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Isolation and identification of infectious bursal disease virus
1. Isolation and Identification of
Infectious Bursal Disease Virus
Presented by
Md. Saiful Islam
MS in Microbiology
Bangladesh Agricultural
University
2. Introduction
The causal agent of infectious bursal disease (IBD)
IBD is an acute, highly contagious viral disease of
young chickens.
Characterized mainly by severe lesions in the bursa
of Fabricius (BF) followed by immunosuppression
and mortality generally at 3-6 weeks of age.
IBD is also known as Gumboro Disease, Poultry
AIDS and Infectious Avian Nephrosis.
7. Stability
of Virus
122 days in
non-
disinfected
house
52 days in
contaminated
water or feed
5 hours at
56°C
pH range
2-12
QACs
1000ppm
0.5 %
formalin for 5
hours
11. Collection of Sample
Instruments and
Appliances
Factors to be
consideredSamples to be
collected
Depends on type
of sample to be
collected and
must be sterile
Timing Source
Live Birds Dead Birds
12. Samples to be collected
In case of
Sick Birds
In case of
Dead Birds
1. Blood
2. Feces
1. Bursa of Fabricius
2. Lymphoid Tissue
3. Thigh and Breast
Muscle
4. Spleen and Kidney
At the early
stage of disease
13. Sampling site for IBDV
Enlarged, hemorrhagic bursa of Fabricius Small, atrophic bursa of Fabricius
Hemorrhages in thigh muscle Swollen Kidneys with urates
14. Transportation of Sample
For short distance
(≤ 1 hour)
For long distance
And longer period of time
Transport media
Balanced salt solution
Phosphate buffer saline
10% fetal calf serum
Frozen
condition
Use of
preservatives
Using ice flask
or bag at 4°C
- 50% buffered
glycerine
- 10% DMSO
- Virus transport
medium
15. Preservation of Sample
Freezing Lyophilization In chemicals
At lower
temperature
0°C up to -70°C
Using
glycerin
Water portion of the sample are
removed through dehydration
and sealed under vacuum
16. Inoculum Preparation
Solid Sample (BF) Liquid Sample (Blood)
Selection of area of sample
Trimming
Grinding
Prep. Of tissue suspension
Filtration Centrifugation
Supernatant collection and Centrifugation
Addition of antimicrobial agents
Waiting
Purity or sterility test
Sterile inoculum if no growth
Finally storage
17. Virus Isolation in Embryos
CAM is the most sensitive route for IBD virus propagation.
Yolk sac route also can be used.
Inoculation of IBD virus in 9-11 days old embryonated egg
through CAM route
Classical isolated will kill the embryo 3-5 days post-inoculation
The dead embryos will be congested with petechial and
echymotic hemorrhage evident along the feather tracts, toe joints
and cerebral areas
Liver may be necrotic but pale appearance
Spleen frequently pink or lacking in color
CAM usually uninfected, hemorrhage may be found
18. Piercing the shell in one side of egg Piercing the shell over the air sac
Injecting IBD virus
20. Swollen liver with patchy congestion and pale yellow-green coloration
Hemorrhage in embryo
21. Virus Isolation in Cell Culture
Most commonly, Infectious bursal disease virus can be
propagated in
Chicken embryo fibroblast cultures
Chicken embryo bursal cells
Chicken embryo kidney cells.
Cytopathic effects (CPE) occurs such as small round
refractive cells.
IBDV also produce CPE in the avian and mammalian
continuous cell lines such as Rabbit RK-13 cells and
Monkey Vero cells.
22. Virus Isolation in CEF Culture
Inoculation of 0.5 ml of sample into each of four freshly confluent
chicken embryo fibroblast (CEF) cultures from a specific pathogen
free (SPF) source in 25 cm flasks
Adsorbed at 37°C for 30-60 minutes
Washing twice with Earle’s balanced salt solution and addition of
maintenance medium to each flask
Incubated at 37°C, observing daily for evidence of CPE
Characterized by small round and aggregation of refractive cells
If no CPE is observed after 6 days, discarding the medium, then
freezing-thawing the cultures and inoculation of the resulting lysate
into fresh cultures
23. Fig : Confluent monolayer of CEF cells Fig : Cytopathic Effect on CEF cell culture
Continued……
24. Cytopathic effect of IBDV in DF-1 cell monolayer. (A) Uninfected
control monolayer. (B) DF-1 monolayer in passage 3rd, days 5
pi. Note the arrow shows DF-1 cells rounding and clumping
Continued……
25. (A) DF-1 monolayer in passage 4th, days 5 pi affected cells
more concentrated with cytoplasmic granularity (B) DF-1
cells passage 5th, 4 day pi note that the arrow shows the
cells detached from the substrate.
