This study aims to identify the presence of avian adenovirus (IBH) in broiler chickens from Diyala Governorate, Iraq, using both histopathological examination and conventional PCR as a molecular detection method by amplification of partial 1300 bp hexon gene. Additionally, the study also aimed to determine the genetic sequence of the adenovirus serotype (IBH) and investigate the genetic relatedness between local Iraqi strains from broiler chickens recorded and reference strains documented in the NCBI Gene bank. Post-mortem tissue samples were collected from broiler chickens across seven regions of Diyala Governorate, including Baqubah, Al-Khalis, Al-Muqdadiyah, Al-Wajhiyah, Kanaan, Al-Galibia, and Baladrose.
A total of 105 tissue samples (liver, spleen, and kidney) were taken for molecular detection and others 35 samples for histopathological examination were randomly collected from five commercial broiler chicken flocks across seven regions in Diyala Governorate, displaying signs of IBH virus infection. Results of the current study exhibit that the clinical signs of the infected birds are characterized by lethargic of the most birds in the flocks, huddling with ruffled feathers with mucoid yellowish droppings covering the vent of the birds. Whereas the post-mortem lesions observed in the broiler chickens infected with IBH involved, enlarged liver with a mottled appearance, pale to icteric (yellowish) of the skin, swollen and pale kidneys, whereas the skeletal muscle showed moderate to severe hemorrhages. Of the 105 samples, 41 (39%) tested positive for IBH using PCR, while 64 (60.9%) were negative. All positive samples demonstrated uniform band mobility on a 1.5% agarose gel, with the amplified DNA yielding the expected target amplicon size of 1300 bp. Using BLAST tool analysis, the obtained sequences were verified and compar with existing sequences in the GenBank database.
The obtain field sequence were subsequently submitted to the GenBank database and assigned accession numbers for 5 local strains Diyala 1 to Diyala 2 as follow (OR661247, OR661251, OR661248, OR661249 and OR661250). Concerning the 1300 bp amplicons of the IBH viruses, the NCBI BLASTn engine showed that nucleotide sequences of the five local hexon genes from FAdV displayed limited diversity and were closely related to each other, demonstrating 99.14–99.44% DNA sequence identity and 98.48–98.74% amino acid homology. By comparing the observed nucleic acid sequences of the investigated local sample with the retrieved nucleic acid sequences (GenBank acc. MK572860), several nucleic acid variants were identified when field virus sequences (5 local strains) were compared with the hexon gene of the reference strain.
The analysis revealed a total of 26 nucleic acid sequence were variants. The majority of these variants (18) were giving the silent mutations that did not affect on amino acid substitution. Further analysis showed that 8 variants cause an amino acid substitutions (missense mutations), with most of these changes occurring in the Diyala 1 strain. Eight significant variations were identified, leading to direct missense mutations causing eight amino acid substitutions. These include P.F>L (Phe>Leu 219) and P.R>C (Arg>Cys 221), found in all Diyala strains; P.T>S (Thr>Ser 306), P.E>G (Glu>Gly 307), and P.N>T (Asn>Thr 324), present only in Diyala 1 strain. Additionally, P.P>Q (Pro>Gln 310) was observed in Diyala 1, 4, and 5 strains; P.D>K (Asp>Lys 234) in Diyala 2 and 4; P.D>E (Asp>Glu 234) in Diyala 3 and 4; and P.P>S (Pro>Ser 246) in Diyala 2, 3, 4, and 5 strains. Phylogenetic algorithms were employed to generate a phylogenetic tree, and the neighbor-joining (NJ) tree. The determination of the genotypes of local FAdV and their phylogenetic relatedness was performed by analyzing their HEXON gene region.
Histopathological examinations of the liver in IBH-infected broilers revealed coagulative necrosis, basophilic intranuclear inclusion bodies in hepatocytes, fatty infiltration, and subacute periportal hepatitis. In the spleens vascular congestion, hemorrhage, and lymphocyte depletion were noticed.
The kidneys showed signs of nephritis, tubular degeneration and mild peritubular mononuclear infiltration. Glomeruli displayed mesangial hyperplasia and proliferative glomerulonephritis with thickening of Bowman’s capsule. Intranuclear inclusion bodies were rarely observed.This study is the first research regarding IBH and the phylogentic analysis in Diyala province from broiler chicken.
