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The technique used to cultivate viruses in embryonated eggs is referred to as In-ovo technique.

Embryonated eggs i.e. Fertilized Hens Eggs form a good medium to cultivate viruses. They are as good as live animal since they as maintained sterile inside the shell and have natural resistance against contaminating bacteria. They are also easily available, contain sufficient food and provide a constant environment for the growth of viruses.

Fertile chicken eggs incubated for 5-12 days can be inoculated through the shell aseptically. The opening is sealed with paraffin wax and the egg is incubated at 37-38oC for the time required for the growth of virus.

The virus cultivation in embryonated eggs involves three phases:

(A) PRE-INOCULATION PHASE:

This involves

  1. Candling i.e.; the fertile eggs are candled in a dark room in an egg candler. The cracked, infertile eggs having dead embryos are discarded. The undulating movements of the living embryo are observed. A point is made with the help of a pencil as to where the inoculation is to be done.
  2. Sterilization: This is done with a swab lightly squeezed in tincture iodine.
  3. Piercing( Drilling) the shell: A square opening is drilled in the shell at the selected point using a dentist drill.

(B) INOCULATION PHASE:

This involves:

  1. Yolk-sac inoculation
  2. Chorioallantic membrane (CAM) inoculation
  3. Allantoic sac inoculation
  4. Amniotic cavity inoculation
  5. Intravenous inoculation
  6. Intracerebral inoculation

(C) POST INOCULATION PHASE:

This includes:

  1. Sealing
  2. Incubation
  3. Humidity at 60%
  4. Position of the egg
  5. Harvesting

Depending on the type of virus, different types of viruses are inoculated into different parts of the embryonated eggs and it is incubated for hatching.

 

Part of the embryonated egg

Age of the fertile egg

Virus inoculated

1.

Yolk Sac

5-8 days old

Mumps virus, Marek’s disease virus, infectious anemia virus, Avian encephalomyelitis virus

2.

Chorioallantic membrane (CAM)

10-12 days old

Canine distemper virus, Herpes zoster, Newcastle disease and Pseudo rabies virus

3.

Allantoic sac

9-12 days old

Influenza virus, mumps virus, Parainfluenza virus, and Ranikhet disease virus.

4.

Amniotic cavity

7-15 days old

Mumps and Influenza virus

5.

Intravenous

10-15 days old

Blue tongue virus

6.

Intra cerebral

8-14 days old

Herpes simplex and Rabies virus

 

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