Abstract
Background: H9N2 influenza virus has a great potential for causing severe economic loss in ducks especially in backyard ducks. Objective: The current study was conducted to isolate H9N2 influenza virus from backyard ducks and preparation of antiserum against isolated H9N2 influenza virus in rabbits and further evaluation by neutralization test. Methods: Samples from liver, spleen, brain and lung were collected from clinically infected ducks during 2014 and 2015. The viruses were isolated and propagated by inoculation of 10 days old embryonated chicken eggs via the allantoic sac. The allantoic fluid was harvested and tested for the titration of the HA activity using chicken erythrocytes. Avian influenza virus type A antigen was detected by flu detect strip test. The RNA was extracted from HA-positive allantoic fluid and identified by RT-PCR using specific primers for H9N2 virus. Antiserum against H9N2 virus was prepared in rabbits. The produced antibody was tested and titrated by HI test against H9N2 isolates from ducks and chickens as well as field samples from ducks and H5N1 isolated from chickens. Neutralization test was also done for evaluation H9N2 antibody protective titer. Results: Ten samples from ducks were HA positive with titer values of 2- 5 to 2- 10. The HA titer of H9N2 isolates from chickens ranged from 2- 4 to 2- 11. Also, 16 field samples from ducks were HA positive with titer values of 2- 3 to 2- 7 and the HA titer of H5N1 isolates from chickens ranged from 2- 7 to 2- 12. The results of RT-PCR showed that the 10 samples from ducks were positive for H9N2 HA gene. The HI results showed complete inhibition of H9N2 isolates from ducks and chickens. HI test for field samples from ducks showed complete inhibition of 16 field samples. On the contrary, HI test for H5N1 isolates from chickens showed no inhibition of 13 isolates using anti-rabbit H9N2 specific antiserum. The neutralization index using H9N2 virus was 2 -5. Conclusion: The presence of LPAI H9N2 may add another risk factor to the poultry industry in Egypt together with the endemicity of HPAI H5N1, especially that backyard ducks and chickens are reared in direct contact allowing easy virus transmission and adds more stress to the poultry populations.

Key words: Avian influenza; H9N2; Backyard ducks; H5N1; HI.