| Summary: | 碩士 === 國立屏東科技大學 === 獸醫學系 === 90 === Infectious bursal disease (IBD) is a B lymphoblast necrotic immunosuppress disease in young chickens causing by a member of Birnaviridae, IBD virus (IBDV). Preventine and control of the disease has been mainly relayed on vaccinating young chicken with a variety of live IBD vaccine. Safety and the immunogenicity of the vaccine has not been clear indicated. An evaluation on the commercial available 23 live IBD vaccines was then designed and done orally in each of the 23 groups, 20 birds per proup, 3-week-old SPF chickens. A nonvaccinated control was also included. Five birds per group was removed for serological enzyme-linked immunosorbent assay (ELISA), body weighing and bursa weighing in calculating bursa/ body weight ratio (BBR), bursa gross lesion (BGL) grading and bursa histological lesion (BHL) grading. Only few (6) vaccinated group had ELISA antibody titer exceed 1:100 at 5 days post vaccination (DPV). Most (13) vaccinated groups had the highest titer (1:71-1:10748) at 21 DPV, but 3 and 7 groups were at 10 and 15 DPV. The BBR value of 3(13.04%), 5(21.74%), 6(26.09%), and 9 (39.13%) vaccinated groups was classified as intermediate plus (IM+; <1.5), intermediate (IM; 1.51-2.00), intermediate minus (IM-; 2.01-3.00) and low (>3.01) virulent level respectively. Only 2 vaccinated groups had moderate (1.13 and 1.30) BGL value at 10 DPV, the other BGL values in different DPV were all under 0.78 and most of the values are quite closed. The BHL value of 3 (13.04%), 2 (8.7%), 5 (21.74%) and 13 (56.52%) vaccinated groups was classified as IM+ (>2.0), IM (1.51-2.00), IM- (1.01-1.50) and low (<1.0) virulent level respectively. By using the above BBR and BHL virulence grading, 16 (69.57%) live IBD vaccines got the same virulent level. Also the BBR and BHL virulent level of the other 7 (30.43％) vaccines was all quite closed. Therefore the BBR and BHL value, but not the BGL value are good pathogenicity indice for live IBD vaccine.
Seven most popular use live IBD vaccines were individually taken to immunize a group of 10 3-week-old chickens then challenge at 21 DPV with a 1995 isolate (V97/TW95) of local very virulent IBDV (vvIBDV). A control group was also included. At 21 DPV and 7 day post challenge (DPC), 5 birds per group were removed for serological ELISA, body weighing and bursa weighing in calculating BBR, BGL grading and BHL grading. All the vaccinated groups had reasonable good titer (2261-4315) of ELISA antibody response. At 21 DPV, only 1 group had the BBR and BHL fall in the IM+ virulent level, the others were under IM virulent level. Three vaccinated groups were found not with good protection by showing much higher of BHL value and moderated lower of BBR value after challenge.
Each 24 groups 3-week-old SPF chickens, 10 birds per group, was individually given with one IBDV of the above 19 vaccines and 5 local vvIBDVs for virus propagation in the bursa. The bursa of each group was collected at 4 days post inoculation for reverse transcription-polymerase chain reaction (RT-PCR) amplifying a 643 bp nucleotide belong to the viral protein 2 hypervariable region (hvVP2) of IBDV. All the nucleotides were then cloned and sequenced. A computer DNASTAR software was used to figure out the relationship of the virus in the phylogenic tree, the identity of nucleotide and amino acid of the virus hvVP2. By using the amino acid sequence of V97/TW95 strain as standard, 3 (60%) local isolates and 4 (21.0%) vaccine strains had the amino acid over 97.2% identity as V97/TW95 stain. By using the amino acid sequence of a 2002 local isolate-V263 as standard, 4 (80%) local isolates and 5 (26.3%) vaccine strains had the amino acid over 97.2% identity as V263 stain. The 5 vaccines are the ones now most popular using in the local chicken industry. A comparison of the hvVP2 of IBD vaccine and local isolate strains comes to be a very valuable reference for practical protective evaluation of the live IBD vaccine.
A whole length of IBDV V97/TW95 strain VP2 gene, 1492 bp, was successfully cloned in pET32a Escherichia coli system and a expressied VP2 protein of 70kDa was verified by positive reaction in Western blotting and immuno-dot blotting assay with V97/TW95 strain immunized SPF chicken polyclonal antibody. The immunogenicity of the subunit vaccine need to be further studied.
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