الفهرس 
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Abstract
The aim of this work are:
propagation and isolation of NzdV from supected samples collected from infected chickens using the embryonating chicken eggs
biological and serological detection and identification of different loacl isolated on the basis of haemagglutination (HA) property and haemagglutination inhabition test using NDV specific antiserum.Newcastle disease (ND) is a highly contagious disease of birds and one of
the major causes of economic losses in the poultry industry. Despite routine
vaccination programs, several outbreaks have occurred in Egypt in the last 2
years and remain a constant threat to commercial poultry. Hence, the present
study was aimed to characterize NDV isolates obtained from clinical cases in
various locations of South Valley Provinces (Sohag, Qena and Luxor) during the
period of 2011 - 2012.This study was carried out on 126 samples (liver, proventriculus,
intestines, brain, spleen, lung, trachea and cecal tonsils), were collected from
freshly dead and diseased live commercial broiler and backyard chicken flocks
showing respiratory and/or nervous manifestations.Samples were prepared and inoculated into embryonating chicken eggs of
9 -11 days old via allantoic cavity for virus propagation. Inoculated eggs were
observed for 5-7 days post-inoculation (PI) then the allantoic fluid was collected
and tested for haemagglutination (HA) character by rapid and slow HA tests to
confirm the virus growth. The positive samples for HA were identified by
haemagglutination inhibition (HI) test using NDV specific antiserum to confirm
the presence of the virus. The obtained results indicated that 45 (35.7 %)
samples were NDV positive.Molecular confirmation of the virus was performed by one step reverse
transcriptase polymerase chain reaction (RT-PCR) after the RNA extraction
from the allantoic fluids of 45 ND positive samples. RT-PCR was conducted
using specific primers to amplify 695 base pair (bp) fragment targeting region at
the end of matrix (M) gene and Carboxyl terminus (C- terminus) of Fusion (F)
gene, including cleavage site. RT-PCR results showed that all ND positive
samples produced the specific PCR product at 695bp.The pathotyping of the isolated virus was carried out biologically by
intracerebral pathogenicity index (ICPI) in 1-day-old chicks. The clinical
manifestations and post mortem picture of NDV were observed on the
inoculated chicks. The results revealed that The ICPI ranged from 1.4- 2 index
which referred that all NDV isolates were ranged from moderate to velogenic
typesIn order to understand the evolution of NDV in Upper Egypt and confirm
the pathotyping, 8 out of 45 samples were selected from different regions for
DNA sequencing and phylogenetic analysis. The post sequencing analysis of the
cleavage site of the fusion gene showed that all the isolates have multiple basic
amino acids at the C- terminus of the F2 protein and phenylalanine at residues
117 of the F1 protein, with cleavage site motif 112R-R-Q-K-R-F117, classifying
them as velogenic which correlated to the results of ICPIThe obtained nucleotide sequences were aligned in compare with the
LaSota and Egyptian strains previously isolated and the results indicated that all
(8 strains) were 22 – 20% different from LaSota (vaccine strain) and 1-2 % from
Egyptian one (2011), 16-22 % from other Egyptian one (2006, 2010). However,
the 8 isolated were clustered into 6 clades based on nucleotide differences as
well as away from the LaSota strain. The predicted amino acid sequences of
these isolates were 21% different from Lasota also; some strains had a unique
amino acid in different positionsThe nucleotide sequences were then compared with available sequences
deposit in the Genebank and used to construct the phylogenetic tree.
Phylogenetic analysis indicated that the current isolated strains belonged to
genotype VII which is the predominant genotype responsible for most ND
outbreaks since the end of the last century and it is currently circulating in
Egypt, causing very high mortalities in chicken flocks. The obtained isolates
were genetically distinct and phylogenetically distant from the LaSota strainEgyptian isolates (2006, 2010) and Israel (2003) while grouped with ND strains
isolated from Egypt (2012) and Israel (2011).
Far phylogenetic distance between vaccines and current obtained virulent
strains which indicate to the occurrence of new ND stains different from the
vaccine strains and other Egyptian strains isolated from chicken in 2006. These
strains developed from the accumulation of point mutations in different sites of
the F gene as well as this evolution may be a major reason for the continuous
outbreaks of ND in Egypt.from the previous results we can conclude that:
1. Newcastle disease is still a major problem for the commercial and
backyard poultry.
2. Presence of new NDV strains which differ from the vaccinal strains used
for vaccination programs in Egypt so, studies on comparison of antigenic
structure of the newly pathogenic NDV strains isolated from different
outbreaks with that of the viruses used for the vaccination are of crucial
importance to understand the causes of frequently occurring vaccine
failure.
3. Poultry farmers need to be educated to adopt the biosecurity programs
4. A timely reporting mechanism should be developed that can be used to
control an ND epidemic in the country