A Basic Laboratory Manual for the Small-Scale Production

and Testing of I-2 Newcastle Disease Vaccine

By: Sally E. Grimes

Introduction

Chickens are susceptible to many infectious diseases. One of the most important of these is the viral
disease known as Newcastle disease, which causes devastating losses in both commercial and village
chickens. Reducing losses of large numbers of village chickens to virulent Newcastle disease is an
essential first step to improving their productivity. Newcastle disease can be controlled by the use of
vaccines. There are many Newcastle disease vaccines suitable for use in commercial chickens. These
are available on the international market. The I-2 Newcastle disease vaccine has been developed for
local or regional production and use in controlling Newcastle disease in village chickens.

Many Newcastle disease vaccines deteriorate after storage for one or two hours at room temperature.
This makes them unsuitable for use in villages where the vaccine may need to be transported for
hours or in some cases days at ambient temperature. The I-2 Newcastle disease vaccine is more
robust and is known as a thermostable vaccine. Thermostable vaccines still require long-term storage
in the refrigerator. However during transportation of the vaccine to the field, the vaccine will not
deteriorate as quickly as the traditional vaccines. Evaporative cooling provided by wrapping the
vaccine in a damp cloth will be adequate for maintaining the viability of the vaccine during
transportation to remote villages. However if it is stored in direct sunlight or allowed to reach high
temperatures (above 37°C) for more than a few hours it too will deteriorate and be unsuitable for use
as a vaccine.

Immunity to Newcastle disease virus

Chickens that survive infection with virulent Newcastle disease virus develop a long lasting immunity
to further infection with Newcastle disease virus.

The basis of this immunity is:

1. Circulating antibodies.
2. Secreted antibody producing mucosal immunity.
3. Cell mediated immunity.

1. Collection of blood from chickens

Introduction

Blood is collected from chickens for two purposes:

1. To obtain serum which will be tested for Newcastle disease virus antibodies, no anticoagulant is
required and the blood is allowed to clot. The levels of antibody detected in individual birds and flocks
give an indication of the response to a vaccination. It also indicates whether birds have been
challenged by field strains of Newcastle disease virus.

2. To obtain red blood cells, the blood is collected into anticoagulant. The cells are washed and used
to test for the presence of virus in the haemagglutination test. They are also used in the
haemagglutination inhibition test for the presence of antibodies.

Wing vein bleeding

Materials
 • 2.5 mL syringes
• 25 gauge needles for small chickens
• 23 gauge needles for larger chickens
• Cotton wool
• 70 percent alcohol solution
• Labels or marking pen to label each syringe

Method

1. Ask an assistant to hold the chicken horizontally on its back. The assistant uses one hand to hold
the legs and places the other hand under the back to support the chicken.

2. Pull a wing of the chicken out towards you.

3. Note the wing vein, clearly visible running between the biceps and the triceps muscles. The wing
vein forms a V (bifurcates). Note the tendon of the pronator muscle that runs across the V.

4. Pluck away any small feathers that obscure the vein.

5. Disinfect the area around the bleeding site by swabbing with 70 percent alcohol.

6. Insert the needle under the tendon. Direct the needle into the wing vein in the direction of the flow of
blood. Do not insert the needle too deeply. Keep clear of the ulnar nerve.

7. Once the tip of the needle is in the vein, gently pull the plunger of the syringe. Blood will flow into
the syringe. If blood does not flow, release the plunger and make a very slight adjustment to reposition
the end of the needle.

8. Be patient and use a gentle suction to withdraw the blood. Chicken veins collapse readily.

9. If a haematoma forms, try bleeding from the other wing.

10. After removing the needle, apply pressure to the vein for a few seconds to discourage further
bleeding.

11. Ideally the needle should be removed into a needle disposal container and the cap place on the
end of the syringe to prevent leakage of the serum. However in many places these containers are not
available and the cap will be placed over the needle.

TAKE CARE! Do this very carefully to avoid a needle stick injury.

12. Pull the plunger back approximately 1 cm and place the syringe at an angle with the needle end up
in a rack facilitate clotting.
 2. Preparation of washed chicken red blood cells
Introduction

Chickens used for the supply of blood for the preparation of red blood cells should be housed
separately from chickens used for other purposes. Usually they are not vaccinated with Newcastle
disease vaccine. Blood from vaccinated chickens is acceptable if that is all that is available. Collect
blood from more than one chicken. A collection of 1.0 mL from each of three chickens will usually give
between 8 to 10 mL of a 10 percent solution of washed red blood cells. If you require a larger volume
of washed red blood cells, collect more blood and adjust the volume of anticoagulant accordingly.

