liver, (b) oedema and haemorrhage in the lungs and (c) enlarged kidneys with haemorrhagic
spots. The embryos showed (d) stunted growth and diffuse haemorrhaging. (e, f) Severe cytopathic effects were observed in duck and chicken kidney
cells infected with strain SDSX. Histopathological analysis of lesions in the heart (g) showed cardiac myocyte necrosis and disordered arrangement,
moderate neutrophils and monocytes infiltration, whereas the liver (h) showed necrosis and fatty degeneration of hepatocytes, intranuclear inclusions
in liver parenchymal cells, and severe haemorrhage accompanied by lymphocyte infiltration, and the kidneys showed (i), degeneration and necrosis of
renal tubular epithelial cells, a protein-like substance in the proximal convoluted tubules and extensive congestion in the renal interstitium (Scale bar,
50 lm). [Colour figure can be viewed at wileyonlinelibrary.com]

prevalence of HHS in pericardial effusion samples of in length. RFLPs analysis indicated that the two isolates
ducks in Shandong province was confirmed. RFLPs by were classified into FAdV-4. Phylogenetic analysis of loop
Hae II in fragment A of hexon gene generated two seg- 1 and multiple alignments of fragment A showed that the
ments of 982 bp and 237 bp, respectively, and three seg- two isolates in our study shared high homology with
ments in fragment B were of 132 bp, 356 bp and 862 bp FAdV-C isolates.

© 2016 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 64 (2017) 1262–1267 1265
 18651682, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.12500 by Cochrane Poland, Wiley Online Library on [28/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Hydropericardium Hepatitis Syndrome in Ducks H. Chen et al.

Fig. 2. Phylogenetic tree of loop 1 in hexon gene sequences of the present isolates SDSX, SDJX (included in rectangle) and other 22 fowl adenovirus
strains. The tree was constructed by the neighbour-joining method with 1000-bp replicates using MEGA 5.0 software. Scale bar indicates amino acid
substitutions per site.

A survey based on PCR assay showed that the infection group while the mortality with the field isolates was up to
of FAdV-C in breeder ducks and meat-type ducks was 12% 100% (Mazaheri et al., 1998). Moreover, effective oil emul-
and 6%, respectively. Latent FAdV-C infection in healthy sion vaccines have been introduced in breeder and broiler
ducks revealed that HHS might be caused by FAdV-C flocks for disease prevention and control (Mansoor et al.,
infection co-effected with some conditional pre-disposing 2011; Kim et al., 2014).
factors. Also, silent infection ducks might promoted the In summary, the results of this study indicated an out-
horizontal transmission throughout the entire duck flocks. break of HHS in ducks in China caused by FAdV-C infec-
Experimental infection and virus isolation confirmed the tion. The virus is highly pathogenic in commercial Cherry
pathogenicity of FAdV-C in ducks. In field cases, a certain Valley ducks and breeder ducks. However, no mortality
number of ducklings with pericarditis were found to be occurred in little ducklings flocks (≤1-week-old), in which
FAdV-C positive and HHS often occurred in surviving FAdV-C could be detected.
ducks of diseased flocks at approximately the age of
25 days. Other pathogens (AIV et al.) or stress factors
Nucleotide sequence accession numbers
(temperature, transportation and vaccination) and FAdV-
C might jointly lead to death in ducks. It was reported that Sequence data have been deposited in the GenBank
different FAdV-C isolates showed different pathogenicity to database under accession numbers KT899324 and
1-day-old chicks. No chicks died in the KR5 strain infected KT899325.

1266 © 2016 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 64 (2017) 1262–1267
 18651682, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.12500 by Cochrane Poland, Wiley Online Library on [28/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Chen et al. Hydropericardium Hepatitis Syndrome in Ducks

adenoviruses isolated from chickens with hydropericardium

Acknowledgements
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This work was supported by China Agriculture Research Mazaheri, A., C. Prusas, M. Voss, and M. Hess, 1998: Some
System (CARS-43-10) and National Natural Science Foun- strains of serotype 4 fowl adenoviruses cause inclusion body
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McFerran, J., and T. Connor, 1977: Further studies on the classi-
Conflict of interest fication of fowl adenoviruses. Avian Dis. 21, 585–595.
McFerran, J., J. Clarke, and T. Connor, 1972: Serological classifi-
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