15. Isolation of virulent Newcastle disease virus

Introduction

Virulent Newcastle disease virus is used in challenge trials of vaccinated chickens to test the efficacy or potency of the I-2 Newcastle disease vaccine. There are many strains of Newcastle disease virus and they vary in pathogenicity. This is reflected in the severity of disease in chickens infected with isolated Newcastle disease virus strains.

Virulent stains of Newcastle disease virus are not stored or used in the research projects at the John Francis Virology Laboratory at the University of Queensland. This information and procedures in this section are based primarily on two sources.

1. “Manual of Standards for Diagnostic Tests and Vaccines” published by the Office International des epizooties (OIE) at http://www.oie.int/eng/normes/MMANUAL/A_00036.htm

(Sometimes this address sometimes opens up the OIE homepage only. In this case look under OIE publications.)

2. “Newcastle Disease” edited by D.J. Alexander, 1988.

There are three designations used to describe Newcastle disease virus strains that cause disease.

1. Velogenic
2. Mesogenic
3. Lentogenic

A fourth designation, “avirulent” has been introduced and includes strains that do not cause disease (Spradbrow, P.B., 1987).

These designations reflect the severity of the disease and are used to describe Newcastle disease pathotypes. In the laboratory, the designations are applied to strains that are grouped according to chicken embryo mortality. This is measured by the mean death time (MDT) of embryos inoculated with the virus by the allantoic cavity route. The MDT is the mean time in hours for the minimal lethal dose to kill inoculated embryos.

Table 8: Designations of virulence of Newcastle disease virus

Designation	Virulence in Chickens	Approximate MDT
Velogenic	Highly virulent strains that kill a high percentage of chickens	Less than 60 hours
Mesogenic	Moderately virulent strains that cause serious disease, some deaths and loss of egg production	Between 60 and 90 hours
Lentogenic	Strains of low virulence that cause respiratory disease especially in young chicks and loss of egg production.	More than 90 hours
Avirulent	Does not cause disease	Does not kill embryos

Overview

Newcastle disease virus isolates are replicated by inoculation in the allantoic cavity of ten-day old embryonated eggs.

The virus grows in the cells of the allantoic membrane that lines the allantoic cavity. It is shed from these cells into the allantoic fluid, which can then be harvested.

Velogenic and Mesogenic strains will grow in the cells of the three layers of the allantoic membrane. These strains rapidly infect the cells of the embryo itself. This causes the death of the embryo in less than four days of incubation.

Some Lentogenic strains do not cross the allantoic membrane and consequently the embryo remains alive after four days of incubation.

An indication of the virulence of the isolates can be derived from observing whether embryos inoculated with and isolate die and if they do, how long it takes.

Protocols for establishing the mean death time (MDT) of embryos are available. They require that eggs are free of maternal antibody which influences the death time of embryos inoculated with mesogenic and velogenic isolates. For the purposes of preparation of a challenge strain for experimental purposes approximate mean death times are an adequate indication of virulence.

Other procedures for determining pathogenicity are the intracerebral pathogenicity index and the intravenous pathogenicity index. Protocols for these tests are not included in this manual.

A local velogenic strain of Newcastle disease must be used to challenge chickens to test the efficacy of Newcastle disease vaccine. The velogenic strain must be isolated from a chicken that shows clinical signs of Newcastle disease or a carcass where the cause of death has been diagnosed as Newcastle disease.

Isolation of virulent Newcastle disease virus in the laboratory

Isolation of Newcastle disease virus from field samples should not be carried out in the vaccine production laboratory as this would introduce a risk of virulent Newcastle disease virus contaminating the vaccine. If a separate facility is not available, a separate room should be set up with an incubator used only for eggs used to isolate Newcastle disease virus from field samples. Make sure the incubator is carefully cleaned and disinfected after use.

Field samples

Use aseptic technique to collect a sample of brain, liver, spleen or marrow from a long bone from a bird showing clinical signs of Newcastle disease or a carcass from which Newcastle disease has been diagnosed.

Detailed protocol

Use the following Materials and Method to prepare a protocol for use in your own laboratory. The protocol will be a detailed step by step description of the procedures involved in isolating virulent Newcastle disease virus in your laboratory. Further reading is recommended.

Materials

Sterilized glass homogenizer, mortar and pestle or needle and syringe.
Centrifuge tubes and centrifuge. A microfuge is suitable.
0.45 mm filter (not essential).
10-day-old embryonated eggs. If possible, purchase the eggs from a layer flock free of high levels of Newcastle disease virus antibodies.
Egg incubator.
Candling lamp.
Antibiotic solution (PSG)
Materials for haemagglutination test. See Section10.

Isolation of virulent Newcastle disease virus in the laboratory

Method

1. Use aseptic technique to homogenize the sample with 2 to 3 mL of PSG buffered antibiotic solution.

2. Transfer the homogenized sample to an appropriate tube and centrifuge.

3. Filter the supernatant through a 0.45 mm filter if available.

4. Candle, mark and disinfect the inoculation site of 10 ten-day old embryonated eggs. Use five eggs per sample. See Section 5.

5. Inoculate the allantoic cavity of the embryonated eggs with 0.1 mL of the supernatant. See Section 6.

6. Place the inoculated eggs in the incubator.

7. Candle eggs daily post inoculation. Record deaths and note the time it takes for the embryo to die. Deaths within the first day are regarded as non-specific and discounted. Discard these embryos. Deaths on subsequent days are likely to be due to Newcastle disease virus.