There are four steps in the preparation of washed red blood cells.

1. Collection of the blood.

2. Washing the red blood cells.
3. Measuring the packed cell volume.
4. Preparation of a 10 percent suspension of red blood cells for storage.

Anticoagulant

Blood is collected from the wing veins of three chickens as described in Section 1. It is mixed with an
anticoagulant

Two techniques for collection of blood into anticoagulant

Technique 1

1. Place 1 mL of ACD or 3 mL of Alsever's solution into a sterile bottle with a lid.

2. Bleed the first chicken and take 1 mL of blood. Immediately remove the needle from the syringe,
gently push down the plunger and transfer the blood to the bottle. Replace the lid on the bottle and
rotate it gently to mix.

3. Repeat Step 2 and take 1 mL of blood from a second chicken. Transfer the blood to the bottle of
blood and anticoagulant and rotate it gently to mix.
4. Repeat Step 2 and take 1 mL of blood from a third chicken. Transfer the blood to the bottle of blood
and anticoagulant and rotate it gently to mix.

The bottle now contains either 3 mL of blood mixed with 1 mL of ACD OR 3 mL of blood mixed with 3
mL of Alsever's solution.

Technique 2

1. Have the correct volume of anticoagulant in the syringe before the blood is taken. Allow 0.33 mL of
ACD or 1 mL of Alsever's solution per mL of blood.

2. Bleed each of three chickens and take 1 mL of blood into the syringe.

3. After collecting the blood from all three chickens, remove the needles from the syringes and pool
the blood samples in a sterile bottle with a lid.

Washing the red blood cells

After collection of the blood into an anticoagulant, the cells are washed and stored. It is best to use
dextrose gelatin veronal (DGV) to wash the cells. If you do not have all the reagents to prepare DGV,
use phosphate buffered saline (PBS).

Always treat blood gently to avoid heamolysis.

Mix gently with the anticoagulant.

Do not squirt the blood through the needle.

Materials

• Blood in anticoagulant
• DGV or PBS solution for washing.
• Sterile 20 mL centrifuge tube with a lid or a 20 mL bottle with a lid, to fit the centrifuge bucket.
• Pasteur pipette or 10 mL graduated pipette with pipette filler.

Method

1. Transfer the blood to a container suitable for centrifugation.

2. Add DGV to fill the container. Mix gently.

3. Centrifuge at 500 g for 10 minutes.

4. Use a Pasteur pipette or a 10 mL glass pipette to remove the supernatant. Take care not to disturb
the pellet of red blood cells.

5. Repeat Steps 2, 3, and 4 twice.

The cells have now formed a pellet after being washed three times and centrifuged. The next steps
are the measurement of the packed cell volume (PCV) and preparation of a 10 percent solution of
cells for storage.

Preparation of 10 percent red blood cells without using a micro-haematocrit centrifuge to

measure packed cell volume (PCV)

There are two methods.

1. Use a micropipette or glass pipette to remove one mL of the pellet of packed red blood cells after
they have been washed as described above. Add 9 mL of DGV to dilute to a 10 percent suspension.

2. Use a graduated centrifuge tube to prepare the washed red blood cells. Measure the volume of the
pellet of washed red blood cells. Calculate the volume of DGV storage solution required to prepare a
final suspension of 10 percent. Use a glass pipette to add or remove DGV to achieve the calculated
volume.

Storage of 10 percent suspension of red blood cells

Store the suspension at 4°C. As the cells settle under gravity, the storage solution should remain
clear. Any red colour in the storage solution indicates haemolysis of the cells and the suspension is
not suitable for use and should be discarded. Normally a 10 percent suspension in DGV can be kept
for one week at 4°C.

Storage of red blood cells in phosphate buffered saline.

Red blood cells can be stored as a 10 percent suspension in phosphate buffered saline. These
suspensions can be expected to haemolyse after one or two days at 4°C. The red blood cells will
settle under gravity. If the storage solution appears pink, the cells have started to lyse and a fresh
suspension of washed red blood cells should be prepared.

