Fifty five tissue samples were collected aseptically from 5 flocks suspected to be naturally infected with NDV in EL- Ismalia, Al-sharkia, El- Gharbia, kafr-Elshiekh & El-Geza and subjected to trials of virus isolation. Nine field samples from 2 flocks (Kafr-Elshiekh and Giza) were positive for virus isolation. Inoculation of SPF chicken eggs induced death of the embryo within 48-72 hours post inoculation. HA test applied on the obtained allantoic fluids showed positive results suggesting that inoculated samples contained NDV. It was noticed that highest infectivity titer of the virus was 9.5 log¹⁰ EID₅₀/ml by the 5^th passage in SPF chicken eggs. Cell culture passage of the obtained virus isolate from the allantoic fluid through ten consecutive passages in BHK cell line. the CPE developed at the 3^rd passage within 24-48 hours post cell infection where polykaryocytosis, cell syncitia, rounding of cells. Tissue cultured fluids (TCF) were found to be direct HA possitive. The virus titer was 3 log10 TCID₅₀ /ml at the 3^rd passage and reach to 7 log10 TCID₅₀ /ml by the 6^th passage. At the 9^th passage, the virus titer was reached to 8.5 log10 TCID₅₀ /ml. Identification of the obtained virus isolate was confirmed by VNT using specific anti-NDV in BHK cell.
The isolate of NDV with HA-positive allantoic fluids of El-Giza flockes were virulent NDVand the F gene give expected size of 724 bp. The isolate of this study also compared against the reference and vaccinal strains from gene bank and the results revealed that the isolate NDV/chicken/Egypt/Giza/2015 was velogenic type resembling the genotype VIId strains. F gene analysis of the NDV/chicken/Egypt/Giza/2015 revealed that the most nucleotides identity from gene bank with this isolate were NDV/Chicken/GC/IS/2010/1224 and NDV/buzzard/Israel/714/2011 with a nucleotide identity of 92.7%. It was also closely related to Chicken/China/SDWF07/2011; Chicken/China/Shandong/01/2012 and NDV/Turkey/Israel/111/2011 with a nucleotide identity of 92.5%. Otherwise nucleotides identity between the selected NDV field isolate in this study and NDV/Lasota was 72.4%. Phylogenetic analysis of NDV/chicken/Egypt/Giza/2015 with other reference and vaccinal strains of NDV revealed that this isolate was related to NDV/B7/RLQP/CH/EG/12 and present in the same group with NDV/F388/RLQP/CH/EG/14 and NDV/F460/RLQP/CH/EG/13.
Regarding evaluation of the prepared experimental vaccines, it was found that the NDV infected tissue culture fluid, were completely inactivated with 3% BEI at 30 °C for 18 hrs post treatments showing no pathological changes in the ECE and no CPE in the BHK cell culture. Humoral immune response of chickens to the prepared vaccines showed that vaccinated chicks with NDV-Montanide ISA 70 adjuvanted vaccine exhibited antibody titer of 8.3 log2 three weeks post vaccination recording the higher titer of (10.6 log2) at the 6^th week post vaccination with and 3^rd week post challenge test. The high antibodies values were persist till the 12^th weeks post NDV-Montanide ISA 70 vaccination followed by gradual decrease till the end of the experiment (24^th weeks). On the other hand, vaccination of chicks with inactivated NDV isolate without adjuvant failed to induce protective HI antibodies (3 log2) all over the experiment. Challenge test showed that chickens vaccinated with ISA-70 adjuvant vaccine showed no sign of disease and no mortalities while the antigen without adjuvant could not provide any protective efficiency to SPF chickens.