DISCUSSION
PiAdVs are classified into two species pigeon aviadenovirus A and pigeon aviadenovirus B within the genus aviadenovirus. (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 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.
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.