3. Haemagglutination test
Introduction

All strains of Newcastle disease virus will agglutinate chicken red blood cells. This is the result of the
haemagglutinin part of the haemagglutinin/neuraminidase viral protein binding to receptors on the
membrane of red blood cells. The linking together of the red blood cells by the viral particles results in
clumping. This clumping is known as haemagglutination.

Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the
presence of viral particles. The test does not discriminate between viral particles that are infectious
and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of
red blood cells.

Note that some other viruses and some bacteria will also agglutinate chicken red blood cells. To
demonstrate that the haemagglutinating agent is Newcastle disease virus, it is necessary to use a
specific Newcastle disease virus antiserum to inhibit the haemagglutinating activity.

Substances that agglutinate red blood cells are referred to as haemagglutinins.

Note:

The abbreviation HA is used in this manual for haemagglutinin and haemagglutination.

Two tests are described, the rapid test which takes one minute and the micro test which takes 45
minutes.

Figure 19: Principle of the haemagglutination test

A. –ve control well (no haemagglutinin) B. +ve control well (contains haemagglutinin)

Micro haemagglutination test in a V-bottom microwell plate

This method is convenient when testing allantoic fluid from a large number of embryonated eggs for
the presence or absence of haemagglutinin. A 1 percent solution of red blood cells is used. The cells
settle faster in V-bottom plates and there is a better contrast between positive and negative results
than observed in U-bottom plates.

Materials

• Inoculated eggs, chilled for at least 2 hours, preferably overnight

• Negative and positive control samples
• V-bottom microwell plate and lid
• Micropipette and tips to measure 50 µL
• 1 percent suspension of red blood cells
• 70 percent alcohol solution
• Cotton wool
• Forceps and/or small scissors
• Absolute alcohol
• Discard tray
• Microwell plate recording sheet. See Appendix 9

Method

1. Fill in the details of samples being tested on a recording sheet. Samples and controls will be
distributed into the wells as indicated on this sheet.

2. Use a micropipette to remove 50 mL of allantoic fluid from each egg and dispense into a well of the
microwell plate. Use a separate tip for each sample.

3. Include negative and positive control allantoic fluid samples on one of the plates.

4. Dispense 50 mL of PBS into two wells. These wells will be the red blood cells controls for auto-
agglutination.

5. Add 25 mL of 1 percent red blood cells to each well.

6. Gently tap sides of the plate to mix. Place a cover on the plate.

7. Allow the plate to stand for 45 minutes at room temperature.

8. Observe and record the results.

Results
 The settling patterns of single and agglutinated red blood cells are different. Single cells roll down
the sides of the V-bottom well and settle as a sharp button. Agglutinated cells do not roll down the
sides of the well to form a button. Instead they settle as a diffuse film.

Negative HA result = a sharp button

Positive HA result = a diffuse film

Red blood cell control = a sharp button

Mark the HA results on microwell recording sheet.

See Appendix 10 for an example completed microwell plate recording sheet.

Table 2: Summary of Haemagglutination tests

Tests Result Interpretation

Rapid HA, Micro Positive Presence of viral particles that may or may not be infectious.
HA
Rapid HA, Micro Negative Absence of viral particles or presence of viral particles in levels too low
HA to detect.

Titration to establish haemagglutinin (HA) titre of a suspension of virus

The haemagglutination test is used to quantify the amount of Newcastle disease virus in a suspension.
This is done by carrying out two-fold serial dilutions of the viral suspension in a microwell plate and
then testing to determine an end point. This result can then be used to determine the amount of
haemagglutinin in the suspension and is expressed as a HA titre.

Materials

• 96 well V-bottom microwell plate and cover

• 25 mL single and multi-channel micropipettes and tips
• PBS
• 1 percent chicken red blood cells
• Sample to be titrated
• Reagent troughs
• Microwell plate recording sheet. See Appendix 9.

Method

1. Record on recording sheets how samples will be dispensed in microwell plate.

2. Dispense 25 µL of PBS into each well of the microwell plate.

3. Place 25 µL of test samples in first well of each row of column 1. Samples can be tested in duplicate
or triplicate if necessary.

4. Use a multichannel pipette to carry out two-fold serial dilutions across the plate until Column 11.
See Appendix 4 for instructions on carrying out two-fold serial dilutions.

