Abstract
Background: The role of free living birds in the spread and epidemiology of H5N1 Highly pathogenic avian influenza is often neglected. Cattle egret is one of the free-living birds that is often present near commercial chicken farms with a possibility of catching avian viral infections specially H5N1 AIV; this is augmented by broken biosecurity which is often in small and medium sized poultry production facilities. This study investigates the possible isolation of H5N1 AIV from cattle egrets in the spread and epidemiology and to try virus isolation if possible.
Methods: rapid test strip for detecting avian influenza antigen was used to test 15 tracheal pool samples collected from 15 commercial broiler flocks showing rapid increase in mortality, respiratory and enteric manifestation in Desouk and Qallin, Kafr-elshiekh Province, Egypt from 2014 to 2015. Another 60 swab samples (oropharyngeal as well as cloacal swabs) were collected from 30 cattle egrets in the vicinity of these flocks. Trials for isolation of AIV was conducted on collected samples via allantoic sac inoculation in 10 days old specific pathogen free (SPF) embryonated chicken eggs (ECEs) followed by hemagglutination (HA) and hemagglutination inhibition (HI) testing of allantoic fluids using specific polyclonal antisera against AIV H5 and H9 and confirmation by RT-PCR.
Results: The rapid field test revealed detection of AIV in one broiler flock (6.67 %). Isolation revealed 2/15 (13.3 %) AIV H5 isolates from broiler chicken flock samples, whereas 1/60 (1.67 %) AIV H5 isolate from cattle egret samples. Two positive AIV H5 (cattle egret isolate and one broiler chicken isolate) were selected for partial sequencing of HA. Phylogenetic analysis, that revealed clustering with Egyptian highly pathogenic avian influenza virus isolates from 2011-2015 in clade 2.2.1.2. Sequences were submitted to NCBI GenBank with accession numbers KT454807 for chicken H5 sequence and KT454808 for cattle egret H5 sequence.
Conclusion: Isolation of HPAIV H5 from cattle egret shed the light on the possible epidemiological role of this resident wild bird in transmission of AIV to susceptible chicken. Further detailed analysis is required to detect the complete antigenic/ genetic characteristics of cattle egret H5 influenza isolate.

Key words: broiler chickens, cattle egret, Egypt, Highly Pathogenic Avian influenza virus, HI, RT-PCR, Egypt.