Download Assiut university researches Some Molecular Biological Studies on

Assiut university researches
Some Molecular Biological Studies on Field
Infectious Bursal Disease Virus isolates from
Broiler Chickens in Upper Egypt
‫ب عض ال درا سات ال جزي ئ ية ال ب يول وج ية ع لى‬
‫م عزوالت مرض ال جم بورو من دجاج ال ت سم ين ف ى‬
.‫ص ع يد م صر‬
Marwa Mohamed Safwat Mohamed
‫مروه محمد ص فوت محمد‬
Kamal El Zanaty,Bakheet Salem,Moemen Abd Elazeem Mohamed
‫ مؤمن ع بدال عظ يم محمد‬،‫ ب خ يت سال م‬،‫ك مال ال زن ات ى‬
Abstract:
In order to identify and characterize the agent of a suggestive
clinical case of IBD that affected several broiler farms in
Assiut province in year 2009, we carried out a combination of
classic and modern molecular methods. - A sharp precipitin
line was observed among 15 pooled local samples by using
AGID. -Inoculation of embryonated chicken eggs with
macerated bursa suspension that was positive for IBDV
resulted in embryo mortality and lesions (edema, distention of
abdominal region, cutaneous congestion, haemorrhages on
cerebral regions, mottled necrosis and echymotic
haemorrhages on the liver) which were compatible with those
caused by IBDV. - Regarding to genotypic identification The
RNA extracts from positive samples with AGID of pooled
bursal suspension were submitted to RT-PCR procedure to
amplify the hypervariable region of the VP2 gene (723bp). The amplicon which was obtained from these samples was
digested with the restriction enzyme sac1 that revealed a
pattern similar to that observed with very virulent IBDV strains
in all examined samples. - Furthermore, molecular
characterization was performed by direct sequencing of a
723-bp cDNA corresponding to the VP2variable domain of
the polyprotein gene, synthesized by a RT-PCR. - All
examined isolates demonstrated close relationship to very
virulent strains European and Asian strains of vv IBDVs. Our
examined strains presented 12 nucleotide changes that are
observed in vvIBDV strains. - Deduced sequence analysis of
the VP2 gene indicated that all the isolates from examined
viral encode amino acids A222, I242, I256, I294, S299 of
VP2, which are conserved among strains of very virulent
IBDV. - The serine rich heptapeptide sequence ’SWSASGS’
was conserved in all examined isolates. - Phylogenetic
analysis placed our vvIBDV strains in a clade of very virulent
reference strains from European, Japanese, and Hong Kong
and divided them into three subclusters 1A, 1Band1C that
indicates the different origin of our isolated strains. - Antigenic
index results suggest that our isolates could be antigenically
distinctive compared to vvIBDV reference strains so
confirmation of the presence of antigenic variation request
additional immunologic studies to elucidate the exact nature
of the viral epitopes. Conclusion The work of this study shows
that RT-PCR followed by RFLP analysis using sac1 for VP2
allowed an accurate classification of a causative agent of our
samples as very virulent IBDV. - All examined strains from
Assiut province were found to be very virulent IBDV strains. There are changes in nucleotide and aa sequences of the
major hyDROPhilic peaks of the isolated local strains that
may lead to a genetic mutations and changes in the shape of
neutralization epitope which resulting in failure of vaccination
and reemergence of the disease. - Based on the phylogenetic
analysis, our local strains are grouped in the clusters of very
virulent strains and subclusters into three subgroups related
to three different reference strains which indicating that our
strains have three origins (European, Japanese and Hong
Kong). - According to antigenic index we found three different
strains in the topography and nature of antigenic peaks as
potential antigenic sites which may lead to failure of
vaccination if infection happens with those strains. - These
viruses were not genetically similar and derived from the
vaccine strains which are used in poultry field. -from
nucleotide sequence analysis we found that Bursavac was
nearly similar to the isolated samples then CEVACIBDL,
Bursin plus and Nobilis Gumboro.