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EMBRYONATED
EGG
EGG
INOCULATION
INOCULATION
By:Chinithung ngullie(M.sc 2nd
year)
MODERATOR:Dr.Varsha A singh
1
virus cultivation
isolate and identify viruses
viral structure, replication, genetics &
pathogenesis
vaccine production
TECHNIQUES OF VIRUS
CULTIVATION
EMBRYONATED EGG INOCULATION
TISSUE CULTURE
ANIMAL INOCULATION
Introduction
Cultivation of viruses in chick
embryo
different type of approach.
For all practical purposes they
behave as tissue cultures
History
First used for cultivation of
viruses by Ernest GoodPasteur
and Burnet (1931)
F.M. Burnet in the
laboratory in the
early 1950's, was
experimenting
on influenza
virus genetics,
using the
developing hen's
egg.
Burnet
goodpasture
EMBRYONATED EGG
state of a
fertilized egg
containing an
embryo
Selection of egg
must be sterile
shell should be intact and healthy.
should be obtained from nonvaccinated, disease-free flocks
EGG
INCUBATOR
Egg incubator
artificially hatched - controlled and favourable conditions
maintain favourable incubation/ environment - constant
temperature over a specified period.
electrically heated thermostat
intelligent control system - correct measurement of heat
quantity ,
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ADVANTAGES OF EMBRYONATED
EGG
Ideal for viruses to grow, offers several sites for virus
cultivation
Isolation and cultivation of many avian viruses and few
mammalian viruses
Sterile and wide range of tissues and fluids
Economical and Readily available
Maintenance easier
Less labour (not need feeding and caging)
They do not have immune mechanism like animals to
counteract virus infection.
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DISADVANTAGES
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DISADVANTAGES
Used much less now a days.
routes of inoculation
1.
2.
3.
4.
Allantoic cavity
Yolk sac
Chorio- allantoic membrane (CAM)
Amniotic cavity
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1. CANDLING OF
EGG:
It is a process of
holding a strong
beam of light
above or below the
egg to observe the
embryo.
Candling is done in
a darkened room or
in an area shielded
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CANDLING
BOX
A candling lamp
consists of a strong
electric bulb
covered by a
plastic or
aluminium
container that has
a handle and
aperture.
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19
Inoculated
eggs are
candled
daily to see
the chicken
embryos
inside
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21
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MARKING OF AIRSAC
1. Hold the blunt end of the egg
against the aperture of the
candling lamp and note the
position of the head of the
embryo.
2.Draw a line on the shell marking
the edge of the air sac.
3.Draw an x approximately 2mm
above this line.
4.The x marks the inoculation site.
23
Embryonated
Egg
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1.
ALLANTOIC
9-12
days old egg required
INOCULATION
INFLEUNZA
NEWCASTLE
DISEASE
VIRUS
VIRUS WHICH
CAN BE
HARVESTED
AVIAN
ADENOVIRU
S
MUMPS
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STEPS
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28
29
STEPS
Candle the egg and mark on the shell
the position of the air space.
Puncture the shell over the centre of
the air cell.
Insert a 22 gauge needle,2 inches in
length on a syringe, into the egg
through the puncture in the shell.
Point the needle straight down for
depth of about 1-1/2 inches.
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35
36
STEPS
Candle the egg and locate an area on
the side of the egg that is free of large
blood vessels. Mark this area with a
pencil.
With a small grinding wheel, grind a
hole through the shell, but not the shell
membrane , at the site marked.
Puncture a second hole at the air space
end, this time penetrating the outer
shell membrane.
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CAM ROUTE OF
INOCULATION
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STEPS
Candle the egg and mark the
position of the embryo and the
outline of the airspace on the shell.
Punch a hole through the shell at the
edge of the airspace directly above
the embryo.
Using a 23- gauge, 1 inch needle on
a syringe make a short jab through
the punched hole, towards the
embryo.
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HARVESTING OF AMNIOTIC
FLUID
Remove the shell and shell
membrane below the air space.
Remove the fluid from the allantoic
cavity , the amnion should then be
clearly visible.
Remove the amniotic fluid with a 20gauge needle and syringe.
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CYTOPATHOGENIC
EFFECTS
49
1
2
Viral
growth
and
multiplicatio
n in the egg
embryo is
indicated by
antigen
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53
PRESENCE OF
BASOPHILIC
INCLUSION
BODIES
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PURPOSE OF INOCULATION
Diagnostic- Poxvirus
Herpes simplex virus
Mumps virus
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REFERENCES
Principles of virology: Molecular
biology, pathogenesis, and control;
S.J Flint, L.W. Enquist, R.M. Krug, V.R.
Racaniello, A.M. Skalka.
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THAN
K
YOU
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