12. Titrating Newcastle disease virus for infectivity

Introduction

The aim of the titration is to measure the concentration of infectious Newcastle disease virus in a suspension. Examples of suspensions of Newcastle disease virus that will need to be titrated are wet vaccine and freeze dried vaccine that has been reconstituted in diluent.

Measuring the concentration of Newcastle disease virus in a suspension

An overview

The concentration of Newcastle disease virus in a suspension is expressed as an Infectivity Titre. The Infectivity Titre is established by carrying out a titration.

The unit of measurement of infectivity of avirulent Newcastle disease virus is the 50 percent Embryo Infectious Dose or EID₅₀. One EID₅₀ unit is the amount of virus that will infect 50 percent of inoculated eggs.

The unit of measurement of infectivity of virulent Newcastle disease virus is the 50 percent Lethal Dose or LD₅₀. If the assay is carried out in embryonated eggs, it is 50 percent Embryo Lethal Dose or ELD₅₀.

One LD₅₀ is the amount of infectious virus that will cause the death of 50 percent of inoculated chickens.

One ELD₅₀ is the amount of infectious virus that will cause the death of 50 percent of inoculated embryonated eggs.

The Infectivity Titre of a suspension of Newcastle disease virus is the number of infectious units of virus per unit volume, usually expressed per mL.

To determine the Infectivity Titre of a suspension of I-2 Newcastle disease virus, a series of ten-fold dilutions are carried out on the suspension. Five embryonated eggs are inoculated with each dilution. After incubation for 4 days, the HA test is used to determine whether or not the virus has infected and multiplied in each of the eggs. This data is used to calculate the Infectivity Titre.

An adaptation of the mathematical technique devised by Reed and Muench is used to calculate the dilution of the suspension of virus being tested that produced the end point. The end point contains one unit of infectivity (1 EID₅₀). This dilution is then used to calculate the Infectivity Titre. (Reed and Muench, 1938.)

Measuring the concentration of I-2 Newcastle disease virus in a suspension

Method

1. Carry out ten-fold serial dilutions of the test suspension of virus. See Appendix 5 Materials and Method are below. The range of dilutions required will be determined by the estimated infectivity titre of the suspension in each 0.1 mL of inoculum.

2. Estimate the infectivity titre in the suspension by considering previous titrations and storage conditions. Is the titre is likely to have dropped during storage and if so by how much? The range of dilutions should include at least two ten-fold dilutions above and below the dilution expected to contain the end point. If the titre is unknown, dilute from 10^-1 to 10^-10.

3. Inoculate 5 eggs with each dilution. Use a separate needle and syringe for each dilution. If needles and syringes are in short supply and will be used to inoculate more than one set of eggs, it is very important to start with the most dilute sample. See Section 6 for Materials and Method for inoculation of eggs. Usually inoculation of 30 eggs is sufficient to determine the endpoint.

4. Incubate eggs for 4 days at 38°C according to instructions in Section 4.

5. After 4 days incubation, harvest allantoic fluid from each egg and test for haemagglutinin to determine the presence or absence of Newcastle disease virus. See Sections 9 and 10 for Materials and Method.

6. Tabulate the results in the daybook.

7. A description of the application of the Reed and Muench mathematical technique to calculate the infectivity titre of the original suspension is included in this section. A recording sheet is provided in Appendix 10.

Ten-fold serial dilutions of a suspension of I-2 Newcastle disease virus

Overview

100 µL of a Newcastle disease virus suspension mixed with 900 µL of diluent will be a 1 in 10 or 10^-1 dilution. This will be one-tenth the concentration of the original suspension.

100 µL of the 1 in 10 or 10^-1 dilution mixed with 900 µL of diluent will be a 1 in 100 or 10^-2 dilution.

By repeating these ten-fold dilutions in series, the original suspension can be diluted to a point where there is no longer enough virus present to infect embryonated eggs.

See Appendix 5 for detailed instructions in carrying out ten-fold serial dilutions.

Materials

• Sterile diluent. Use the standard antibiotic solution, PSG.
• Sterile glass tubes in a rack for carrying out the dilutions. It is preferable if the tubes are fitted with lids.
• One mL micropipette and tips for dispensing diluent.
• 100 mL micropipette and tips.
• Vortex mixer.
• Samples to be titrated.

