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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 c) Transgenic animals In - vivo 2. Embryonated Eggs – In - vivo & In - vitro 3. Tissue Culture System – In - Vitro ANIMAL INOCULATIONDISADVANTAGES • • • • • 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 •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 Influenza Virus Hemorrhagic lesions in the proventriculus, seen at necropsy in fowl with avian influenza 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