Detection of infectious bursal disease virus (IBDV) from bursal tissue by RT-PCR and its comparative efficacy with conventional precipitation assays
Makadiya, N.R. (2004) Detection of infectious bursal disease virus (IBDV) from bursal tissue by RT-PCR and its comparative efficacy with conventional precipitation assays. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Infectious bursal disease (IBD), an economically important infectious viral disease of poultry, is caused by Infectious bursal disease virus (IBDV) belonging to Avibirnavirus genus of Birnaviridae family. The disease causes considerable morbidity and mortality mainly by immunosuppression. Emergence of very virulent IBDV (vvIBDV) strains in different parts of the world including India during the last couple of decades, have demanded further research efforts in understanding the added complexity of the disease process and the means to diagnose and control it. To establish the proper control procedures, it is important to characterize the antigenic and virulent properties of the viral strains prevalent in a geographic area, and hence, it is necessary to develop rapid and accurate methods suitable for identifying as well as characterizing the virus. The present study was aimed at screening IBD suspected bursal samples by Reverse transcription-polymerase chain reaction (RT-PCR). Conventional precipitation assays viz. Agar gel immunodiffusion (AGID), Single radial immunodiffusion (SRID) and Counter immunoelectrophoresis (CIE) were also employed so as to compare their relative sensitivity with RT-PCR as well aamong themselves. Two viral RNA extraction protocols viz. phenol-chloroform and Tri Reagent® method were compared for their efficacy and suitability for RT-PCR. Incidence of IBDV in relation to epidemiological factors like age, breed and strain of the birds was also studied. Processing of the 147 collected samples by AGID, SRID and CIE revealed 70.07, 72.79, and 78.23 per cent positivity respectively. CIE gave the highest positivity among the precipitation assays and hence was used for comparison of incidences as well as of sensitivity and specificity between the tests. Thirty four samples were from layer birds and 113 from broiler birds, of which, 29 (85.29%) and 86 (76.11%) were positive for IBDV antigen respectively by CIE. The strainwise incidence revealed the maximum incidence in C&M chicks (100%), followed by BV-300 (85.29%), Cobb (78.26%) Hybro (68.75%), Hubchix (57.14%) and Croiler (50.00%). The highest incidence was found in the age group > 3 4 weeks, followed by > 5 6 and > 2 3 weeks age group, while, the least incidence was found in > 4 5 weeks of age. Thirty seven bursal samples and two live IBD vaccines were used for viral RNA extraction by phenol-chloroform method (protocol 1) and Tri Reagent® method (protocol 2). RNA extracted by protocol 1 was of poor quality and quantity, and failed to yield targeted amplification in all the samples and vaccines by RT-PCR, where as protocol 2 yielded RNA of good quality and quantity, and produced targeted amplicon of approximately 643 bp and 557 bp respectively for primer pairs P1, P2 and U2, L2, specific for hypervariable region of IBDV VP2 gene, for 31 (83.78%) samples and two vaccines. All the positive samples behaved similarly to the two pairs of primers, which were equally sensitive in detecting the IBDV. Sensitivity of AGID, SRID and CIE with RT-PCR was 74.19, 77.42 and 87.10 per cent, while that of AGID and SRID with CIE was 89.57 and 93.04 per cent respectively. Overall agreement of AGID, SRID and CIE with RTPCR was 78.38, 81.08 and 89.19 per cent, while that of AGID and SRID with CIE was 91.84 and 94.56 per cent respectively. Specificity of all the precipitation assays used with RT-PCR was found 100 per cent.
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