9. Harvesting and storage of allantoic fluid

Introduction

Allantoic fluid from inoculated eggs will be harvested for three reasons.

1. To prepare I-2 Newcastle disease working seed or vaccine.

2. To use as antigen in the haemagglutination inhibition test.

3. To be tested for the presence or absence of Newcastle disease virus by the haemagglutination test. These results are then used to calculate the infectivity titre of a suspension of virus.

Minimizing contamination

During the harvesting of allantoic fluid for use as working seed or vaccine, steps must be taken to minimize the risk of exposure of the allantoic fluid to environmental contaminants. The following list contains appropriate steps to be taken.

Use aseptic technique to harvest the allantoic fluid.

Harvest the allantoic fluid in a designated room or area of the laboratory if possible. Many laboratories use a laminar flow cabinet for harvesting vaccine. Routine servicing of the cabinet should be carried out to ensure sterile conditions inside the cabinet.

Remove all materials not associated with the harvesting process from the room or laminar flow cabinet.

Thoroughly clean the room or cabinet with disinfectant. Irradiation with UV light prior to harvesting vaccine will help kill contaminating organisms.

Technicians should wear clean laboratory coats, clean hair covers, facemasks and scrub their hands with an antimicrobial soap.

Harvesting allantoic fluid to prepare working seed, vaccine or antigen

Materials

• Forceps or a small pair of scissors
• Absolute alcohol for flaming forceps
• Cotton wool
• 70 percent alcohol solution in water
• Discard tray
• 50 mL micropipette and tips or a wire loop
• Sterile Pasteur pipettes with short blunt ends
• 96 well microwell plate
• 10 percent washed red blood cells
• Sterile containers for receiving the harvested allantoic fluid.

Method

1. Chill eggs at 4°C for at least two hours to kill the embryo and to reduce the contamination of the allantoic fluid with blood during harvesting.

2. Remove stationery tape (if used to seal the eggs) and swab each egg with cotton wool soaked with 70 percent alcohol to disinfect and remove condensation from the shells.

3. Dip the forceps or scissors in disinfectant OR if using a Bunsen burner, dip the forceps or scissors in absolute alcohol and flame to sterilize. Remove the eggshell above the air space.

4. Discard embryos that are visibly contaminated.

5. Remove a sample of allantoic fluid from each egg. Use a micropipette and sterile tip, sterile glass pipette or a flamed loop. Test each sample for the presence of Newcastle disease virus by the haemagglutination (HA) test. See Section 10.

6. Discard embryos that do not test HA positive for Newcastle disease virus.

7. Use sterile glass Pasteur pipettes to harvest the allantoic fluid from the eggs. The pipettes can be either hand held or used unplugged and connected to a vacuum pump. Collect the fluid into sterile containers. See Figures 16 and 17.

Preliminary Quality Control

This step involves the inoculation of a general purpose broth culture. See Section 14 for more information about quality control.

1. Test each container for bacterial contamination by inoculating tryptic soy broth with test samples and incubation at 37°C overnight.

2. Centrifuge the samples of allantoic fluid or stand overnight at 4°C to allow particles including red blood cells to settle. The allantoic fluid should appear clear after centrifugation or standing overnight.

3. After 24 hours, read the results of the tests for bacterial contamination.

4. Use aseptic technique to transfer the clear allantoic fluid supernatant from containers that showed no bacterial growth into a sterile container for storage. This step pools the fluid and ensures homogeneity.

Figure 16: Harvesting allantoic fluid using a hand held Pasteur pipette

Figure 17: Harvesting allantoic fluid using a vacuum pump

Harvesting allantoic fluid to test for presence of haemagglutinin

The following method describes harvesting a small sample of allantoic fluid for testing for the presence of haemagglutinin using the rapid or micro tests described in Section 10.

Materials

• Forceps or a small pair of scissors
• Absolute alcohol for flaming forceps
• Cotton wool
• 70 percent alcohol solution
• Discard tray
• 50 mL micropipette and tips, a wire loop or sterile Pasteur pipettes
• 96 well V-bottom microwell plate and cover or glass slide

Method

1. Chill eggs at 4°C for at least two hours to kill the embryo and to reduce the contamination of the allantoic fluid with blood during harvesting.

2. Remove sticky tape (if used to seal the eggs) and swab each egg with cotton wool soaked with 70 percent alcohol to disinfect and remove condensation from the shells.

3. Dip the forceps or scissors in absolute alcohol and flame to sterilize. Remove the eggshell above the air space.

4. Discard embryos that are visibly contaminated.

5. Remove a sample of allantoic fluid from each egg. Use a micropipette and sterile tip, sterile glass pipette or a flamed loop and dispense the sample according to method being used for the test

Note:

When harvesting allantoic fluid from each inoculated egg to establish infectivity titres, additional allantoic fluid is usually not harvested for further use. In this case, rigorous steps to minimize contamination are not necessary.

Figure 18: Harvesting allantoic fluid for HA test using a micropipette

Storage of allantoic fluid

The optimum temperature for storage of allantoic fluid containing live Newcastle disease virus is -70°C. Storage at -20°C is not as effective and the infectivity titre will slowly decrease. The action of the freezing and thawing also decreases the infectivity titre of the virus. Allantoic fluid containing Newcastle disease virus has been stored for up to 6 weeks at 4°C without significant loss of titre.

Allantoic fluid will be stored for two purposes.

1. Antigen for use in the haemagglutination inhibition test as described in Section 11. Prepare 1 mL aliquots of undiluted allantoic fluid in vials.

2. Preparation of vaccine.

Storage of allantoic fluid at 4°C for use as a wet vaccine

Diluents containing a stabilizing agent are used in the preparation of wet Newcastle disease vaccine. Suitable stabilizing agents are gelatin and skim milk powder. Diluents containing 2 percent gelatin solution or 8 percent skim milk powder in phosphate buffered saline are sterilized prior to use and mixed one part diluent with one part allantoic fluid. A further dilution step in two parts of PSG antibiotic solution will reduce the risk of growth of contaminating bacteria during storage. Trials at John Francis Virology Laboratory have shown that 1 percent gelatin is a superior storage agent to 4 percent skim milk.

(Bensink Z. and P. Spradbrow, 1999)

For more details see Section 13: The production of I-2 Newcastle disease vaccine.