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\begin{document}

\title{Young Pigeon Disease Syndrome (YPDS) in Turkish Pigeons (Columba livia
domestica) stemming from coinfection with Pigeon aviadenovirus A and
Pigeon circovirus: The first isolation and molecular characterization}



\author[1]{Ismail Sahindokuyucu}%
\author[2]{Merve Biskin Turkmen}%
\author[2]{Tugce Anteplioglu}%
\author[2]{Eray Alcigir}%
\author[3]{Zafer YAZICI}%
\author[4]{Gerald Barry}%
\author[3]{Mustafa Yavuz Gulbahar}%
\author[2]{Oguz Kul}%
\affil[1]{Izmir/Bornova Veterinary Control Institute}%
\affil[2]{Kirikkale University Faculty of Veterinary Medicine}%
\affil[3]{Ondokuz Mayis University Faculty of Veterinary Medicine}%
\affil[4]{University College Dublin School of Agriculture Food Science and Veterinary Medicine}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Pigeon aviadenovirus A and Pigeon circovirus are both viruses that can
cause disease in pigeons. This study reports the identification of a
natural co-infection of Pigeon aviadenovirus A and Pigeon circovirus in
a breeding pigeon flock in central Anatolia, Turkey. Both viruses were
isolated from pooled internal organs of pigeons using primary chicken
embryo kidney cell cultures (CEKC) and specific pathogen free (SPF)
embryonated chicken eggs. Both viruses were identified by PCR
amplification followed by Sanger sequencing, while histopathological
examination showed degenerated hepatocytes with basophilic intranuclear
viral inclusions. The viruses have similar transmission characteristics
and common clinical manifestations, but it is possible that coinfection
may exacerabate disease. This is the first report of its kind in Turkey
and is important for the protection against disease in pigeons.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Viral infections of pigeons, particularly under the age of 1, are
associated with high morbidity and mortality. It has been reported that
Pigeon aviadenovirus A (Duchatel et al., 2000; De Herdt et al., 1995)
and Pigeon circovirus (Todd, 2000) are associated with an immuno
supression syndrome called ``Young Pigeon Disease Syndrome'' (YPDS).

Pigeon aviadenovirus A (PiAdV-A) is a member of the genus Aviadenovirus
with in the family \emph{Adenoviridae}(Raue et al., 2002). To date, the
Aviadenovirus genus is made up of 14 different members incluiding, Duck
aviadenovirus B, Falcon aviadenovirus A, Fowl aviadenovirus A-E, Goose
aviadenovirus A, Pigeon aviadenovirus A-B, Psittacine aviadenovirus B
and Turkey aviadenovirus B-D.

Pigeon circovirus (PiCV) is a member of the Circovirus genus, family
Circoviridae (Mankertz et al., 2000) and within this family there are 10
viruses that infect and cause disease in birds; incluiding, Pigeon
circovirus, Beak and feather disease virus, Canary circovirus, Goose
circovirus, Duck circovirus, Gull circovirus, Raven circovirus, Swan
circovirus, Chicken associated cyclovirus 1 and Duck associated
cyclovirus 1.

Pigeons with adenovirus infection were originally reported by McFerran
et al. in 1976 (McFerran et al., 1976a), although PiAdV infection was
firstly isolated and identified in 1984 in Belgium. Pigeons infected
with PiAdV have since been observed all around the world (Vereecken et
al., 1998). Although pigeons of any age can be infected, young pigeons
under one year of age are particularly badly affected by PiAdV-A,
showing acute watery diarrhoea, vomiting and anorexia. PiAdV-B affects
pigeons of all ages and is characterized by sudden death and intensive
hepatic necrosis (Duchatel et al., 2000; De Herdt et al., 1995;
Vereecken et al., 1998). The annual pigeon mortality rate is very high,
with approximately 30\% due to PiAdV, but in some cases it can reach
100\% in pigeon lofts with necrotizing hepatitis infections (Vereecken
et al., 1998).

