الفهرس 
Introduction and Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
The present study was performed in order to identify a non-synonymous dimorphism G/A in exon 14 of chicken Mx gene (this position correspond to nucleotide number 2032 in Mx cDNA) which is responsible for altering the Mx protein’s antiviral activity. Chicken posses the A allele at this nucleotide position have been reported to have higher antiviral activity than those posses the G allele. Therefore the aim of the present study was to identify Egyptian chickens that carry the Mx gene resistant allele with the high frequencies using the PCR-RFLP genotyping and sequencing techniques. The obtained data could help in improving poultry breeding in Egypt by producing infectious disease-resistant chickens.
For achieving this purpose blood samples were collected on EDTA as anticoagulant matter, from 246 Egyptian chickens representing two pure breeds (Dandarawy and Fayoumi) and seven strains (El-Salam, Golden Montazah, Dokki-4, White egg commercial, red egg commercial, Gemmizah and Baladi).
Details of the breeds and strains as well as the sampled birds are as follows:
1) Dandarawy breed, 38 bird (8 males & 30 females) Fayoum Poultry Station, ”El Fayoum”.
2) Fayoumi breed, 38 bird (10 males & 28 females) Fayoum Poultry Station, ”El Fayoum”.
3) El-Salam strain, 36 bird (6 males & 30 females) Fayoum Poultry Station, ”El Fayoum”.
4) Golden Montazah strain, 32 bird (14 males & 18 females) Fayoum Poultry Station, ”El Fayoum”.
5) Dokki-4 strain, 20 birds (unknown type, since they are young aged birds). Sakha, Kafr El Sheikh, ”Animal Production Research Center”.
6) White egg commercial strain, 17 birds (all are females). El Noubaria farm, El Behera governate.
7) Red egg commercial strain, 19 birds (all are females). El Noubaria farm, El Behera governate.
8) Gemmizah strain, 25 birds (1 male & 24 female). Collected from 3 different farms, Gemizah poultry station, ”El Gharbia, Tkamoly farm, El Azab village, El Fayoum” and Sabahia Farm in Alexandria.
9) Baladi strain, 21 birds (11 males & 10 females). El Noubaria farm, El Behera governate.
DNA was extracted and purified from the collected blood samples. DNA concentration was measured at 260nm UV spectrophotometer and then diluted for the suitable concentration for performing Polymerase Chain Reaction (PCR). For amplification of the targeted region a specific primer set for avian Mx gene was used. For the detection of PCR success, some of the PCR products were run horizontally on agarose gel electrophoresis and stained with ethidium bromide, visualized on UV transilluminator, images and were captured using gel documentation system. For allele identifications, the products of the successful PCRs were subjected to be cut with the restriction enzyme HPY81, the cutting results were visualized on agarose gel electrophoresis after running horizontally and ethidium bromide staining. The images were captured for fragment size determination and subsequently allele and birds genotypes.
The results showed that amplification of the targeted region produced a band of interest around 299 bp lengths. The restriction enzyme cutting to the PCR products produced two bands, their size lengths are ~200+ 100 bp for the allele G, while the allele A was no cut. Results of the PCR-RFLP were confirmed after DNA sequencing for some known samples representing the three genotypes (AA& AG and GG).
The results also showed the presence of the substitution in all the Egyptian breeds and strains with different percentages except the Baladi strain which showed 100% of the AA genotype means that this strain is highly resistant to avian flu virus.
Regarding the frequency of the A allele which is linked with disease resistance, it was high in all the breeds and strains studied. The average frequency of this allele was: 0.667 while the frequency of the other allele (G) was 0.332. The highest frequency of this allele was 100% in the Baladi strain while its lowest frequency was 41.7% in El Salam strain and the frequency was higher than the half in the rest of the breeds and strains studied. The frequency of the G allele was generally low except El Salam strain which its frequency was 58.3%.
The increase of allele A frequency is reflected to an increase in the genotypes AA and AG when compared with the genotype GG. The average frequency of the AA genotype was 0.441; the lowest frequency was 0.105 in the commercial red egg producing strain. Regarding the AG genotype, the average frequency was 0.453, and the highest frequency of this genotype was 0.842 it was in commercial red egg producing strain, while this genotype AG was absent in Baladi strain.
The results of DNA sequencing for some samples confirm the PCR-RFLP methodology. The obtained sequences were successfully deposited at the International Gene Bank with accession numbers: KY584063, KY584064, KY584065, KY584066 and KY584067. In addition an insertion of TT bases was observed in the intronic region of some birds which is reflected in the PCR size length to be ranged from 299-301 bp.
In conclusion, applying the PCR-RFLP technique in the breeding programs to select chickens that carry the A allele with high frequencies could help in improving poultry breeding in Egypt by producing infectious disease-resistant chickens. It will also save the expenses paid in purchasing vaccines, drugs used for treating the infected birds. Moreover it will protect the humans near the infected birds from the infection transfer to them.