Materials and Methods
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,
Kırıkkale 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.
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 μg/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 °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-μm 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 μg
of streptomycin (Sigma-Aldrich, USA) antibiotics per ml was used. The
cells were incubated at 37 °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% CO2 for 7 days. Once
strong CPE was observed, the cultures were freeze thawed,centrifuged at
4000×g for 10 minutes, and the supernatants were stored at -20 °C
until DNA extraction was performed.
Pathologic Examinations