الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
In the present study, a total of 72 samples including 32 bursa of Fabricius, 10 thymus, 10 spleen, 10 kidney and 10 liver were collected from 32 chicken farms at Dakahlia Governorate, Egypt during the period from March 2014 to December 2015. Out of these 32 farms, 27 farms with a history of previous vaccination against Infectious Bursal Disease (IBD) and 5 non- vaccinated farms. Each sample was pooled randomly from three to five birds (14 to 44 days old). Trials for isolation of the suspected virus from the collected samples were carried out via chorioallantoic membranes (CAMs) of 9 days embryonated chicken eggs (ECEs), collected from hens free from infectious bursal disease virus (IBDV) antibodies. Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard IBDV. IBDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Also our work was directed to study molecular characterization of a field isolate of IBDV circulating at Dakahlia Governorate depending on detection of IBDV nucleic acid by reverse-transcriptase - polymerase chain reaction (RT- PCR) in inoculated CAMs (after 3rd passage) using primers were designed to amplify 620 bp fragment of VP-2 encoding gene of IBDV and nucleotide and amino acid sequence analysis of amplified region of VP2 gene that containing major conformational and neutralizing epitopes and responsible for virulence and cellular tropism. The results revealed that out of 72 collected samples, 27 samples (84.37 %) bursa of Fabricius, 9 (90 %) samples from thymus, spleen and liver and 7(70%) samples from kidney gave positive results of virus isolation appeared in form of stunted embryos, mottled and ecchymotic hemorrhages or swollen greenish pale liver as well as pale heart and congested and thickened CAMs after 3rd passage in ECEs. AGPT and IFAT were used for identification of virus in prepared field samples and in 3rd egg passaged samples and the results indicate sensitivity of AGPT and IFAT in virus detection from field samples is equal while IFAT is superior over AGPT in virus detection after 3rd passage in ECEs. RT- PCR can be used for detection of IBDV in CAMs inoculated with virus (after 3rd passage) and results revealed the presence of the amplified products at the correct expected size of the VP2 encoding gene (620 bp). Results of genetic analysis of the nucleotide sequence of amplified hypervariable region of VP2 gene revealed that the four isolates in this study (IBD- S13, IBD- S14, IBD- S16, IBD-S20) showed close relationships between the previously isolated Egyptian very virulent IBDV strains but they were grouped at a far from vaccinal strains although these isolates collected from vaccinated flocks while one isolates (IBD- S18) was grouped with CEVAC IBD L vaccine strains. Analysis of deduced amino acids sequence of hypervariable region of VP2 protein by protean analysis (DNASTAR,Lasergene) confirmed the results of nucleotides sequances. The conclusion, the results of nucleotide and amino acid sequence analysis confirmed the continuous evolution and mutation of IBDV in Egypt in spite of vaccination.