1. EGG INOCULATION TECHNIQUESBy
DR. R. KALAIMATHI
M.V.Sc. (Micro), Ph.D. (Biotech)
Mobile –

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 Disadvantages 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 Growth of virus detected by haemagglutination
Primary isolation of influenza and mumps viruses
Growth of virus detected by haemagglutination
Influenza Virus
Hemorrhagic lesions in the proventriculus, seen at necropsy in fowl with avian influenza
Mumps Virus

31. INTRAVENOUS ROUTE Cherry red embryo Highly cumbersome
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