5. Add 25 µL of PBS to each well.

6. Add 25 µL of 1 percent red blood cells to each well including Column 12. The wells in this column
are control wells that contain only PBS and red blood cells.

7. Gently tap sides of the plate to mix. Place a cover on the plate.

8. Allow the plate to stand for 45 minutes at room temperature.

9. Read and record the results in each well. All the control wells should be HA negative.

10. HA negative: A sharp button of red blood cells at the bottom of the V-bottom well.

11. HA positive: A hazy film of red blood cells, no button or a very a small button of red blood cells at
the bottom of the V-bottom well.

12. Identify the end point. This will be the last well to show complete haemagglutination and contains
one haemagglutinating unit.
Figure 21: Titration to determine HA titre of allantoic fluid sample
Definition of one HA unit

One HA unit in the haemagglutinin titration is the minimum amount of virus that will cause complete
agglutination of the red blood cells. The last well that shows complete agglutination is the well that
contains one HA unit.

Calculation of the HA titre of the test sample

The HA titre is the reciprocal of the dilution that produces one HA unit.

Example of HA titration shown in Figure 21.

A 1 in 64 (1/64) dilution contains 1 HA unit.

The HA titre of the test sample is therefore the reciprocal of 1/64 = 64 = 26

The titre of the suspension of Newcastle disease virus can be expressed as 64 or 26 HA units in
25 mL.

4. Serology
Introduction

The presence of antibodies to Newcastle disease virus in chickens is detected by serological testing.
The results of these tests are used for three purposes.

1. To assess the efficacy of Newcastle disease vaccine in laboratory and field trials.

2. To assess the level of Newcastle disease virus antibodies in the field.

3. Serum known to contain antibodies to Newcastle disease virus is used to confirm the presence of
Newcastle disease virus in a test sample of allantoic fluid. Such a sample would be obtained during
the isolation of virulent Newcastle disease virus. See Section 15.

There are two assays commonly used to carry out serological testing for Newcastle disease virus
antibodies.

1. Haemagglutination inhibition (HI) test. The HI test is a convenient and commonly used assay that
requires cheap reagents and is read by eye.

2. ELISA (Enzyme linked immunosorbent assay). This is a colourimetric assay and requires the use of
a sophisticated instrument to read the optical density of the reactions. ELISA kits for Newcastle
disease virus antibody detection are prepared and sold commercially. Detailed instructions are
supplied with the kits. They are usually quite expensive.

In this manual a protocol for the HI test based on the test described by Allan and Gough will be used
for serological testing. (Allan and Gough, 1974 a.)

Preparation of Newcastle disease virus antigen for use in HI tests

Antigen is prepared by inoculating embryonated eggs with a sample of Newcastle disease virus and
harvesting the allantoic fluid four days later. Part of the first batch of I-2 Newcastle disease vaccine
prepared from the I-2 working seed can be set aside for storage as antigen. A volume of 50 mL to 100
mL is adequate for a large number of tests. Centrifuge the sample at 1 200 g to clarify and remove any
contaminating red blood cells. Store the antigen in one mL aliquots at -20°C.

See Sections 6 and 9.

Preparation of 4HA units of Newcastle disease virus antigen

The standard amount of Newcastle disease virus used in the haemagglutination inhibition (HI) test is
4HA units. It is necessary to prepare and test a suspension of Newcastle disease virus containing 4HA
units in order to carry out the HI test. This involves a series of following steps.

1. Titrate the stored suspension of virus to be used as the antigen in the HI test. See Section
10 Calculate the HA titre.

2. Calculate the dilution factor required to produce 4 HA units. A simple way is to divide the HA titre by
4.

3. Apply the dilution factor and dilute the original suspension of antigen in PBS to produce an
adequate volume of 4HA antigen to carry out the HI test. Allow 2.5 mL for each microwell plate.

4. Titrate the diluted (4HA) suspension of virus. This is a back titration to check the diluted antigen
contains 4 HA units.

5. Read HA titre. It should equal 4HA units. If not adjust the dilution and titrate again.

6. Use the 4HA unit dilution of antigen in an HI test to test the standard positive and negative serum.
The HI titre of the laboratory standard positive serum should equal the predetermined titre.

Interpretation

The results of the back titration of the diluted antigen and the HI titre of the standard positive are
both used to confirm the antigen has been diluted to a concentration equivalent to the standard 4 HA
units.

