Molecular characterization of infectious bursal disease virus isolates from field cases in broiler chicken
Abstract
This study was conducted to understand the epidemiology of infectious bursal disease (IBD) in broiler chicks in parts of Haryana as well as to characterize the infectious bursal disease virus (IBDV) by reverse transcription-polymerase chain reaction (RT-PCR), restriction enzyme analysis (RE analysis) and sequencing. The data related to IBD outbreaks for the period from July, 2005 to June, 2008 was analyzed. During this period, 483 outbreaks occurred in broiler chicks with overall morbidity, cumulative mortality and case fatality rate of 4.54, 2.34 and 51.69%, respectively. The disease was recorded through out the year and approximately 86% of the outbreaks were
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in the birds of age 21-40 days. Of these outbreaks, 334 were recorded in vaccinated flocks while 149 were in unvaccinated flocks. Per cent morbidity and cumulative mortality were comparatively higher in unvaccinated flocks than in vaccinated flocks.
Detection of IBDV by RT- PCR was carried out in 30 field samples and 4 vaccine strains by amplifying the VP1 gene and hypervariable region of VP2 gene. The IBDV was detected in 28 field samples and all the 4 vaccine strains. The 432 bp PCR products of VP1 gene and 479bp PCR products of VP2 gene of 19 field isolates and 4 vaccine strains were subjected to RE analysis'. Sca1 and Pst1 restriction enzymes were used for VP1 and BstN1, Ssp1 and Pst1 were used for VP2 gene. No restriction site was observed for Pst1 and Sca1 restriction enzymes in PCR products of VP1 gene. However, in case of VP2 gene, 13 of the 19 field isolates were Ssp1+ , Pst1+ and BstN1+ , suggestive of vv types. Three fields isolates were Ssp1- , Pst1- and BstN1+ similar to classical strains. Remaining 3 field isolates were Ssp1+ , Pst1- and BstN1+ and were similar to MB and I+ vaccine strains. Phylogenetic analysis based on the nucleotide sequence of VP1 gene of four IBDVs revealed that all four were of vv type and were close to the already reported very virulent field isolates from India and abroad (KT1/99 India, OKYM, Japan, UK661, TL2004 China, 9109 USA). The nucleotide sequence analysis of VP2 gene of 10 IBDVs revealed that 9 were of vv nature due to the presence of substitutions at 795G, 827T, 833C, 857C, 897A, 905T, 908T, 1011A and1094G , specific to vvIBDV strains. Remaining one was classical/ avirulent type. There was a unique change of amino acid from alanine (A), serine (S), glutamic acid (E) to threonine (T) at amino acid position 270 in IBDVs of this study. The phylogenetic analysis suggested further divergence of IBDVs. Nucleotide and deduced amino acid sequencing revealed that the presence of vvIBDV strains in Haryana which are closely reported to the vvIBDVs from UK and Japan and also suggested continuation of changes at genetic level in these strains.