جداسازی و شناسایی مولکولی ویروس های برونشیت عفونی طیور در مرغداریهای صنعتی ایران

Objective: Detection of infectious bronchitis viruses by RT-PCR/RFLPs in poultry farms of Iran.
Design: Longitudinal study from 1997 to 2003.
Samples: Tracheas, lungs and kidneys of suspected flocks to respiratory diseases.
Procedure: From 1997-2003, tissues samples including lung, trachea and kidney, had been prepared from broiler and layer flocks were submitted to avian virology laboratory of poultry diseases section in order to isolate respiratory viral diseases viruses. A total number of 50 infectious bronchitis viruses (IBV) were isolated in embryonated chicken eggs. The infected embryos displayed stunting, urate deposition in the mesonephros or death after three passages. Twelve isolates that had showed typical signs in embryos, were selected for molecular identification. Viral RNA was extracted by RNXTM plus (CinnaGen Co.) using chloroform / Isoamyl alcohol, Isopropanol, Ethanol and DEPC water. cDNA was prepared from extracted RNA with RT enzyme, RH primer, RT buffer, dNTP and Rnase inhibitor. For PCR reaction, buffer PCR lOX, MgCI2, Sloligo5’ & 3’ primer, ampli Taq DNA polymerase and dNTP were added to cDNA and then PCR was conducted in thermal cycler. The PCR products were analyzed on a 1% agarous gel and Ethidium Bromide staining. The Si glycoprotein genes of IBV strains appeared to be above 1600 bp in size. PCR products were digested by HaeIII, Ecorl and Hindill, according to the manufacture’s
recommendation.
Results: Base on RFLPs patterns and comparison with RFLP references patterns (793/B, D274, M41, H120), 8 of 12 strains showed 793/B pattern and the rests (4 of 12) showed Mass pattern in RFLPs.
Clinical implications: Regarding to low homology and weak cross protection between 793/B serotype and vaccinal strain (Massachusetts), prevention and a controled strategy against lB should be altered.
J. Fac. Vet. Me’L Univ. Tehran. 59, 3: 259-264, 2004.