If the HI titre of the positive control serum is less than the standard titre, the antigen is too
concentrated. Prepare a new dilution and test again.

Conversely if the HI titre of the positive control serum is too high the antigen is too dilute. Prepare
a new dilution and test again.

Worked example

Preparation of 4HA units of antigen for HI test in 10 microwell plates:

The HA titre of the antigen was tested according to the protocol described above. The HA titre =
128

Calculation of dilution factor to prepare 4 HA units: 128/4 = 32

Calculation of volume of 4HA unit dilution of antigen required:

Allow 2.5 mL per plate; total volume required = 10 × 2.5 mL = 25 mL

Apply dilution factor = 25 mL/32 = 0.781 mL = 781 µL

Preparation of the diluted antigen: mix 781 µL of original virus suspension with 24.219 mL of
diluent. PBS is a suitable diluent.

Note: In this case it would be easiest to prepare 32 mL of the 4HA antigen. This would use 1 mL of the
original suspension diluted in 31 mL of PBS.

Haemagglutination inhibition

Materials

• Thawed serum samples in racks

• V-bottom microwell plates and covers
• PBS
• 1 percent washed red blood cells
• V-bottom reagent trough
• 25 µL single and multichannel pipettes and tips
• Microwell plate recording sheet.
• Newcastle disease virus antigen diluted to 4 HA units per 25 µL
• Standard positive and negative serum

Method

1. Fill in recording sheets to record how samples will be dispensed into microwell plates.

2. Calculate the number of plates required and number each plate.

3. Dispense 25 µL of PBS into each well of the plates.

4. Shake each serum sample and dispense 25 µL into the first well and the last (control) well of a row
of a microwell plate.

5. Use a multichannel pipette to make two-fold serial dilutions along the row until the second last well
from the end. The last well is the serum control. Do not dilute this well. See Appendix 4 for
instructions on carrying out two-fold serial dilutions.

6. Add 25 µL of the 4HA dilution of antigen to each well excluding the control wells in the last column.
See Section 10 for preparation of 4HA units of antigen

7. Gently tap the sides of the microwell plates to mix the reagents. Cover plates with a lid. Allow to
stand for 30 minutes at room temperature.

8. Add 25 µL of 1 percent washed red blood cells to each well including the control wells in the last
column.

9. Gently tap the sides of the microwell plates to mix the reagents. Cover the plates with a lid. Allow to
stand at room temperature for 45 minutes.

10. Read the settling patterns for each serum sample. Read the control serum well first then read the
patterns in the other wells.

11. Record the pattern observed in each well on a microwell plate recording sheet. Determine the
endpoint. This is the point where there is complete inhibition of haemagglutination.
12. Record the antibody level for each serum sample. This is expressed as a log base 2. For
convenience, the titre is often recorded as just the log index. For example a titre of 26 would be
recorded as 6.

Interpretation of results

In the wells where antibodies are present there will be haemagglutination inhibition. The red blood
cells will settle as a button.

In the wells where antibodies are absent, the red blood cells will agglutinate.

The end point of the titration is the well that shows complete haemagglutination inhibition.
Sometimes it is not easy to determine. Look at the size of the button as an indication of the degree of
haemagglutination inhibition. Use the control well as a point of comparison. Be consistent in
determining the endpoint.

Elution

The neuraminidase enzyme present in the virus particle will eventually break the bond between the
virus and red blood cells. This process is called elution.

When elution occurs, the red blood cells are no longer agglutinated. They roll down the side of V-
bottom microwell plates to resemble the negative settling pattern, a tight button.

Some Newcastle disease virus strains elute more rapidly and the test must be read before this occurs.

Usually elution takes longer than 45 minutes. A control well with virus and red blood cells is useful to
determine elution time.
Figure 22: Titration to determine HI titre

Non-specific inhibitors
Some sera may contain substances other than antibodies that inhibit viral haemagglutinin. These
substances are described as non-specific inhibitors and are rarely observed with chicken serum and
Newcastle disease.

Natural agglutinins

Some chicken sera contain substances that will agglutinate chicken red blood cells. The settling
pattern of the agglutinated cells is similar to that produced by Newcastle disease virus. These natural
agglutinins are present in low concentration. The control serum well will indicate the presence of
natural agglutinins.