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).
Results
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.
TABLE 2. Semiquantitative scoring of histopathologic and
Immunoperoxidase test findings; (-) None, (+) Mild, (++) Moderate, (+++)
Severe
IPT: Immunoperoxidase test