PiCV was first diagnosed in Canada in 1986, although it is known to be
present across the World (Hess et al., 1998, Woods et al., 1993) and,
similar to PiAdV-A, it is younger pigeons that are mainly affected by it
(2 months - 1 year of age) (Pare et al., 1999; Takase et al., 1990;
Tavernier et al., 2000; Todd, 2000; Woods et al., 1994). PiCV infection
can induce a wide spectrum of non-specific clinical signs including
lethargy, weight loss, respiratory distress and diarrhoea (Woods et al.,
1994). Until recently, it was unclear what effect many aviadenoviruses
had on animals, but now the impact of viruses such as the Fowl
aviadenoviruses (FAdV) is well recognised (Hess, 2013). These FAdV
strains can cause gizzard erosion (GE), hydropericardium syndrome (HS)
and severe liver damage leading to the condition known as inclusion body
hepatitis (IBH) and serotypes 2, 4, 5, 6, 8, 10 and 12 have been
isolated from both diseased and healthy pigeons (Goryo et al., 1988;
Hess et al., 1998a; Hess et al., 1998b; McFerran et al., 1976a). In this
study we report the isolation and analysis of both PiAdV and PiCV from a
co-infection in pigeons in central Anatolia, Turkey.

\textbf{Materials and Methods}

\textbf{History, Gross Findings and Sampling}

A pigeon flock (N=45, 4-5 month old domestic pigeons) had a history of
increased mortality (20-25\%) during November-December 2019. According
to the pigeons' owner, the first sign was inapppetance followed within a
couple of days by vomiting. This was followed within 24 hours by dark
green watery diarrhea that continued for approximately 2 days with many
pigeons dying during this time. A total of 5 pigeons were submitted to
the Pathology Department of the Faculty of Veterinary Medicine,
K\selectlanguage{polish}ı\selectlanguage{english}r\selectlanguage{polish}ı\selectlanguage{english}kkale University, Turkey for examination. All pigeons were
necropsied, following natural death after a period of chronic weight
loss. The birds had severe intestinal ascariasis and the intestinal wall
presented with gross lesions. Tissue samples from different internal
organs, including liver, kidney, spleen, gut and pancreas, were
collected for virus isolation and identification under aseptic
conditions.

\textbf{Isolation of Viruses}

Two different groups of pigeons' internal organs were pooled and mixed
with PBS (Sigma-Aldrich) containing penicillin (2000 units/ml),
streptomycin (2 mg/ml), and gentamicin (50 \selectlanguage{greek}μ\selectlanguage{english}g/ml) antibiotics and
Mycostatin (1000 units/ml). The organs were homogenized, followed by
centrifugation (4000 rpm for 10 min). The supernatant was collected for
screening by PCR using primers specific for PiAdV-A and PiCV (Freick et
al., 2008; Raue et al., 2002). Supernatants that were positive for
PiAdV-A and PiCV were firstly inoculated into the chorioallantoic cavity
of 10 day-old specific pathogen-free (SPF) embryonated chicken eggs and
the yolk sac of 6 day old SPF eggs to observe and compare the viruses
adaptation and propagation according to the protocol of the Villegas
Laboratory Manual (Villegas, 2006). The eggs were incubated at 37 \selectlanguage{ngerman}°C for
5 to 10 days according to inoculation route, respectively. The
inoculated eggs were examined daily until embryos stopped moving and
were presumed dead under ovoscope light. At this point the allantoic
fluid was removed and inoculated into a fresh egg following the same
procedure as before. This was repeated until each supernatant passaged
through 5 eggs.

Cell culture was also used. The supernatants were passed through a
0.22-\selectlanguage{greek}μ\selectlanguage{english}m microfilter directly before the inoculation. Following
filtration, the samples were inoculated at the same time into primary
chicken embryo fibroblast (CEF) and primary chicken embryo kidney cell
(CEKC) cultures to compare cytopathic effects (CPE) and virus
propagation.

