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