molecular characterisation, attenuation and inactivation of very virulent infectious bursal disease...
TRANSCRIPT
Molecular characterisation, attenuation and inactivation of very virulent
infectious bursal disease virus for development of tissue culture based
vaccines
MAJED H. MOHAMMED [email protected]
Hypothesis of this study
Very virulent IBDV can be adaptated and attenuated in mammalian tissues culture (Vero cells and DF-1 cells)
IBDV vaccine developed from vvIBDV could provide high protection against vvIBDV field challenge
Objectives
To adapt and attenuate vvIBDV isolate in tissue culture for vaccine development
To determine the molecular characteristic of the adapted and attenuated local isolate of vvIBDV
To determine the pathogenicity and immunogenicity of the attenuated vvIBDV in SPF chicks
To determine the pathogenicity and immunogenicity of the inactivated vvIBDV in SPF chicks
UPM0081UPM94/237 UPM04190
Vero cellsVero cellsVero cells
CPE / 4 passages No CPE / 6 passages No CPE / 6 passages
Propagation of vvIBDVin 10-day-old embryonated SPF eggs
Adaptation and attenuation of vvIBDV isolate in tissue culture for vaccine development
Cytopathic effect of isolate UPM0081 in Vero cells monolayer. (a) Uninfected control monolayer. (b) Vero cell monolayer in
passage 4th , days 10 pi. Note The arrows shows Vero cells rounding and aggregation. 10 x .
IBDV replication and adaptation
Vero cell lineResults
Vero cells monolayer in passage 6th, days 8 pi (a). Vero cell monolayer in passage 12th, days 6 pi (b). Note the arrows shows Vero cells rounding and aggregate in
clumps and granulated in cytoplasm. 10x.
IBDV replication and adaptation
Vero cells line
Results
Vero cells monolayer in passage 13th, days 3 pi (a). Vero cells monolayer in passage 20th, days 3 pi (b). Note the cells detached from the substrate, with the eventual destruction of the entire monolayer.
10 x.
IBDV replication and adaptation
Vero cells line
Results
Cytopathic effect of isolate UPM0081 in DF-1 cell monolayer. (a) Uninfected control monolayer. (b) DF-1monolayer in passage 3rd, days 5
pi. Note the arrow shows DF-1 cells rounding and clumping. 10 x.
IBDV replication and adaptation
DF-1 cell lineResults
DF-1 monolayer in passage 4th, days 5 pi affected cells more concentrated with cytoplasmic granularity (A). DF-1 cells
passage 5th day 4 pi the arrow shows the cells detached from the substrate. 10 x (B).
IBDV replication and adaptation
DF-1 cell lineResults
IBD titration (TCID50/ml)
passage TCID50
Vero cells DF-1 cells
P6 103.5 101.5
P9 104.5 102.5
P10 104.7 -
P15 106.7 -
P20 107.4 -
Virus titer determined by Tissue Culture Infective Dose 50 (TCID50)
Results
Identify of CPE forming agent in Vero cells culture using IIPS infected cell cultures stained with HRP-conjugated
antibody. (a) Uninfected control Vero cells. (b) Vero cells infected with B0081T20 2 days pi.
Note specific intracytoplasmic brownish coloration. 10 x.
Indirect Immunoperoxidase Test (IIPS)Results
Identify of CPE forming agent in DF-1 cells culture using IIPS infected cell cultures stained with HRP-conjugated antibody. (a)
Uninfected control DF-1. (b) DF-1 infected with B0081T7 2 days pi. Note specific intracytoplasmic brownish coloration. 10 x
Indirect Immunoperoxidase Test (IIPS)Results
Hypervariable region (643pb) amplification of IBDV VP2 genesLane 1- Negative control; Lane 2 positive B0081TD5; Lane 3 positive B0081TD7; Lane 4 positive B0081TV5 and Lane 5 positive B0081TV7; M- 100 bp DNA marker (Promega, USA
PCR screening on white colonies amplification of IBDV VP2 genesLane 1,2 and 3 White colonies positive for VP2 gene passages (B0081TD5, B0081TV5 and B0081TV7) respectively, Lane 4 Negative control; M- 100 bp DNA marker (Promega, USA).