Primary CEF and CEKC cultures were prepared from 10 day-old and 18
day-old SPF embryonated chicken eggs according to the protocol of the
Villegas Laboratory Manual, respectively (Villegas, 2006). For cell
propagation, MEM (Gibco, UK) containing Earle's balanced salts, 10\%
fetal calf serum, 100 IU of penicillin (Sigma-Aldrich, USA) and 100 \selectlanguage{greek}μ\selectlanguage{english}g
of streptomycin (Sigma-Aldrich, USA) antibiotics per ml was used. The
cells were incubated at 37 \selectlanguage{ngerman}°C for 24-48 hours until a confluent
monolayer of CEFs and CEKCs had formed. For inoculation, the cell
culture supernatants were removed and filtered supernatants from two
groups of organ samples were overlaid onto the cell cultures, which were
then incubated at 37 °C for 1 hour. Following this, fresh MEM
supplemented with 2\% fetal bovine serum was added to the cultures, and
they were incubated at 37 °C in 5\% CO\textsubscript{2} for 7 days. Once
strong CPE was observed, the cultures were freeze thawed,centrifuged at
4000×\emph{g} for 10 minutes, and the supernatants were stored at -20 °C
until DNA extraction was performed.

\textbf{Pathologic Examinations}

\textbf{c.1) Necropsy and Histopathological Examinations}

After necropsy of the pigeons, collected systemic tissue samples were
fixed in 10\% neutral formalin (pH 7.4) and processed according to
routine tissue processing procedures (Leica TP1020). After embedding in
paraffin wax, tissue sections were cut at 5 \selectlanguage{greek}μ\selectlanguage{english}m thickness from paraffin
blocks and stained with Haematoxylin-Eosin (H\&E) (Luna, 1968). The
histopathological changes of the lesioned organs were semiquantitatively
scored and their photomicrographs were taken (Olympus BX51, DP25 digital
camera).

\textbf{c.2) Fowl Adenovirus-4 Specific Immunoperaxidase Test}

The avidin biotin complex peroxidase (ABC-P) method was performed
according to manufacturer's instructions (ab64264, Abcam, HRP/DAB, MA,
USA). Briefly, tissue sections were deparaffinized, hydrated and then
boiled in antigen retrieval solution (citrate buffer pH 6.0) for 20
minutes. The sections were incubated in 3\% hydroperoxide-methanol
mixture at room temperature for 20 minutes before blocking was performed
using normal goat serum at 37\selectlanguage{ngerman}°C for 7 minutes. The sections were
incubated with polyclonal rabbit anti chicken FAdV-4 hyperimmune serum
(kindly provided from Prof. Dr. Hafez) diluted at 1:40 at 37°C for 1
hour. After this, the sections were incubated with a biotinylated, HRP
labelled anti-rabbt secondary antibody (Abcam kit, USA) followed by
exposure to the AEC substrate chromogen solution (Dako). For
counterstaining, sections were kept in Gill's haematoxylin (Sigma) for 2
minutes and cover slipped with aquous mounting medium. Washing
procedures were carried out with Tris-HCl twice for 5 minutes excluding
the blocking step. As a negative control, the sections were treated with
Phosphate Buffered Saline (PBS) instead of primary antibodies. As a
positive control, the same protocol was performed on chicken liver and
brain sections that are obtained hyperimmun serum from `'Hafez Mohamed
Hafez in Freie Universität Berlin-Germany'', against FAdV-4 virus
infection study in chickens.

\textbf{DNA extraction and PCR}

DNA was extracted from 200 \selectlanguage{greek}μ\selectlanguage{english}L of supernatant from two groups of samples
separately, using a High Pure Viral Nucleic Acid Kit (Roche) according
to the manufacturer's protocol. The extracted DNAs were stored at -20 \selectlanguage{ngerman}°C
until PCR was performed.

PCR for PiAdV-A and PiCV was carried out in a total volume of 25 \selectlanguage{greek}μ\selectlanguage{english}l
containing 5 \selectlanguage{greek}μ\selectlanguage{english}l of DNA, 0.4 \selectlanguage{greek}μ\selectlanguage{english}M of each primer, and PCR master mix
(Grisp, Portugal). The PCR for PiAdV-A was carried out in a thermocycler
(Techne, UK) with an initial denaturation at 95 \selectlanguage{ngerman}°C for 5 min, followed
by 35 cycles of 95 °C for 20 seconds, 58 °C for 30 seconds, and 72 °C
for 1 min with a final elongation step at 72 °C for 5 min. The PCR for
PiCV was carried out in the same thermocycler with an initial
denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 20
seconds, 62 °C for 30 seconds, and 72 °C for 30 seconds with a final
elongation step at 72 °C for 5 min. The PCR amplicons were visualised by
agarose gel electrophoresis. The PCR amplicons were purified according
to the manufacturer's protocol (ExoSAP-IT PCR Product Cleanup Reagent,
USA), before sequencing.

