6. Inoculation of embryonated eggs by the allantoic cavity route

Introduction

The most convenient method of propagating Newcastle disease virus in the laboratory is by the inoculation of the allantoic cavity of embryonated eggs.

All strains of Newcastle disease virus will grow in the cells lining the allantoic cavity. The virus enters these cells where it multiplies. As the cells are disrupted the virus is shed into the allantoic fluid.

Virulent strains of the virus will invade cells beyond the lining of the allantoic cavity and kill the embryo. The time taken for this to occur is the basis of the “Mean Death Time Assays”, which indicate the level of virulence.

The avirulent I-2 strain of Newcastle disease virus will not kill embryos inoculated by the allantoic cavity.

Inoculation of the allantoic cavity of embryonated eggs is a technique used in the following procedures:

1. Newcastle disease vaccine production

2. Establishing the infectivity titre of a suspension of Newcastle disease virus.

3. Isolation of Newcastle disease virus from field specimens for laboratory diagnosis.

Inoculation of the allantoic cavity

Materials

• Eggs: 9-day old or 10-day old embryonated eggs. Candle the eggs and mark the inoculation sites as described in Section 5. Eggs should be placed in an egg rack with the inoculation site uppermost.
• Egg shell punch.
• Cotton wool.
• A 70 percent alcohol solution in water.
• Syringe 1 mL.
• Needles preferably 25 gauge, 16 mm.
• Stationery tape (also called cello or sticky tape) or melted wax to seal the inoculation site.
• Inoculum. This must be free of microbial contamination.
• Discard tray.

Method

1. Use cotton wool and 70 percent alcohol to swab the end of the eggs to be inoculated. Allow the alcohol to evaporate.

2. Swab the eggshell punch with 70 percent alcohol solution. Place used cotton wool in discard tray.

3. Pierce a hole in the end of the egg at the marked inoculation site.

4. Attach needle to 1 mL syringe.

5. Draw inoculum into 1 mL syringe.

6. Keeping the needle and syringe vertical, place the needle through the hole in the eggshell. The needle will need to penetrate approximately 16 mm into the egg to reach the allantoic cavity.

7. Inject 0.1 mL of inoculum into the egg.

8. Withdraw the needle from the egg.

9. Seal the hole in the shell with stationery tape or melted wax.

10. Discard the used needles and syringes.

11. Place the inoculated eggs into a second incubator. Check the temperature and humidity of incubator.

Figure 7: Inoculation of the allantoic cavity

Tools for piercing a hole in the egg shell

A dental drill can be used if it is available. In most laboratories a tool called an eggshell punch can be improvised using materials that are cheap and easy to procure. Two examples are described, the first being the eggshell punch made in the workshop at the John Francis Virology Laboratory.

Figure 8: Egg shell punch

Example 1:

Materials: A small piece of copper approximately 3 cm by 1 cm. A length of 4 mm diameter stainless steel rod or steel wire. Brazing alloy

A hole is made in the middle of the piece of copper, which is then curved to the shape of an egg. A file is used to create a small spike at the end of the rod or wire, which is then pushed through the hole in the copper until the point extends one mm beyond the copper. The rod or wire is then brazed in place where it passes through the copper. The other end of the steel wire or rod is bent to form a handle.

Figure 9: Improvised egg shell punch

Example 2:

Materials:

A needle (20 gauge is suitable)

Rubber stopper

Insert the needle into the rubber stopper so that only 1 mm of the tip is showing. Use and store carefully as the tip of the needle will be very sharp.