EGG INOCULATION TECHNIQUES By DR. R. KALAIMATHI M.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 sac b) Allantoic sac c) Chorioallantoic membrane d. Amniotic cavity e. Intravenous

CANDLING BOX

EMBRYONATED EGG

BLOOD VESSELS

YOLK SAC ROUTE

YOLK SAC ROUTE Advantages Disadvantages 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 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

INTRAVENOUS ROUTE Cherry red embryo Highly cumbersome 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