The PiAdV-A primers (F1-s and F2-as) and PiCV (PiCV2-s and PiCV2-as)
primers were used in the study (Table 1) bind to a conserved region of
the fiber 2 gene of PiAdV-A and capsid gene of PiCV. The samples were
also tested by other primers which are listed in Table. 1, to
investigate FAdV, PiAdV-B and PiHV to detect the presence of other
possible viruses.

\textbf{TABLE 1.} Primers used in this study\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{The viruses}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Target gene}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Primer names}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Sequence (5'--3')}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{product size (bp)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Reference}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Fowl aviadenoviruses\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hexon\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Hexon A\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
CAARTTCAGRCAGACGGT\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
897\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Meulemans et al., 2001).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Hexon B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TAGTGATGMCGSGACATCAT\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Fowl aviadenoviruses\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hexon\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
H1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TGGACATGGGGGCGACCTA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1219\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Raue \& Hess, 1998).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
H2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
AAGGGATTGACGTTGTCCA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Fowl aviadenoviruses\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hexon\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
H3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
AACGTCAACCCCTTCAACCACC\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1319\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
H4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TTGCCTGTGGCGAAAGGCG\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PiAdV-1 (PiAdV-A)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
fiber-2\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PiAdV-1 F1-s\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
ATCAACTACGACAACGAAGGC\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
967\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
(Raue et al., 2002).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiAdV-1 F2-as\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
CGGTAGAGTTACGGGGAAATT\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PiAdV-2 (PiAdV-B)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
hexon\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PiAdV-2 Hex-3-F\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
GTAACATGAGCGTGCTGTTTG\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
643\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
(Teske et al., 2017).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiAdV-2 Hex-3-R\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
CTGAGAAACGAAACCCGAATTG\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiHV\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polymerase\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiHV-s\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
GGGACGCTCTGATTAAGGAAT\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
242\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Raue et al., 2005).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiHV-as\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
CTTGGTGATCAGCAGCAGCTTG\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiCV\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Capsid\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiCV2-s\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TTGAAAGGTTTTCAGCCTGGC\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
325\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Freick et al., 2008).\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PiCV2-as\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
AGGAGACGAAGGACACGCCTC\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{DNA sequencing and phylogenetic analysis}

The PCR products of the fiber 2 gene of PiAdV-A and capsid gene of PiCV
were sequenced with PCR primers by Microsynth (Switzerland). Sequencing
was performed using the Sanger dideoxy sequencing method with an ABI
3730XL automatic sequencing apparatus and a Big Dye Terminator Cycle
Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) in
each direction (forward and reverse) for each isolate to ensure the
validity of the data. Partial nucleotide sequences of the fiber 2 gene
and capsid gene were aligned and edited using MEGA X (Molecular
Evolutionary Genetics Analysis, version 10.1.8) software (The Biodesign
Institute, Arizona, USA). The sequence data were then compared to
complete genome reference sequences of aviadenovirus and circovirus
species available from the National Center for Biotechnology Information
(NCBI) and their phylogenetic relationships were investigated.
Phylogenetic trees were built using the neighbor-joining method with the
Hasegawa-Kishino-Yano and Kimura-2 parameter substitution model and
gamma distribution using the maximum-likelihood statistical method in
MEGA X, respectively (Hasegawa et al., 1985; Kimura, 1980; Kumar et al.,
2018).

\textbf{Results}

\textbf{Necropsy and histopathological findings}

Macroscopically,the pigeons were dehydrated and emaciated with pectoral
muscle atrophy while postmortal watery vomite was detected in two
pigeons (Figure 1). The most prominent gross findings were severe liver
necroses and hemorrhages (n=3). Enlargement and fragility of the
affected livers showed association with pale grey-yellow necrotic areas
(Figure 1), and widespread hemorrages. The proventriculus and stomach
mucosae were thickened and oedematous in appearance, and in some
instances they were coated with a sticky mucous. The kidneys were also
enlarged and contained multifocal grey foci that were detectable on the
serosal surfaces.

