ISOLATION AND CHARACTERIZATION OF INDIGENOUS INFECTIOUS BURSAL DISEASE VIRUS ISOLATES IN AND AROUND HYDERABAD
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Date
2009-03
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT :
The present study was taken up with the objective of isolation and characterization of IBDV isolates. Ten pooled bursal samples (each pool consisting of five samples) were screened for IBD virus by AGID, out of which 5 pooled samples were positive for IBD virus. Chicken embryos were used for isolation of IBD virus by CAM route. Out of five samples two samples, PDP-1 and VH-1 yielded IBD virus after three serial passages in 11 day old chicken embryos. These isolates produced characteristic lesions like dwarfing of embryo, cutaneous edema, hemorrhages on thigh muscles, toes and greenish yellow discolouration of the liver in chicken embryos. These isolates could be adapted to BHK-21 cell cultures only after 5 successive passages as evidenced by stabilization of typical cytopathic effects. At 3rd passage level, CPE started with rounding of cells 72 h, PI. Large numbers of cells showed rounding and clumping, increase in cytoplasmic granularity at 96 h, of PI in 4th passage. In 5th passage, CPE started early i.e., 48 h, of PI and by 72 h, 60% of the cells were involved. There was extensive detachment of cells by 96 h, PI. Cell culture adapted isolates were characterized for physico-chemical properties. They were resistant to chloroform but inhibited by 1.0% formalin for 6 h, 0.1% formalin for 24 h. They were viable even after exposure to 560C for 90 min. They were resistant to acidic pH of 2.0 but completely inactivated at pH 12.0. Immunological characterization of cell culture adapted IBDV isolates by AGID and CIE revealed clear precipitation lines.
The polypeptide analysis by SDS-PAGE revealed that two isolates contained identical viral polypeptides with similar molecular weights and band pattern. Both isolates revealed 5 poly peptides, in addition to these two faint bands. The molecular weights of the 5 polypeptides were: (91 KD) VP1, (52 KD) VPx, (42 KD) VP2, (32 KD) and VP3, (27KD) VP4 and molecular weights of two faint bands were 70 KD and 66 KD. In this study, RT-PCR was performed on cell culture fluids and corresponding bursal tissues to detect the virus. RT-PCR was performed using P1 and P2 primers specific to hyper variable region of VP2 gene pertaining to IBDV. These studies resulted in generation of specific amplification of 643 bp from samples tested. Pathogenecity studies with the cell culture adopted field isolates inoculated in 3 week old chicks revealed 50 % mortality with PDP-1, 37.5 % mortality with VH-1.The dead birds revealed characteristic lesion i.e., haemorrhages in thigh and pectoral muscles, enlarged and haemorrhagic bursae. Histopathological changes were more pronounced in bursa of Fabricius. The changes were characterized by lymphoid necrosis, follicular degeneration and proliferation of connective tissue.
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