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