SERO-SURVEILLANCE AND MOLECULAR CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE FROM POULTRY OF ASSAM
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Date
2016-01
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Assam Agricultural University, Khanapara, Guwahati
Abstract
Infectious Bursal disease (IBD) is a highly infectious and contagious disease that primarily affects chicks of 3-6 weeks of age causing immunosuppression by affecting the immune system of poultry where it damages the Bursa of Fabricius, thymus etc. The disease has great economic importance in both broiler and pullet growers as the affected birds are susceptible to minor environmental pathogens leading to high morbidity and mortality. The disease is caused by double stranded bisegmented IBD virus (IBDV) that comes under the genus Avibirnavirus of family Birinaviridae. The best way to prevent the disease is by vaccination and good managemental practices. However, there are frequent reports of the occurrence of the disease from different parts of India including Assam. So the present study was aimed to assess the infection by detecting presence of antibodies in the serum samples through indirect Enzyme linked immuno sorbent assay (ELISA) which were randomly collected from unvaccinated local birds from different parts of Assam and detection of virus from clinically affected tissue samples by direct Reverse Transcription- Polymerase Chain Reaction (RT-PCR) and by integrated cell culture PCR (ICC-PCR) after isolating it in the chicken embryo fibroblast (CEF) and Vero cell line.
A total of 1093 sera samples were randomly collected from 19 different districts of Assam and screened for presence of antibodies against IBDV using commercial Indirect- ELISA kit. Out of which, 306 samples (27.99%) were found positive. Based on different age groups, collected serum samples were categorized out of which 2-4 weeks of age group meaning young chicks were found to contain antibodies against IBDV.
Clinical samples were collected from different places of Assam which shown characteristic necropsy lesion for IBD infection. Total 23 numbers of clinical samples were collected for diagnosis of IBDV antigen through RT-PCR with specific set of primers. Out of which, 12 samples (52.173%) were confirmed for the presence of IBDV. Samples positive in PCR were stained with DNA loading dye and run under polyacrylamide gel electrophoresis along with DNA ladder. A distinct band was observed at 643 bp size region.
Representative three PCR amplicon from Nalbari, Hajo and Bijoynagar were further sequenced via an out source and a phylogenetic tree was constructed by maximum likelihood method along with other IBDV isolates reported from various part of the world. Percent identities were analyzed within the isolates reported from our study, from different parts of India and other parts of the world.
IBDV can be well adapted in CEF and Vero cells. Virus was passaged for five times in primary chicken embryo fibroblast cells before adaptation in Vero cells. Cytopathic effects (CPE) were observed from second passage onwards in both the cell line. IBDV was passaged in both Vero and CEF cells for at least five times and the cell lysates of each passage were checked by performing Integrated Cell Culture -PCR. All positive samples gave similar results to the pairs of primers which were used for the detection of IBDV nucleic acid in the tissue samples. The present study confirms the prevalence of the disease in poultry population of Assam.
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