1. EGG INOCULATION
TECHNIQUES
By
DR. R. KALAIMATHI
M.V.Sc. (Micro), Ph.D. (Biotech)
Mobile – 94447 72414

2. VIRUS CULTIVATION
SYSTEMS
1. BIOLOGICAL SYSTEM
2. EMBRYONATED EGGS
3. TISSUE CULTURE SYSTEM

3. 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

4. ANIMAL INOCULATION-
DISADVANTAGES
• Cost
• Maintenance
• Interference of immune system
• Individual variations
• Difficulty in choosing of animals for
particular virus

5. 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

6. •Cost- much less
•Maintenance-easier
•Less labour
• Readily available

7. • 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.

8. METHODS OF CULTIVATION
• Various routes of inoculation
a)Yolk sac
b) Allantoic sac
c) Chorioallantoic membrane
d. Amniotic cavity
e. Intravenous

9. CANDLING
BOX

11. EMBRYONATED EGG

12. BLOOD VESSELS

13. YOLK SAC ROUTE

14. YOLK SAC ROUTE
•Advantages
•Simplest method
•Mostly mammalian viruses
•Immune interference for most of avian
viruses
•Disadvantages
•Not suited for avian viruses

15. ALLANTOIC ROUTE – INOCULATION
SITE DETERMINATION

16. CHORIOALLANTOIC SAC ROUTE
OF INOCULATION

19. ALLANTOIC ROUTE
•Most popular
•Most of avian viruses
•High titered virus
•Simple technique

20. CHORIOALLANTOIC
MEMBRANE ROUTE
• Pox and Herpes viruses.
• ‘Pock Lesions’
• Suitable for plaque studies

25. CAM ROUTE OF INOCULATION

27. EGG INOCULATION ROUTES -
COMBINED

29. 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

31. INTRAVENOUS ROUTE
• Blue tongue virus
• Cherry red embryo
• Highly cumbersome
• Most sophisticated
procedure

33. EGG HOLDERS

38. HARVEST OF ALLANTOIC
FLUID

39. HARVEST OF EMBRYO

41. INFECTIOUS BRONCHITIS

42. IBV EMBRYOS

43. NDV – OCCIPITAL
HAEMORRHAGE

44. ND VIRULENT VIRUS

45. NDV NEUTRALIZATION

46. DUCK PLAGUE – VIRULENT
VIRUS

47. DUCK PLAGUE VACCINE VIRUS

48. BLUE TONGUE VIRUS – CHERRY
RED EMBRYO

49. BLUE TONGUE VIRUS –
CHERRY RED EMBRYO

50. Egg Holders

52. ‘POCK’ LESIONS ON CAM