Embryonated Egg culture Technique for Viruses
Goodpasture and Burnet
in 1931 first used the embryonated hen’s egg for the cultivation of virus. The
process of cultivation of viruses in embryonated eggs depends on the type of
egg being used. Eggs provide a suitable means for:
•
the primary isolation and identification of
viruses
•
the maintenance of stock cultures
•
and the production of vaccines
Terms most often refer
to eggs:
•
Embryonated:
having an embryo,
•
Unembryonated: not having an embryo
•
De-embryonated: having lost an embryo
Embryonated
egg are refered to an advanced stage of development and not merely after fertilization.
Advantages:
•
An embryo is an early developmental stage of
animals marked by rapid differentiation of cells.
•
Birds undergo their embryonic period within the
closed protective case of an egg, which makes an incubating bird egg a nearly perfect
system for viral propagation.
•
It is an intact and self-supporting unit,
complete with its own sterile environment and nourishment.
•
It furnishes several embryonic tissues that
readily support viral multiplication
•
Defense mechanisms are not involved in
embryonated eggs
•
Cost- much less, Maintenance-easier, Less labor
and Readily available
Inoculation of Virus:
Chicken, duck, and
turkey eggs are the most common choices for inoculation. The egg used for
cultivation must be sterile and the shell should be intact and healthy. Rigorous
sterile techniques must be used to prevent contamination by bacteria and fungi
from the air and the outer surface of the shell.
Detection of viral
growth:
Viruses multiplying in
embryos may or may not cause effects visible to the naked eye. The signs of
viral growth include:
•
Death of the embryo
•
Defects in embryonic development
•
and localized areas of damage in the membranes,
resulting in discrete opaque spots called pocks
If a virus does not
produce obvious changes in the developing embryonic tissue, virologists have
other methods of detection. Embryonic fluids and tissues can be prepared for
direct examination with an electron microscope. Certain viruses can also be
detected by:
•
their ability to agglutinate red blood cells
•
or by their reaction with an antibody of known
specificity
Parts of Embryonated Egg:
The air sac is
important to the developing embryo for respiration and for pressure adjustments.
The shell and shell membrane function both as a barrier and as an exchange
system for gases and liquid molecules. The chorioallantoic sac and its contents
(allantoic fluid) remove waste products produced by the developing embryo. This
Membrane and its contents increase in size as the embryo grows. The yolk sac is
the source of nourishment for the developing Embryo. As the embryo develops,
the yolk sac decreases in size until it is completely absorbed into the
digestive system of the mature embryo. The amnion is a thin membrane that
encloses the embryo and protects it from physical damage. It also serves as an
exchange system and is best seen in the younger embryos.
An embryonated egg offers various sites for the cultivation of viruses;
1. Chorioallantoic
membrane(CAM)
2. Amniotic
Cavity
3. Allantoic
Cavity
4. Yolk sac
Figure 1a & 1b: An Embryonated egg showing sites of inoculation
The chosen route of
inoculation and age of the embryo are determined by the given virus selectivity
for a certain membrane or developmental stage of the embryo. For example Infectious
bronchitis virus is propagated in the yolk sac of a 5-6 day old embryo. Whereas
Rous-sarcoma virus is inoculated on the chorioallantoic membrane of a 9-11 day
old embryo and will produce pocks 5-10 days post-infection.
Candling of Egg: It
is the process of holding a strong light above or below the egg to observe the
embryo. A candling lamp consists of a strong electric bulb covered by a plastic
or aluminum container that has a handle and an aperture.
Figure 2a & 2b: Candling of egg to observe embryo
1. Chorioallantoic membrane (CAM): This method has been widely used in veterinary
virology. Many viruses grow readily or can be adapted to grow on the CAM. Viruses
produce visible foci or ‘pocks’, inclusion bodies, oedema or other
abnormalities. Each infectious virus particle forms one pock. Viruses which can
be grown include: Herpes viruses and poxviruses
2. Amniotic Cavity: The
virus is introduced directly into the amniotic fluid that bathes the developing
embryo. The volume of
fluid in the
infected amniotic sac
is small (1-2 ml). The amniotic
route is recommended for the
primary isolation of human viruses: mumps virus, and influenza A, B and C
viruses. It has little application in veterinary virology. Newly isolated
influenza viruses may require several passages before they adapt to growth by
other routes, such as allantoic.
3. Allantoic Cavity: Many
viruses such as Newcastle disease virus can grow readily. Other viruses such as
influenza, may require repeated amniotic passages before becoming adapted to
the egg and grown in the allantoic cavity. Allantoic inoculation is a quick and
easy method that yields large amounts (8–15 ml) of virus-infected egg fluids.
4. Yolk sac: It is also a simplest method for growth
and multiplication of virus. Mostly mammalian viruses are isolated using this
method. Immune interference mechanism can be detected in most of avian viruses.
This method is also used for the cultivation of some bacteria like Chlamydiae
and Rickettsiae.