\textbf{TABLE 2.} Semiquantitative scoring of histopathologic and
Immunoperoxidase test findings; (-) None, (+) Mild, (++) Moderate, (+++)
Severe

IPT: Immunoperoxidase test\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Case\#}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Severity of Histopathologic Findings}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Severity of Histopathologic Findings}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Severity of Histopathologic Findings}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{IPT} \textbf{(FAdV-4)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Liver}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Kidneys}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Lungs}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+++\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{3}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{5}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
++\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
+++\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURE 1.} a) Vomiting characterized by mucous and watery
content; shrunken eye indicating dehydration and poor condition of the
diseased pigeon. b) Widespread discoloration indicates severe necroses
(white arrows) and hemorrhages on the liver.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Histopathologically, multifocal coagulation necroses characterized by
acellular homogenous eosinophilic appearance and mononuclear cell
infiltrations surrounded necrotic cell debris. In severe cases,
widespread hemorrhages showed association with hepatic necroses.
Hepatocytes mostly showed parenchymatous degeneration, cytoplasmic
swelling and they had clear, hyperchromatic nuclei. In some nuclei of
the hepatocytes, intranuclear basophilic viral inclusions were observed
(Figure 2). In kidneys, multifocally localized interstitial lymphocytes
and plasma cell infiltrations in the cortex, tubular hydropic
degenerations were detected. In two cases, interstitial pnemonia,
alveolar epithelia hyperplasia, and bronch/iolitis were seen. In
proventriculus mucosae, numerous heterophilic leucocyte infiltrations
were present in the submucosae. FAdV-4 antigen specific immunoreactions
were observed in degenerated hepatocytes and in the necrotic areas,
kidney tubul epithelia, interstitital inflammatory cells and in
urethelial epithelia, alveolar and bronchial epithelia in the lungs.

\textbf{FIGURE 2.} a) Severe coagulation necrosis, homogeneous
eosinophilic appearance and some structural remnants of hepatic cords
were visible but the hepatocytes were completely necrotic (arrows). At
the periphery, focal lymphocyte and macrophage accumulations (arrow
heads) were seen in the liver, H\&E, Bar= 300 \selectlanguage{greek}μ\selectlanguage{english}m.

b) Clear nucleus due to characteristic circular margination of the
chromatin in the hepatocytes. Some degenerated hepatocytes had
basophilic intranuclear viral inclusions (arrow heads and inset). Liver,
Hematoxylin-eosin, Bar= 45 \selectlanguage{greek}μ\selectlanguage{english}m.

c) Hydropic degeneration and necrosis of the cortical tubule epithelia,
and in healthy tubular epithelia nuclei seemed clearbut contained
numerous intranuclear basophilic inclusions. Hematoxylin-eosin, Bar= 90
\selectlanguage{greek}μ\selectlanguage{english}m.

d) FAdV-4 antigen immunopositive reactions, red dots, in the necrotic
areas and degenerated hepatocytes (arrowheads). Avidin biotin complex
immunoperoxidase test, FAdV-4 antibody, Gill's hematoxylin
counterstaining, Bar= 180 \selectlanguage{greek}μ\selectlanguage{english}m.

e) Immunoperoxidase positive reactions in the degenerative and necrotic
tubule epithelia of the kidney. Avidin biotin complex immunoperoxidase
test, FAdV-4 antibody, Gill's hematoxylin counterstaining, Bar= 180 \selectlanguage{greek}μ\selectlanguage{english}m.

f) Strong viral antigen immunopositivity on the bronchiolar epithelia
(arrowheads= and alveolar walls. Avidin biotin complex immunoperoxidase
test, FAdV-4 antibody, Gill's hematoxylin counterstaining, Bar= 300 \selectlanguage{greek}μ\selectlanguage{english}m.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Virus isolation}

The supernatants of PiAdV-A and PiCV positive samples from a pigeon
flock were inoculated into SPF embryonated chicken eggs. All samples
were passaged 5 times in SPF chicken eggs. It was not possible to
propogate PiAdV-A virus from the PiAdV-A PCR positive sample in eggs,
but PiCV virus was propagated from the PiCV PCR positive sample in the
SPF embryonated chicken eggs and caused extensive damage to chicken
embryos regardless of the inoculation route (chorioallantoic cavity and
yolk sac) (Figure.3). PiAdV-A and PiCV positive samples did not lead to
any propogation of virus in primary CEFCs, but both samples caused
extensive rounding, clumping and detatchment of cells in the CEKC
cultures 48-72 hours post-inoculation (Figure.~4).

