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Molecular Characterization of Infectious Bursal Disease Virus Isolated from Broiler Chickens in Bangladesh

M. A. lslam
Department of Microbiology and Hygiene, BAU, Mymensingh-2202, Bangladesh.

M. M. Rahman
Department of Microbiology and Hygiene, BAU, Mymensingh-2202, Bangladesh.

M. R. Islam
Department of Pathology, BAU, Mymensingh-2202, Bangladesh.

M. M. Khatun
Department of Microbiology and Hygiene, BAU, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

An attempt was undertaken for molecular characterization of infectious bursal disease virus (IBDV) field isolates. In order to isolate the virus, bursae of thirty five dead chicken with clinical infectious bursal disease (IBD) were collected from Bangladesh Agricultural University (BAU), Mymensingh. Isolation of field strain of IBDV was carried out in chickens of 5-week-old. Five IBDV isolates were obtained from chicken inoculation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was preformed to detect IBDV isolates in the bursal tissues. RT-PCR couple with restriction enzyme (RE) analysis was carried out for molecular characterization of IBDV isolates to determine the pathotype. 677 bp fragments from IBDV genome segment A corresponding to the hyper variable domain of outer capsid protein VP₂ was amplified by RT-PCR. Two restriction endonuclease (REs), SspI and SacI were used for digestion of RT-PCR products. RT-PCR product was digested by SspI but not SacI. The presence of SspI restriction site in the 677 bp RT-PCR fragment indicated that IBDV isolates belonging to very virulent (vv) pathotype.

Keywords: Molecular characterization, Infectious bursal disease virus, Broiler chickens

Location: Department of Microbiology and Hygiene and Department of Pathology, Faculty of Veterinary Science, BAU, Mymensingh

Start Date: 00-05-2003

End Date: 00-04-2004

Program Area: Pest Management

Commodity: Chicken, Diseases

Objectives:

To characterize IBDV field isolate at molecular level.

Methodology:

The experiment was conducted in the Department of Microbiology and Hygiene and Department of Pathology, Faculty of Veterinary Science, BAU, Mymensingh during the period from May 2003 to April 2004. Bursa of Fabricius of thirty five dead birds suspected to be infected with IBO were collected from BAU poultry farm, Mymensingh and stored at -20°C. 10% bursal homogenate was prepared from five bursae in phosphate buffered saline (PBS). The suspension was centrifuged at 3000 rpm for 30 minutes. The supernatant was treated with broad spectrum antibiotic (Gentamycin) @ 50 µg/ml and kept at room temperature for 30 minutes. In order to isolates the IBDV field isolates five chickens of 35 days old were inoculated with 10% bursal homogenate @ 100 µl through intranasal, intraocular and intra cloacal routes. Five chickens of same age were kept as uninfected control. At day 3 post infection (P.I.) all chickens were Sacrificed and bursae were collected aseptically. BD-3wt representing a vvlBDV isolate and a vaccine virus Nobilis D-78 representing an attenuated classical strain were used as reference virus. Total RNA was isolated from bursal tissues. The procedure was based on guanidine lysis, phenol-chloroform-isoamyl alcohol extraction, and isopropanol precipitation. For isolation of RNA from the vaccine virus Nobilis D-78, the lyophilized vaccine (1000 dose) was resuspended in 1 ml distilled water and processed as the tissue homogenate. One- tube RT-PCR in 50 µl volume was performed following the procedure after some modifications. RT -PCR products were analyzed by electrophoresis in 1.2% agarose gel. PCR products were subjected to restriction enzyme (RE) analysis without further purification. PCR products were digested with the use of restriction enzymes Sspl and Sacl. The cleavage of ONA by RE digestion was analyzed in 2% agarose gel by electrophoresis. The results were examined and recorded by using the image documentation system.

Source of Information: International Journal of Poultry Science 3 (10): 364-667, 2004

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Five IBDV isolates were obtained from chicken inoculation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was preformed to detect IBDV isolates in the bursal tissues. RT-PCR couple with restriction enzyme (RE) analysis was carried out for molecular characterization of IBDV isolates to determine the pathotype. 677 bp fragments from IBDV genome segment A corresponding to the hyper variable domain of outer capsid protein VP₂ was amplified by RT-PCR. Two restriction endonuclease (REs), SspI and SacI were used for digestion of RT-PCR products. RT-PCR product was digested by SspI but not SacI. The presence of SspI restriction site in the 677 bp RT-PCR fragment indicated that IBDV isolates belonging to very virulent (vv) pathotype.

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