AVIAN VIRUS ISOLATION (VI) TECHNIQUES

Purpose: To isolate and identify common avian viruses

Method A: Embryonating chicken eggs

  1. Specific Pathogen Free embryonating chicken eggs provide an excellent medium in which most avian viruses will readily propagate.

  2. Infected tissues or swabs of tissues may be used. The material, suspended in a medium which contains antibiotics, is filtered through a millipore filter. The antibiotics and filtration are both used to eliminate bacterial contamination.

  3. Embryonating chicken eggs are inoculated with the prepared sample.

  4. The three most common routes of embryo inoculation are:

  1. allantoic sac (9-11 day embryos)

  2. chorioallantoic membrane (9-11 day embryos)

  3. yolk sac (5-7 day embryos)

  1. The route of inoculation depends on the suspected virus.

  1. allantoic sac - IB, AI and NDV

  2. chorioallantoic membrane - LT and Pox

  3. yolk sac - AE and REO

  1. Some of these viruses produce lesions in the embryos (e.g. Infectious Bronchitis - dwarfing and stunting and excessive urates in kidneys) others require further testing such as ether sensitivity, hemagglutination.

Method B: Cell culture

  1. Can be used as an alternative to embryonating chicken eggs.

  2. Various types of tissues may be used (e.g. liver, kidney, fibroblasts). To prepare these cell cultures, organs are removed from specific pathogen free embryonating eggs.

  3. Tissues are prepared as in #2 above (Method A)

  4. Some viruses produce characteristic cytopathic effects (CPE) while others don't and must be examined using fluorescent antibodies (FA) or special dyes.

 

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