الفهرس 
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Abstract
Infectious bronchitis virus had emerged as the cause of severe disease and high mortality in chickens in Egypt and there is no of any vaccine live or inactivated can get sufficient immunity nor cross-protection in flocks experiencing clinical disease due to viral mutation or recombination in the field. The veterinary authorities adopted vaccination as one of the important tools to control the spreading of the disease. So, isolation and identification of IB field viruses is necessary to perform potent local vaccine can face and protect against the circulating IBV. So, the current study was designed to cover the following: 1. Isolation of infectious bronchitis virus from clinically suspected live or freshly dead birds. 2. Adaptation and propagation of suspected IBV samples on embryonated chicken eggs (ECE). 3. Molecular identification and characterization of IB virus using modern molecular techniques. 4. Estimation of the most common isolate circulating in field can be used for preparation of potent local inactivated oil emulsion whole IBV antigen vaccine can compete devastating field virus strain threaten poultry industry in Egypt. 5. Preparing an experimental batch of inactivated vaccine from the selected isolate and completely identified IB virus field local strain. 6. Evaluation of the prepared vaccine using serum neutralization test (SNT) and challenge test (cilliostasis and re-isolation tests). The tracheal swaps and tissue homogenates from 36 Egyptian isolate that were collected were inoculated in 9-11 days old SPF emberyonated chicken eggs (ECE) for isolation of IBV, the specific lesion of infectious bronchitis virus (curling and dwarfing) was appear after third passage, some isolates appear after 4th to 5th passage . The allantoic fluids of all isolated samples were shown strong HA positive when tested by slide HA test. Only one sample was HA negative and 8 samples give faint slide HA. Concerning the Molecular identification and characterization of IB virus using modern molecular techniques, it was also noticed that the nine selected samples showed positive IBV when analyzed by Real time RT-PCR targeting the highly conserved nucleocapsid (N) gene of IBV isolates. Only Four isolates (IBV3, IBV4, IBV8 and IBV9) that gave positive IBV directly analyzed by one step RT-PCR targeting 1200 bp of spike gene (S1) with the same set of primer. The extracted RNA of the four isolates of IBV is directly sequenced targeting the hyper variable region 3 (HVR3) of the amplified part of S1 gene and then the obtained sequences were assembled using BIOEDIT software and blasted to the gene bank and the percent of identity of nucleotides were calculated by Mega Align (DNASTAR software) and the phylogenetic analysis was done using MEGA 5 software which revealed that there are 2 types of variants circulating in Egyptian field which they are variant 2 and Q1 variant, but variant 2 strains is the most dominant in the field rather than other variant strains. Regarding to estimation of the most common isolate circulating in field can be used for preparation of local inactivated oil emulsion whole IBV antigen vaccine can compete devastating field virus strain threaten poultry industry in Egypt we observe that IBV9 (IBV-S1/VSVRI_G9/Egy 013) is the best isolate for vaccine preparation due to its purity (free from extraneous viruses such as Newcastle, Influenza H5, H9, H7 and Infectious bursal disease virus (IBDV) by RT-PCR and also gave high percent of identity to the local Egyptian isolates. The isolate then is directly propagated in SPF emberyonated chicken egg (ECE), titrated and then inactivated using formalin analar 37% (1:1000) then prepared as water in oil form using Montanoid ISA 70 for preparation of inactivated oil emulsion vaccine. Evaluation of the prepared vaccine was carried out after vaccination of a group of 50 SPF chicks vaccinated with inactivated vaccine and the other group of 20 SPF chicks was kept as non-vaccinated control at 14 days old, blood samples were collected up to the six weeks post vaccination, serum was separated and subjected for evaluation of the neutralizing antibody using β method of serum neutralization test (SNT), using the homologous virus (that used for vaccine preparation) as SNT revealed a titers of (1.5, 3, 3.58, 4.78, 5 and 6.17) log2 from the first week to the 6th week in descending order. The cillary protection percent of the vaccinated group by inactivated oil emulsion IBV vaccine after challenge at 28 day after vaccination resulted in 68.72% protection percent. The re-isolation in embryonating chicken eggs revealed that the percent of the virus presence in trachea in the vaccinated challenged group was (33.33%) and in kidney was (20%), indicating that the relative protection percent of the vaccine was (66.67%) for trachea and (80%) for kidney.