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EMBRYONATED

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

foetus in its early


stages of
developments
especially before
it has reached a
distinctively

Selection of egg
must be sterile
shell should be intact and healthy.
should be obtained from nonvaccinated, disease-free flocks

incubation - 38 39C and 60 70%


humidity.
need to be turned at least twice a
day
or
rolled continually in a specially
designed egg incubator.

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 ,

- adjusting hatching control


temperature constantly
variation of temperature - ambient to 70 C

controlled by JUMO/ EGO


German Capillary thermostat having accuracy of + 0.5 c.
Capacity:
50 to 2000 eggs

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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

Some viruses do not show growth on


primary inoculation into the egg.
Slight amount of bacterial
contamination in the inoculum may
kill the embryo.
Eggs may be contaminated with
mycoplasma and latent fowl viruses
which may interfere with the growth
of other virus.
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EGGS USED IN VIROLOGY


HEN EGG
DUCK EGG
TURKEYS EGG

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ADVANTAGES AND DISADVANTAGES


OF HEN EGG

For making teaching material


For characterisation or vaccine
production of influenza virus
An extra method for isolating difficult
wild strains of influenza A and B.
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DISADVANTAGES
Used much less now a days.

Tighter regulations over the use of


embryo tissues????
Eradication of smallpox?????
Development of simpler and more reliable
methods for typing herpes simplex and
development of molecular techniques
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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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The egg is placed


against the
aperture and
illuminated by
light.

19

Inoculated
eggs are
candled
daily to see
the chicken
embryos
inside
20

Under the candling lamp, the embryo


appears as a dark shadow with the head
as a dark spot (eye).
Healthy embryos will respond to the
light by moving.

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Dead embryo-Candling will reveal a small dark


area and disrupted blood vessels.

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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.

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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

Candle the eggs and mark the inoculation site.


Puncture the shell over the centre of the air
sac. A convenient punch can be made by
cutting off a hypodermic needle and fitting it to
a syringe barrel.
Insert a 23- gauge needle, 1-1/2 inches in
length on a 1 ml syringe, into the egg through
the puncture in the shell at a 45 angle to the
long axis of the egg and away from the
embryo.
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ALLANTOIC ROUTE INOCULATION


SITE DETERMINATION

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Inject 2/10th (O.2ml)of fluid into the


egg.
Seal the puncture with nail polish or
cellophane tape.
Position the eggs and incubate at
37o C.

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Harvesting of Allantoic Fluid


Eggs must be chilled to obtain
allantoic fluid free of RBCs.
Clean the upper half of the shell with
70% alcohol.
Cut away the shell above the air
space.
Peel away the white opaque shell
membrane lining the air space,
exposing the transparent allantoic
membrane directly beneath.

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Tear the allantoic membrane with sterile


forceps.
Attach a ballpoint needle to a syringe
and insert the needle into the cavity.
Remove the fluid by suction.
Culture the harvested fluid in a suitable
medium for a sterility check.
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YOLK SAC INOCULATION


6-8 days old eggs required.
Rickettsiae, Chlamydia
trachomatis, C. psittaci,
basophilic viruses( Bedsonia)
can be harvested.
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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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YOLK SAC ROUTE

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Express 0.5 ml of inoculum into the


yolk sac.
Seal the puncture with nail polish or
cellophane tape.
Incubate the eggs.

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HARVESTING OF YOLK SAC


Disinfect the upper half of the shell.
Remove the shell, shell membrane
and underlying chorio-allantoic
membrane.
Lift the embryo up with sterile
forceps to expose the attached yolk
sac.
Pull the yolk sac free with another
pair of forceps and place it in a
sterile petridish.

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Chorio- Allantoic Membrane (CAM)


Inoculation
11- 14 days old eggs required.
Viruses inoculated- Herpes simplex
virus, Pox virus.

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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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Place a drop of sterile physiological


saline on the side hole and gently
tease apart the fibers of the shell
membrane with a 27- gauge needle.
When the shell membrane has been
penetrated, the drop of saline will be
drawn into the egg as a result of
separation of the CAM and shell
membrane.
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Apply negative pressure to the air space


opening by means of mouth suction
with a rubber tube. As the air is
removed the CAM will drop from the
shell around the side hole, creating an
artificial airspace , outline the limits of
artificial airspace.

Express upto 0.2 ml of inoculum through


the side opening onto the CAM.
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41

Seal openings with cellophane tape.


Gently rotate the egg to spread the
inoculum over the entire CAM under the
false air space.
Incubate the eggs on side with false air
space upward.

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Harvesting the CAM


Membrane
Disinfect the shell.
With sterile scissors, cut through the
shell along the longitudinal axis,
about 1/3 down from the upper
surface.
Gently remove the shell to a discard
pan.
With sterile forceps,lift the CAM, cut
free.
Place the CAM in sterile saline and

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AMNIOTIC CAVITY INOCULATION

1. 9-10 days old


egg required.
2.The amniotic
sac is mainly
inoculated for
primary
isolation of
Influenza virus
and Mumps
virus

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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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46

When the amniotic membrane is


penetrated, the embryo will be seen to
follow the movements of the needle.
Express upto 0.2 ml of inoculum.
Seal the puncture with nail polish or
cellophane tape and incubate at 37o C.

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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

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1
2

Viral

growth
and

multiplicatio
n in the egg
embryo is
indicated by

antigen

death of the embryo


embryo cell damage
formation of typical
pocks or lesions
on the egg membranes
oedema of the
developing membranes
inclusion bodies
Presence of viral
in egg fluids

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The process of cultivation of viruses in embryonated


eggs depends on the type of egg which is used.
A hole is drilled in the shell of the embryonated egg,
and a viral suspension or suspected viruscontaining tissue is injected into the fluid of the
egg.
Viral growth and multiplication in the egg embryo is
indicated by the death of the embryo, by embryo
cell damage, or by the formation of typical pocks or
lesions on the egg membranes, oedema of the
developing membranes, inclusion bodies in sections
prepared from embryo tissues or the presence of
viral antigens in the egg fluids.

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GROWTH OF VIRUS ON THE


CAM
Formation of characteristic pocks.
Variola produces small circular pocks, dome
shaped, no surrounding necrosis or
haemmorrhage whereas Vaccinia virus larger
lesions , flattened with necrosis and
haemmorrhage.

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Herpes simplex virus- small , oval


shaped with no evidence of necrosis.

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GROWTH OF A VIRUS IN THE YOLK


SAC

PRESENCE OF
BASOPHILIC
INCLUSION
BODIES

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Viruses which can be harvested by


various routes:An Overview

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PURPOSE OF INOCULATION
Diagnostic- Poxvirus
Herpes simplex virus
Mumps virus

Vaccine Production- Influenza virus

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How Vaccines are Produced in Eggs

In egg culture, flu viruses are injected into


chicken egg embryos, where they multiply. After
several days of incubation a machine opens the
egg and harvests the virus, which is then purified
and chemically killed.
On average it takes one or two eggs to produce a
single dose of annual flu vaccine.

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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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