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Isolation and Identification of Infectious Bursal Disease Virus from Broiler and Layer Chickens During the Outbreak Year 2007 in Bangladesh

Md. Taohidul Islam
Department of Microbiology and Hygiene, Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Mohammad Mohiuddin
Department of Microbiology and Hygiene, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Muhammad Tofazzal Hossain
Department of Microbiology and Hygiene, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Bahanur Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Mostafizur Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Siddiqur Rahman
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Hee-Jong Song
Korea Zoonoses Research Institute, Chonbuk National University, Jeonju 561-756, Korea

Md. Alimul Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

The objective of the present investigation was to isolate and identify infectious bursal disease viruses (IBDVs) from broiler and layer chickens on outbreaks of infectious bursal disease (IBD) in three districts of Bangladesh. A total of 70 bursal samples were collected from dead broiler (n=40) and layer (n=30) chickens showing specific lesions of IBD from seven commercial poultry farms of three different districts (Mymensingh, Chittagong and Tangail) of Bangladesh during the year 2007. Five representative bursal samples from each farm were used for the isolation of IBDVs using 9-day-old embryonated eggs of seronegative flock of layer birds and for identification the samples were subjected to agar gel immunodiffusion test (AGIDT), immuno histochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). Out of 35 bursal samples, IBDVs were successfully isolated from 28 (80%) samples. By AGIDT, 32 (91.4%) samples were found positive for IBDV antigen. Results of AGIDT clearly indicated that IBDVs detected in 29 bursal samples of six affected farms were identical to each other but not to IBDVs present in the remaining three samples of another farm. Indirect immunoperoxidase staining of the bursal sections revealed the presence of IBDV antigen in 32 (91.4%) samples and the IBDV antigen was detected mainly in the cortex of the lymphoid follicles of the bursal tissues. In histopathology, cell depletion, atrophy and necrosis were observed in many bursal follicles with severe edema of interfollicular septa. Of the 35 bursal samples, 34 (97.1%) samples generated 254 bp product by RT-PCR. In conclusion, the results of virus isolation and identification by AGIDT, IHC and the analysis of viral genome by RT-PCR confirmed the outbreaks of acute IBD in commercial poultry of Bangladesh. Moreover, histopathological findings and results of AGIDT gave a clear indication that the isolates from six outbreaks were different from classical strain and it seems to be of very virulent strain. On the other hand, the isolates from the other outbreak were similar to the classical strain.

Keywords: Infectious bursal disease virus, Isolation, Immunohistochemistry, RT-PCR, Broiler, Layer chicken

Location: Mymensingh, Chittagong and Tangail districts

Start Date: 00-00-2007

End Date: 00-00-2007

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To collate samples from the outbreaks of infectious bursal disease (IBD) in broiler and layer chickens
To isolate and identify infectious bursal disease viruses (IBDVs) from the samples of three districts of Bangladesh

.

Methodology:

The vaccine strains Nobilis.Gumboro 228E (Intervet International, Netherlands) and BAU 404 of classical strain (DLS, Dhaka, Bangladeshi) were used as reference IBDVs in agar gel immunodiffusion (AGIDP) test and reverse transcription-polymerase chain reaction (RT-PCR) assay. Bursal samples of dead birds showing specific lesions of IBD were collected from seven field outbreaks (four in broiler farms and three in layer farms) during the year 2007 from Mymensingh, Chittagong and Tangail districts. From each outbreak of IBD, 10 bursal samples were collected. A portion of each bursal sample was preserved at −86^oC while another portion was kept in 10% buffered normal saline for histo pathology and immuno histochemistry. Bursal suspension preserved at the Department of Microbiology and Hygiene, BAU, Mymensingh which were previously confirmed negative for IBDV by AGIDT and RT-PCR were used as negative control. Five representative bursal samples from each outbreak were homogenized with sea sand and 20% bursal homogenates were prepared in phosphate buffered saline (PBS). After centrifugation of the suspension at 3,000 rpm for 15 min, the supernatants were treated with penicillin (10,000 IU/ml) and streptomycin (10,000 μg/ml), which were used as inoculum. 0.2 ml of each sample was inoculated into 9-day-old embryonated chicken eggs through chorioallantoic membrane (CAM). The eggs were collected from seronegative layer birds of 68 to 70 weeks old. Five embryonated eggs were used for each sample. Allantoicfluids, CAM and the embryo proper were harvested aseptically from the embryonated eggs dying after 24 hr and within 5 days after inoculation and were stored at −86^oC until used. Embryos were processed to make 50% suspensions for the extraction of viral RNA.

Source of Information: Korean J Vet Serv, 2012, 35(1), 9-17; ISSN 1225-6552

Article Link (Online Journal): http://kosves.re.kr

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The presence of IBDV antigen was confirmed in 32 (91.4%) bursal samples by AGIDT. This clearly indicated that, IBDVs of six outbreaks were identical to each other but not to IBDVs of another one outbreak and BAU 404 vaccine strain (classical strain) as they formed spurs at the junction between the lines of precipitation. However, IBDVs of that outbreak were identical to Nobilis.Gumboro 228E vaccine strain, which led to assume that there might had occurred vaccine failure. AGIDT had been reported by a number of workers to be useful in easy screening of the field samples, prior to either isolation of the virus or to applying other techniques for virus detection and characterization.

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