8. Harvest some allantoic fluid from each dead egg and check for the presence of haemagglutinin. A positive result is an indication of the presence of Newcastle disease virus. Live eggs are unlikely to contain virulent Newcastle disease virus. See Section 10

9. Use aseptic technique to harvest allantoic fluid from those eggs with positive HA. Dispense fluid into centrifuge tubes.

10. Centrifuge the harvested allantoic fluid, pool and then prepare aliquots of the supernatant. Label clearly and store at -20°C.

11. Confirm the allantoic fluid collected contains Newcastle disease virus. This is done by using harvested allantoic fluid as antigen to test for haemagglutination inhibition (HI) standard Newcastle disease virus antiserum. See the following part.

12. This allantoic fluid must be passaged again in embyronated eggs to establish mean death time.

Confirmation of identity of Newcastle disease virus

The haemagglutination test is not specific for Newcastle disease virus and other viruses will agglutinate red blood cells. Therefore a sample of allantoic fluid testing positive for haemagglutinin will need further testing to confirm the presence of Newcastle disease virus. The presence of Newcastle disease virus in the sample is confirmed by using the haemagglutination inhibition (HI) test.

Harvested allantoic fluid that tests positive for haemagglutinin is titrated for HA titre and diluted to 4HA units in 25 µL. See Section 10.

The allantoic fluid is used as the antigen in an HI test of the standard positive serum containing antibodies to Newcastle disease virus and of the standard negative serum that does not contain antibodies to Newcastle disease virus.

If the standard positive inhibits haemagglutination in the test allantoic fluid sample, this result will confirm the presence of Newcastle disease virus in the test sample. There should be no inhibition by the negative control serum.

See Section 11 for details of HI test.

Mean death time (MDT) in eggs

The following protocol to establish the MDT is taken from the protocol published by Office International des Epizooties. It is available from the OIE website at http://www.oie.int/eng/normes/mmanual/A_00036.htm

Dilute fresh infective allantoic fluid in sterile saline to give a ten-fold dilution series between 10^-6 and 10^-9.

For each dilution, inoculate 0.1 mL into the allantoic cavity of each of five 9-day-old or 10-day-old embryonated SPF chicken eggs and incubate at 37°C.

Retain the remaining virus dilutions at 4°C and inoculate another five eggs with 0.1 mL of each dilution 8 hours later and place at 37°C. This staggers the time of inoculation.

Each egg is examined twice daily for 7 days and the times of any embryo deaths are recorded.

The minimum lethal dose is the highest virus dilution that causes all the embryos inoculated with that dilution to die.

The MDT is the mean time in hours for the minimum lethal dose to kill embryos.

Use of non-SPF embryonated eggs

Eggs from flocks vaccinated with Newcastle disease vaccine can be used to grow Newcastle disease virus. Eggs from these flocks will contain varying levels of antibodies in their yolks. These antibodies are derived from the hen. As the embryos develop, the maternal antibodies enter the blood. Virulent strains of Newcastle disease virus might not kill the embryos if there are high levels of antibodies. However the virus will grow in the cells lining the allantoic cavity, where there are no antibodies and is then shed into the allantoic fluid. The haemagglutination test, described in Section 10, will detect the presence of Newcastle disease virus in the allantoic fluid.

The presence of antibodies in eggs used for the isolation of Newcastle disease virus would be expected to increase the mean death time. Therefore when non SPF eggs are used to propagate the virus, the mean death times as described in Table 3 are not a reliable indication of pathotypes. In this situation, the pathotype of an isolate would have to be established by inoculation of susceptible chickens.

Measuring the concentration of virulent Newcastle disease virus in a suspension

For measuring the 50 percent Embryo Lethal Dose or ELD₅₀, carry out ten-fold serial dilutions and inoculate ten-day old embryonated eggs according to the Material and Methods in Section 12. Virulent virus will kill the embryos and the end point will be determined by the death of the embryos. Calculate the 50 percent endpoint using the Reed Muench method as described in Section 12.

This manual does not include a protocol for measuring the LD₅₀ or 50 percent lethal dose in susceptible chickens. Further reading of books describing virology laboratory techniques will be required for details of the materials and method for carrying out this infectivity assay.

Testing the virulence of a challenge isolate

7. Find a source of chickens that are likely to be free of Newcastle disease virus antibodies. Take serum samples and test for HI antibodies. See Section 11. Select chickens that are free of Newcastle disease virus antibodies. If antibodies are detected, titres of 8 (2³) and higher are considered protective so select chickens with titres of less than 8 for challenge.

8. House a small group of these chickens in isolation and take quarantine measures to avoid spread of virulent Newcastle disease from the chickens after infection.

9. Remove one of the aliquots of the stored allantoic fluid containing virulent Newcastle disease virus. Challenge some of the chickens by eye drop or intramuscular injection with undiluted allantoic fluid.

10. Do not infect the remainder of the birds. Their response will indicate whether contact spread between the birds occurs.

11. Monitor the birds. Infection by eye-drop with virulent Newcastle disease virus will cause clinical signs within three days and mortality in about five to seven days. The response to contact infection will become evident a few days later.

12. Maintain uninfected control chickens from the same source in a separate room. They should remain free of Newcastle disease.

TAKE CARE not to contaminate your vaccine or the working seed with the Newcastle disease virus challenge strain. Label all samples clearly, store separately and preferably carry out bench work in a separate section of the laboratory.

Disinfect materials and benches after handling the virus!