Molecular characterisation of the adapted and attenuated local isolate of IBDV
Amplification of the hypervariable region of VP2 Gene
PCR Analysis of recombinant colonies
Results
Summary of the proposed molecular markers (amino acid residues) of B0081T10, B0081T15 and B0081T20 atIBDV
isolates with other published IBDV strains.Results
Phylogenetic tree based on nucleotide sequence of HPVR of VP2 gene of IBDV isolates, displaying relationship of B0081T10, B0081T15 and B0001T20 passages and other published atIBDV strains
Phylogenetic analysis
Results
Pathogenicity and immunogenicity of the attenuated vvIBDV in SPF chicken
Experimental design
Groups Passages (P) Time of sampling (Days) N0. Of chicken
0 1 3 5 7 10 14
1 (a) Vaccinated sacrificed P15 – 5 5 5 5 5 5 30
(b) Vaccinated and vvIBDV challenged
– – – – – – – 5
(c) Vaccinated mortality – – – – – – – 5
2 (a) Vaccinated sacrificed P20 – 5 5 5 5 5 5 30
(b) Vaccinated and vvIBDV challenged
– – – – – – – 5
(c) Vaccinated mortality – – – – – – – 5
3 (a) Non vaccinated sacrificed control
5 5 5 5 5 5 5 35
(b) Non vaccinated and challenged
– – – – – – – 5
(c) Non vaccinated and non challenged mortality
– – – – – – – 5
Total accumulation death
Days (post
Challenged)
Challenged
group
1 2 3 4 5 6 7 8 9 10 % of % of
Mortality protection
P15 Vaccinated and
vvIBDV challenged
0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0% 100%
P20 Vaccinated and
vvIBDV challenged
0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0% 100%
Non vaccinated and
vvIBDV challanged
0/5 2/5 2/5 3/5 5/5 5/5 5/5 5/5 5/5 5/5 100% 0%
Rate of mortality at 10 days post challenged and the percentage of protection based on the
number of chickens that survivedResults
Body weight of the chickens throughout the experiment
Bursa weight of the chickens throughout the experiment
Results
Group 3: Non vaccinated and
challanged Group 1: (P15) vaccinated & vvIBDV challenge
Gross PathologyGross Pathology
Results
Group 1: (P15) vaccinated & vvIBDV challenge
Group 3: Non vaccinated and challenged
Group 1: (P15) vaccinated & vvIBDV challengebursa of Fabricus day 7 ( lesion scoring
1 slight to mild tissue reaction)
Group 2: (P20) vaccinated & vvIBDV challenge Bursa of Fabricus day 7 pv lesion scoring 1 slight to mild tissue reaction
Histopathology changes
Results
Group 3: Non vaccinated and challenged bursa of Fabricus day 3 pc) (lesion scoring 5
Group 3: Non vaccinated and challenged bursa of Fabricus day 2 pc) (lesion scoring 5
IBD antibody titer of the chickens throughout the experiment
Enzyme Linked Immunosorbent assay (ELISA)Results
pathogenecity and Immunogenicity of the inactivated vvIBDV in SPF chicken
Experimental design
Groups No. of
chicken
Type of vaccines Definition
B1 5 passage15 BEI With vaccine & vvIBDV challenge
B2 5 Passage20 BEI With vaccine & vvIBDV challenge
E1 5 Passage15 ECA With vaccine & vvIBDV challenge
E2 5 Passage20 ECA With vaccine & vvIBDV challenge
CP 5 Control positive Without vaccine & vvIBDV challenge
CN 5 Control negative Without vaccine & without challenge
Total accumulation drath
Days (post
challenged)
Challenged
group
1 2 3 4 5 6 7 8 9 10 % of % of
Mortality protection
Group: Positive
Control
0/4 0/4 1/4 3/4 4/4 4/4 4/4 4/4 4/4 4/4 100% 0%
Group: B1 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0% 100%
Group: B2 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0% 100%
Group: E1 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0% 100%
Group: E2 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0% 100%
Rate of mortality at 10 days post challenged and the percentage of protection based on the number of
chickens that survivedResults
Body weight of the chickens at 2 weeks post challenge
Bursa weight of the chickens at 2 weeks post challenge
Results
Gross Pathology
Results
Group BEI: (P15) vaccinated & vvIBDV challenge
Group PC: Non vaccinated and challanged
Group ECA: (P15) vaccinated & vvIBDV challenge
Group PC: Non vaccinated and challanged
Bursa of Fabricus in the B2 group (with vaccine and challenge) at day14 post challenges
Histopathology changes
Results
Bursa of Fabricus of control PC( without vaccine and challenge) at day 4 post challenges
Results ELISA Ab titer
IBD antibody titer of the chickens at 2 weeks pv and 2 weeks pc
This study has successfully propagate Malaysia vvIBDV strain namely UPM0081 in (SPF) embryonated chickens egg via chorioallontoic membrane (CAM) three times, and infected continuous cell line namely the Vero cells and DF-1cells
Vero cells was used for further study on the development of vaccines due to high titre (104.5
TCID50/0.1ml) when compare to DF-1 cells (102.5 TCID50/0.1ml) on the passage 9 of vvIBDV (UPM0081)
Based on molecular technique the IBDV passages, B0081T10, B0081T15 and B0081T20 were characterised as atIBDV, by having all of the proposed molecular markers for atIBDV strain with an accession No. FJ824699, FJ898321 and FJ898322 respectively
Conclusion
The vvIBDV (UPM0081) adapted and attenuated in Vero cell line passage 15 and 20 conferred full protection to the immunized SPF chickens against vvIBDV challenged.
The water in oil emulsion Freund’s Incomplete Adjuvant with killed vaccine groups (BEI and ECA) could protect the chickens from death against vvIBDV challenged.
Conclusion
