Molecular characterization of field isolates of Gumboro virus

Infectious bursal disease (IBD) is one of the major health problems causing significant economic losses in poultry industry in Egypt. During the period of 2016- 2018, bursal samples were collected from thirty commercial broiler chicken farms of different breeds of age range 20-30 days localized in three governorates (Menofia, Qalubia and Dakahlia) in Egypt. The suspected farms with history of IBD exhibited signs of dullness, diarrhea, dehydration, sudden onset of mortality in addition to edematous and/or hemorrhagic bursal tissues at necropsy. The processed bursal samples were inoculated in specific pathogen free embryonated chicken eggs (SPF-ECEs) for three blind passages via chorioallantoic-membrane (CAM) route in order to isolate the suspected virus. Agar gel precipitation test (AGPT) was applied on processed CAMs to confirm the result of virus isolation. One step reverse transcription-polymerase chain reaction (RT-PCR) was done on RNA extracted from processed CAMs for amplification of VP2 gene of IBDV. Fifteen out of thirty samples (50%) were positive and showed a band at 620 bp by electrophoreses. Five amplified samples named IBDV/Egypt/Menofia/16, IBDV/Egypt/Qalubia/17, IBDV/Egypt/Dakahlia/2/18, IBDV/Egypt/Dakahlia/3/18 and IBDV/Egypt/Dakahlia/4/18 were selected for sequencing then submitted to National Center for Biotechnology Information (NCBI) and are available in GenBank across individual accession numbers MK088025, MK088026, MK396669, MK396670 and MK396671; respectively. Nucleotide sequences analysis and phylogenetic tree proved that the examined IBDV/Egypt/Menofia/16, IBDV/Egypt/Dakahlia/2/18, IBDV/Egypt/Dakahlia/3/18 and IBDV/Egypt/Dakahlia/4/18 isolates were vvIBDV strains while the IBDV/Egypt/Qalubia/17 isolate was vvIBDV strain. Pathogenicity test for IBDV isolates revealed that the vvIBDV isolates caused higher morbidity and mortality rates than vvIBDV isolate accompanied with similar mean bursa to body weight (B:BW) ratio. In conclusion, the phylogenetic and molecular relationships successfully characterized four vvIBDV strains and one vvIBDV strain circulating in chicken broiler flocks in three Egyptian governorates. The presented results highlight the significance of the persistent monitoring of the IBDV field situation, in addition to application of further researches to establish effective vaccination strategies in order to hinder the IBDV infection.