Department of Microbiology, Collage of Veterinary Medicine, University of Diyal, Iraq

Abstract

This study was done at the college of Veterinary Medicine, University of Diyala, from September 2021 to April 2022 to assess the seroprevalence and molecular detection of avian influenza virus (AIV) H5N8 in layer chickens. All samples came from 8 commercial flocks of layer chickens. Clinical sings included, severe respiratory infection with enlargement of the head, neck, and prominent edema of the face, followed by excessive lacrimation, sinusitis, severe rales, and cyanosis in all parts of unfeather skin predominantly the wattles comb, and the legs. In this regard, ELISA was used as a serological test and RT-PCR as a molecular detection technique. Accordingly, (364) serum samples from 8 probable AIV (H5N8) infected layer farms were collected and subjected to ELISA test using commercial kit that specific for IgG antibodies to AIV H5N8. The results showed a significant increase of IgG antibodies in such serum samples at days 70 and 200 of age. According to the instruction manual of ELISA kit manufacturer and to mean titers of layers of present study, all farms are infected with AIVH5N8 strain. However, at the age of 200 days, 48 postmortem tissue samples including trachea, lung and liver of affected birds were collected from clinically and sub-clinically infected flocks from all farms. These samples were processed, RNA extracted and submitted to RT-PCR using specific primers for H5N8 strain. The results showed that 32 out of the 48 tissue samples (66.6%) tested positive for H5N8.The resulted Amplicon (320bp) was commercially sequenced and analyzed. The sequencing of the local AIV H5N8 revealed a 99% sequence identity with the reference sequences. The detected strain was registered in GenBank data (NCBI) under acc.number ON247929.1. Phylogenetic tree for locally detected virus in comparison to data from of NCBI was created and showed that the investigated S1(ON247929.1, AIV (A/laying hens/Iraq/(H5N8) segment 4 hemagglutinin (HA) gene, partial cds, local sample is closely related to reference isolates from the NCBI acc. no. of MW961428.1, MW961444.1, MW961476.1, MW961436.,1 and MW961484.1. These strains of the Influenza H5 virus have been deposited from Nigeria.