\textbf{FIGURE 3.} a-b-c) Extensive malformation and hemorrhaging was
caused in chicken embryos by PiCV (TR/SKPC20) and compared with normal
chicken embryo (d). Livers with multiple petechial haemorrhages (black
arrows)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{FIGURE 4.} CPE formed by PiAdV-A field isolate (TR/SKPA20) and
PiCV field isolate (TR/SKPC20) in CEKC cultures. a) Healthy,
mock-infected CEKCs. b-c) PiAdV-A and PiCV infected cultures displaying
CPE, respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{PCR}

The PiAdV-A fiber-2 and PiCV capsid genes were partially amplified by
PCR using the F1-s/F2-as and PiCV2-s/PiCV2-as primers, respectively. The
expected 967-bp and 325-bp specific DNA fragments were amplified
(Figure. 3). The specific PCR products were identified as PiAdV-A and PiCV
by sequencing and BLAST analysis.

\textbf{FIGURE 5.} a) Lane M, 100 bp ladder marker; lane 1, TR/SKPA20
(liver); lane2, TR/SKPA20 (kidney) ;lane 3, TR/SKPA20 (spleen); lane 4,
TR/SKPA20 (gut and pancreas); lane 5, negative control (Raue et al.,
2002) (967 bp.). Lane 6, TR/SKPC20 (liver); lane7, TR/SKPC20 (kidney) ;
lane 8, TR/SKPC20 (spleen); lane 9, TR/SKPC20 (gut and pancreas); lane
10, negative control (Freick et al., 2008) (325 bp.).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{DNA sequencing and phylogenetic analysis}

The amplified fiber-2 gene and capsid gene fragments were sequenced
directly. The PiAdV-A isolate was named ''TR/SKPA20'' and the PiCV
isolate was named ''TR/SKPC20''. A BLAST search
(https://blast.ncbi.nlm.nih.gov) was used to compare these sequences
with the complete genomes of aviadenovirus and circovirus species. The
newly detected field isolate TR/SKPA20 was found to be related with only
one PiAdV-A `'IDA4'' strain, with 99.03\% similarity at the amino acid
level. The TR/SKPC20 field isolate was found to be closely related with
strains previously isolated in Poland (MK994767, KC691682), Hungary
(JF330097) and Taiwan (GO844278) (Figures. 6 and 7). The nucleotide
sequences of the field isolates presented here (TR/SKPA20 and TR/SKPC20)
have been submitted to the NCBI-GenBank database under the following
accession numbers: MN985817, MT130538.

\textbf{FIGURE 6.} Phylogenetic tree based on the amino acid sequence of
the PiCV capsid gene. The tree shows circovirus species which affect
fowl. The tree shows the names and GenBank accession numbers of each
isolate. The PiCV isolate from this study is indicated by a red dot.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{FIGURE 7.} Phylogenetic tree based on the amino acid sequence of
the PiAdV fiber-2 gene. The tree shows aviadenovirus species which
affect fowl. The tree shows the names and GenBank accession number of
each isolate. The PiAdV-A isolate from this study is indicated by a red
dot.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{DISCUSSION}

PiAdVs are classified into two species pigeon aviadenovirus A and pigeon
aviadenovirus B within the genus aviadenovirus\emph{.} (De Herdt et al.,
1995). PiCV is the only circovirus that only affects pigeons. In the
present study, we detected a PiCV, PiAdV co-infection, in a pigeon flock
in Kirikkale province, Turkey. The genetic material of both PiAdV-A and
PiCV were found in the same flock of young pigeons under six months old
with severe clinical signs (crop vomiting, watery diarrhea, anorexia and
sudden death) that are typically associated with Young Pigeon Disease
Syndrome (YPDS).

Primers based on the PiAdV-A fiber-2 gene (Raue at al., 2002) and PiCV
capsid gene (Freick et al., 2008), were used for the detection and
amplification of the isolates. This was then sequenced and phylogenetic
analyses were performed.