Method

1. Place required number of dilution tubes in the rack.

2. Label the dilution tubes in sequential order. Each tube must be clearly labeled with the dilution factor of the original suspension. For example, to prepare a series of dilutions from 10^-1 to 10^-4; the labels would read 10^-1, 10^-2, 10^-3, 10^-4.

3. Dispense 900 µL of diluent into each tube.

4. Take 100 mL of the virus suspension and transfer to the first tube labeled 10^-1. Discard the tip.

5. Mix well using the vortex mixer or by hand.

6. Use a clean tip to transfer 100 mL of the diluted virus suspension from the tube labeled 10^-1 to the next tube in the series. Discard the tip.

7. Mix well using the vortex mixer or by hand.

8. Repeat steps 6 and 7 until the final dilution is reached.

Application of the Reed and Muench mathematical technique to calculate 50 percent embryo infectious dose (EID₅₀)

The method described above uses a titration of the infectivity of a test virus suspension to quantify the amount of infectious virus in the suspension. The end point of the titration is used to calculate the infectivity titre of the original suspension of virus. The Reed and Muench mathematical technique is used to calculate this end point from the results of the HA test on each of the inoculated eggs. A formula is used to calculate an index (proportionate distance) that is then applied to the appropriate dilution. The infectivity titre is expressed as EID₅₀ per mL.

Use the table provided in Appendix 10 as a working sheet.

Instructions

Tabulate the results of the HA test as follows:

Dilution of inoculum: Include the range of dilutions from 100 percent infection to 0 percent infection. This will be the range 5 out of 5 eggs showing HA positive to 5 out of 5 eggs being HA negative.

For each dilution, record the Number of eggs infected (HA +ve) and the Number of eggs not infected (HA -ve) by the inoculum. An extract from the working sheet is reproduced for clarification.

Table 4: Extract from Reed Muench working sheet (1)

These figures are then accumulated by addition as indicated by the arrows on the recording sheet.

From these accumulated numbers, the percentage of infected eggs for each dilution is calculated.

See extract from working sheet on next page.

Table 5: Extract from Reed Muench working sheet (2)

At this step, the “Percentage infected” column may include the figure of 50 percent. In this case, the dilution in this row has produced the 50 percent end point and contains one EID₅₀ in the inoculum. If the figure of 50 percent has not been calculated it lies between two dilutions. The Reed Muench calculation is then used to establish the dilution by calculating an index. The index is then added to the dilution that produced the percentage infected immediately above 50 percent.

The Reed and Muench formula to calculate the index is:

The index is applied to the dilution that produced the percentage of infection immediately above 50 percent.

We now have the dilution that theoretically produced one EID₅₀ unit.

The reciprocal of this dilution is the number of EID₅₀ in the 0.1 mL volume of inoculum inoculated into each egg. Thus ten times this number will be the number of EID₅₀ in 1 mL of inoculum.

Worked example

The titration of a suspension of I-2 Newcastle disease wet vaccine stored at 4°C for 4 weeks.

The Materials and Method were according to Section 12 of this Laboratory Manual.

Results of HA tests of allantoic fluid from each of the inoculated eggs are recorded in the table below.

Table 6: Extract from completed Reed Muench working sheet (1)

This data is used to calculate the percentage infected. The percentage infected is in the table below.

Table 7: Extract from completed Reed Muench working sheet (2)

From this data, the percentage figures entered in the Reed and Muench formula are as follows:

Calculation of the index

Index = (86% - 50%) ÷ (86% - 33%)

Index = 36 ÷ 53 = 0.7

The index is then applied to the dilution that produced the percentage infected immediately above 50 percent. In this example this is the 10^-8 dilution. The index of 0.7 is applied to this dilution.

In this worked example the dilution that provided the 50 percent infection of eggs or 1 EID₅₀ is 10^-8.7.

The reciprocal of this dilution is the amount virus contained in the 0.1 mL of the original suspension

= 10^8.7EID₅₀/0.1 mL

= 10^9.7EID₅₀/mL

The infectivity titre of I-2 wet vaccine stored at 4°C has been calculated to be 10^9.7EID₅₀/mL.

See Appendix 10 for an example of a completed work sheet.