الفهرس 
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Abstract
Avian influenza is a contagious disease of animals caused by viruses that normally infect only birds, and less commonly, pigs, this virus also may infect humans. This study was conducted on a total of 34 tracheal and lung samples collected from live and/or freshly dead birds suspected to be infected with avian influenza virus. Diseased birds were showing respiratory signs, such as ocular and nasal discharges, coughing and dyspnoea, swelling of the sinuses and/or head, apathy, reduced vocalisation, marked reduction in feed and water intake, cyanosis of the unfeathered skin, wattles and comb, incoordination and nervous signs and diarrhoea. Samples were collected from different farms in Egypt Governorates through the period from winter 2012 to winter 2013 for screening the presence of avian influenza virus subtype H5. These thirty four tracheal and lung samples were collected and prepared then subjected for virus isolation via allantoic cavity of 9 days old ECEs; three passages were done depending on s/c hemorrhage and deaths of embryos. Identification of isolated AIV virus from inoculated fertile eggs was carried out by HA, HI and indirect IF test. The obtained results were confirmed by PCR and gene sequencing. At the last years the RT-PCR has been used for diagnosis of avian influenza as it has many advantages such as the high sensitivity, specificity and the rapidity of diagnosis. The obtained results as follows: 1- The results demonstrated that out of 34 tracheal and lung samples collected from diseased chicken, deaths of inoculated ECEs showing congestion and hemorrhage occured in 14 samples (41.1%) after the first passage and 25 samples (73.5%) after the second passage. At the third passage there is increase in number of positive samples to reach 27 samples (79.4%) 2- Results of haemagglutination test on field samples were 24 positive samples while when done on isolates were 30 positive samples. Results of haemagglutination inhibition test on field samples were 23 positive samples while when done on isolates were 26 positive samples. 3- The results obtained by immunoflourescent for detection of AIV virus in 34 tissue sections were 26 samples (76.4%) gave positive results as yellowish green coloration distributed throughout tracheal and lung tissue. 4- Analysis of the PCR products obtained from amplification reaction of extracted nucleic acid from 10 field samples by agarose gel electrophoresis revealed positive amplification of 320 bp fragment for 8 samples and for positive control while there is no band for negative control and two samples. 5- Then we examine these ten field samples by RT-PCR and Amplification curves appear in all of them and for positive control while there is negative result for the negative control. Then a purified RT-PCR product was sequenced in the forward directions on an Applied Biosystems 3130 automated DNA Sequencer, a comparative analysis of sequences was performed using the CLUSTAL V multiple sequence alignment program. 6- Determine the difference in proteolytic cleavage site (pcs) in HPAI in Egyptian isolates from year 2006 till year 2014 by Sequencing of 229bp of HA gene of AI H5N1 isolates as amplified H5 of selected four samples with that in GenBank from year 2006 till year 2009 revealed substitution of three nucleotides A, G and A instead of T, A and G respectively at positions 42, 66, 203 in which protein formed serine (S) instead of Glycine (G) at position 16 and Lysine (K) instead of Arginine (R) at position 66. 7- The Phylogenetic tree pattern for the alignment of the sequenced selected AI viruses and references GenBank revealed that our isolates were grouped together and more related to A-Ostrich-Egypt-11139F-2011(H5N1) and Chicken-Egypt-1058sf-2010(H5N1) than A-Chicken-Egypt-0865-NLQP-2008(H5N1). 8- The percent nucleotide identity between our isolates and others H5 gene relative to the GenBank in 2010 and 2011 was ranged from 100% to 97.4% and with 2008 was 97.4% to 96.1% and divergence with 2006 was 97.4%.