Several FAdV serotypes 2, 4, 5, 6, 8, 10 and 12 have been previously
isolated from diseased and healthy pigeons (Goryo et al., 1988; Hess at
al., 1998a; Hess et al., 1998b; McFerran et al., 1976a). In this present
study, other viruses such as fowl aviadenovirus (FAdV 1-7, -8a and -8b,
-9-11) and PiAdV-B and PiHV were all screened for but not detected
(Table. 1). In a recent study, adenoviral poultry diseases, especially
IBH were reported for the first time in broiler and broiler-breeder
flocks in Turkey (Sahindokuyucu et al., 2020). PiAdV-A adenovirosis is
sometimes called IBH due to the intranuclear basophilic bodies seen by
histopathology (McFerran et al., 1976b; Abadie et al., 2001) and it is
possible that misdiagnosis has previously occured. This study
illustrates the importance of molecular diagnosis of infection.

The sequence analysis carried out showed that one of the isolates
belonged to the PiAdV-A species. The isolate matched with a strain
called \emph{''} Pigeon adenovirus 1 complete genome, strain IDA4'' in
the NCBI-Genbank database. The percent identity at the amino acid level
between our sample and IDA4 was 99.03\%. To the best of our knowledge,
this study reports the first identification and isolation of PiAdV-A in
a pigeon flock in Turkey.

In this study both PiAdV-A and PiCV positive samples were isolated onto
CEK cell cultures. An attempt was also made to use primary CEF cell
cultures to isolate positive PiAdV and PiCV samples, but it was
unsuccessful. PiAdV serotypes are generally quite challenging to grow in
cell culture (Duchatel et al., 2000; Marlier \& Vindevogel, 2006;
Vereecken et al., 1998). The isolation of pigeon avidenovirus strains
have been achieved in primary chicken embryo kidney cells (McFerran et
al., 1976a) and primary chicken embryo liver cells (Takase et al., 1990;
Vereecken et al., 1998). However, even with these techniques isolation
does not always succeed. The isolates reported in this study can now be
used for further molecular and virulence studies.

Isolation of PiAdV was only successful in CEKC cultures. PCR of the
fiber-2 gene was used for identification. Although little is understood
about the fiber-2 protein of PiAdV, the fiber protein of FAdV is known
to play a crucial role in viral infection and pathogenesis (Lu et al.,
2019; Pallister et al., 1996; Zhang et al. 2018). It is responsible for
initial attachment of the virus to cellular receptor, which is followed
by binding to cell surface integrins (Wickham et al., 1993). It is
likely that the PiAdV fiber-2 protein plays a similar role. When the
region of the fiber-2 gene that was amplified and sequenced was compared
to IDA4 reference strain, there were 6 amino acid differences present
(Table 3). The consequence of these changes is currently unclear and an
area for future investigations.

\textbf{TABLE 3.} Location of amino acid differences between the
TR/SKPA20 (MN985817) and IDA4 (MT130538) strains in the section of the
Fiber-2 protein that was amplified and compared.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Accession} \textbf{No}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\textbf{Sequence of Amino Acids}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{40}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{75}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{91}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{142}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{151}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{162}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
MN985817\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
S\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
D\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
S\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
R\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
A\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
MT130538\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
G\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
N\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
T\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Q\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
E\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

From this study it is clear that co-infections of PiAdV and PiCV is
possible and can lead to serious consequences for the birds infected.
The prevelance of both viruses in pigeon populations needs further
investigation to determine their pathogenic burden. Moreover, further
studies should focus on the whole genome sequence and the virulence of
the PiAdVs and PiCV in diseased pigeons as well as their potential to
jump species into economically important fowl. This study paves the way
for these future studies and offers the potential for a better
understanding of viral infections of birds in Turkey.

\textbf{Acknowledgement}

The using hyperimmun serum against FAdV-4 in the study, the authors
thank to Prof. Dr. Hafez Mohamed Hafez in Freie Universit\selectlanguage{ngerman}ät
Berlin-Germany.

\textbf{Conflict of Interest:} Authors declare that there is no
potential conflict of interest.

\textbf{Funding:} There has been no financial support for this work.

\textbf{Ethical statement:}No culling has been done for the sampling
process.

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11) Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018). MEGA
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12) Lu, H., Shao, H., Chen, H., Zhang, J., Wang, W., Li, T., Xie, Q.,
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