Continued……
26. Virus Isolation in Vero Cell Line
Cytopathic effect of IBDV 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
27. Continued……
(A) Vero cells monolayer in passage 6th, days 8 pi (B) Vero cell
monolayer in passage 12th, days 6 pi. Note the arrows shows
Vero cells rounding and aggregate in clumps and granulated in
cytoplasm
28. Continued……
(A) Vero cells monolayer in passage 13th, days 3 pi (B) 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
29. Histopathological examination
Collection of samples including bursae, spleens and
thymus glands
Fixation of the organs in 10% buffered formalin
Dehydrated in grade alcohol
cleared with Xyline
Embedded in paraffin sections with average sections
of 5 micron
Stained with H&E and observed under electron microscope
30. Continued……
(a) Control negative showing normal follicles (b) Classical strains infected chickens
showing moderate depletion (c) Classical strains infected chickens showing vacuolated
follicles (d) Variant strain infected chickens showing normal tissue in bursae
31. Continued……
(a) Variant infected chickens- leucocytic infiltration (b) Variant infected chickens- vacuolated
follicles and mononuclear leucocytic infiltration (c) vvIBDV infected chickens- follicles with cyst
like space containing cell debris (d) vvIBDV infected birds- atrophied follicles in bursae
32. Continued……
a (negative control) and b (classical strain) are showing normal tissue of thymus and c (vvIBD
and variant strain) is showing necrosed cortex and medulla. d (negative) and e (classical) are
showing slight and severe congestion respectively in spleen and f (vvIBD and variant strain)
is showing necrosed follicles.
33. Pathogenicity test
Inoculation of 0.1 ml of IBD virus sample in 14 days old
chickens through intraocular route
Observation
Characteristic clinical signs like watery diarrhea, soiled vent
feathers, vent picking, and inflammation of the cloaca
4-6 days post-infection, the bursa of fabricius is covered
with a yellowish gelatinous transudate
This is followed by busal atrophy, which occurs 7-10
days after infection
36. Agar Gel Immunodiffusion (AGID)
Figure : Agar gel immunodiffusion test positive wells with a precipitin
between positive control (+ve) and wells A. There was no precipitin
around the negative control (-ve)
37. Enzyme Linked Immunosorbent Assay (ELISA)
A B
CD
ELISA plate with (A) dilution buffer and pre-diluted sample. (B) color reaction after
adding concentrated conjugate 1x solution. (C) color reaction before adding stop
solution. (D) color reaction after adding stop solution.
40. Molecular Detection of IBDV
Gene Primer Primer Design Product Size
VP2
F
5` GCGATGACAAACCTGCAAGAT 3`
642 bp
R
5` AGGTGGGAACATGTGGAGAC 3`
HVR
F
5` TCACCGTCCTCAGCTTAC 3`
604 bp
R
5` TCAGGATTTGGGATCAGC 3`
Vvfp
F
5´-AATTCTCATCACAGTACCAAG -3´
253 bp
R
5´-GCTGGTTGGAATCACAAT -3´
For the detection of IBDV, Reverse transcriptase- polymerase
chain reaction (RT-PCR) is used.
41. Fig : Electrophoretic pattern of PCR product of collected samples:
Lane M: Marker (DNA ladder)
Lane 1, 9, 10 and 11: Showing negative samples
Lane 2, 3, 4, 5, 6, 7, 8, 12 and 13: Showing positive amplification of VP2
at 642 bp.
Continued……
42. Continued……
1000
1000
200
100
M 1 2 3 4 5 6
253 bp
Fig: RT-PCR products of IBDV analyzed using 2% agarose gel electrophoresis.
M = DNA Marker, Lane 1= reference IBDV and Lane 2-6 = positive amplification
of Vvfp at 253 bp.
43. Vaccination
IBD vaccine and MDA
Since IBDV enters through the oral route, and should find
its way to the bursa via the blood stream, any MDA in chick
blood would neutralize the vaccine.
MDA is protective when present at a sufficiently high titre.
Due to metabolism and growth, the antibody titre declines
to half (t1/2) at a rate of:
• Broilers 3.5 days
• Broiler breeders 4.5 days
• Layers 5.5 days
44. 2 to 3 weeks post hatch, the susceptibility of a chicken
flock to an IBDV infection increases.
Breeder flocks are immunized against IBD, so they
would confer protective antibodies to their progenies.
Advantage
Protects chickens from early infection.
Disadvantage:
Neutralizes live vaccines.
Continued……
45. Age
ELISA titre
Maternal antibody will normally protect chicks for 1-3 weeks.
By boosting the immunity in breeder flocks with oil adjuvanted vaccines,
passive immunity may be extended to 4 or 5 weeks
MDA Decrease
48. Timing of Vaccination
Too soon
MDA neutralizes vaccine
Late
Late protection
Optimal
Sooner the better.
49. Vaccination for IBD
Species Age Route Remarks
Broiler 18-21 days Spray/ D.W.
If MDA is low
Live vaccine
Broiler and
Layers
3weeks
16 weeks
Eye drop
IM Killed vaccine
Commercial
layers
3weeks
16 weeks
Eye drop
IM Killed vaccine
