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EGG INOCULATION TECHNIQUES
By
DR. R. KALAIMATHIM.V.Sc. (Micro), Ph.D. (Biotech)
Mobile – 94447 72414
VIRUS CULTIVATION SYSTEMS
1. BIOLOGICAL SYSTEM
2. EMBRYONATED EGGS
3. TISSUE CULTURE SYSTEM
IN-VIVO / IN-VITRO?
1. Biological System
a) Natural host
b) Experimental animals In - vivo
c) Transgenic animals
2. Embryonated Eggs –
In - vivo & In - vitro
3. Tissue Culture System – In - Vitro
ANIMAL INOCULATION- DISADVANTAGES
• Cost
• Maintenance
• Interference of immune system
• Individual variations
• Difficulty in choosing of animals for particular virus
EMBRYONATED EGGS
ADVANTAGES• Isolation and cultivation of many avian and
few mammalian viruses
• Ideal receptacle for virus to grow
• Sterile & wide range of tissues and fluids
•Cost- much less
•Maintenance-easier
•Less labour
• Readily available
• Free from bacteria and many latent viruses.
• Free from specific and non specific factors of defence.
• Sensitive to viruses which do not produce infection in adult birds.
METHODS OF CULTIVATION • Various routes of inoculation
a)Yolk sacb) Allantoic sac
c) Chorioallantoic membraned. Amniotic cavitye. Intravenous
CANDLING BOX
EMBRYONATED EGG
BLOOD VESSELS
YOLK SAC ROUTE
YOLK SAC ROUTE
•Advantages•Simplest method
•Mostly mammalian viruses
•Immune interference for most of avian viruses
•Disadvantages
•Not suited for avian viruses
ALLANTOIC ROUTE – INOCULATION SITE DETERMINATION
CHORIOALLANTOIC SAC ROUTE OF INOCULATION
ALLANTOIC ROUTE
•Most popular
•Most of avian viruses
•High titered virus
•Simple technique
CHORIOALLANTOIC MEMBRANE ROUTE
• Pox and Herpes viruses.
• ‘Pock Lesions’
• Suitable for plaque studies
CAM ROUTE OF INOCULATION
EGG INOCULATION ROUTES - COMBINED
AMNIOTIC ROUTE
• Primary isolation of influenza and mumps viruses
• Growth of virus detected by haemagglutination
Hemorrhagic lesions in the proventriculus, seen at necropsy in fowl with avian influenza
Influenza Virus
Mumps Virus
INTRAVENOUS ROUTE
• Blue tongue virus• Cherry red embryo• Highly cumbersome• Most sophisticated
procedure
EGG HOLDERS
HARVEST OF ALLANTOIC FLUID
HARVEST OF EMBRYO
INFECTIOUS BRONCHITIS
IBV EMBRYOS
NDV – OCCIPITAL HAEMORRHAGE
ND VIRULENT VIRUS
NDV NEUTRALIZATION
DUCK PLAGUE – VIRULENT VIRUS
DUCK PLAGUE VACCINE VIRUS
BLUE TONGUE VIRUS – CHERRY RED EMBRYO
BLUE TONGUE VIRUS – CHERRY RED EMBRYO
Egg Holders
‘POCK’ LESIONS ON CAM