Virus Propagation in Embryonating Eggs

INTRODUCTION The embryonating chicken egg has long been one of the most widely used host system for the isolation, propagation and characterization of viruses and for the production of viral vaccines.

the success or failure of this system for propagating and isolating viruses depends upon several conditions: - route of inoculation. - age of embryo. - Incubation temperature. -Length of incubation time following incubation. -Volume and dilution of the inoculum used.

*The propagation of viruses in embryonating chicken eggs can be detected in most cases, by one or more of the following: 1- Embryo death. 2- Lesions in the chorioallantoic membrane (CAM) such as edema , cutaneous hemorrhage, abnormal development of muscles and abnormalities in the visceral organs including enlargements of the liver and spleen , greenish discoloration of the liver , necrotic foci on the liver or heart.

More direct and definitive method for detection of virus infection chicken embryos include ability of the amniotic allantoic fluid (AAF) to cause hemagglutination (HA) of wash chicken erythrocytes, use of serologic and molecular techniques and electron microscopy. ** Special care should be taken to differentiate lesions that may be the presence of bacterial agents.

Structure of the embryonating egg.

Sample preparation : -The type of specimens submitted for virus isolation will differ depending on the disease suspected but will usually consist of tissues, or swabs. -Suspension of these specimens should be prepared in Tris-buffered tryptose broth, nutrient broth, brain-heart infusion broth or other suitable medium containg antibiotics. -

-The concentration of antibiotics used may depend on the type of sample being processed but could contain, as an upper limit , penicillin (10,000 IU/ml), streptomycin sulphate (2.0 mg/ml), gentamicin sulphate (1.0 mg/ml), kanamycin sulphate (0.65 mg/ml) and amphotericin B (0.02 mg/ml). The antibiotics may vary with local conditions and applications. It is important that the PH of the antibiotics diluent be adjusted to the range of 7.0- 7.4.

Inoculation routes of embryonating egg: The four most common routes for the inoculation of embryonating egg are via the allantoic sac, yolk sac, chorioallantoic membrane (CAM) and amniotic sac.

Allantoic sac inoculation: Candle 8 to 11 day-old embryonating eggs, making sure that all air cells are in the normal position. Place eggs on an egg flat, air cell up, and disinfect the area directly at the top of the egg (the area over the air cell). Drill a small hole through the eggshell along the centre axis at the top of the egg.

Using a syringe fitted with a 25-gauge 5/8-inch (16-mm) needle, inculcate 0.1-0.3 ml of inoculum per egg by inserting the needle vertically through the hole the entire length of the needle and injecting the desired amount. Seal hole and return the eggs to the incubator. Eggs inoculated by the allantoic route are normally incubated 3-7 days post inoculation.

Collection of specimen from embryonating eggs. Inoculated eggs should be candled at least once a day to identify eggs with dead embryos. such eggs are removed from the incubator. Embryos that die within the first 24 hr should be discarded as nonspecific death caused by injury or bacterial contamination

All embryo deaths beyond 24 hr should be considered suspect. If the inoculated eggs have live embryos following the specified incubation period, eggs should be refrigerator for a minimum 4 hr or overnight to kill the embryo and allow the blood to clot before harvesting egg contents. All eggs from which specimens are to be collected should be surface-disinfected with 70% ethanol or other suitable disinfectant

Collection of AAF 1- Crack the shell over the air cell by a sterile forceps. 2- Depress the membranes over the yolk sac with the forceps. 3- using a 5ml pipette or syringe and needle, aspirate the fluid and place into a sterile vial. 4- Culture a loopful of the AAF for bacterial using blood agar or nutrient agar. Incubate plates at 37C overnight and record results. 5- Clarify the AAF by centrifugation at 1500g for 10min and test AAF for HA activity using a standard micortiter procedure for influenza virus. 6- Store AAF at -70C for passage or other use.

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