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.
- “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.)
- “Newcastle Disease” edited by D.J. Alexander, 1988.
There are three designations used to describe
Newcastle disease virus strains that cause disease.
- Velogenic
- Mesogenic
- 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
- Use
aseptic technique to homogenize the sample with 2 to 3 mL of PSG buffered
antibiotic solution.
- Transfer
the homogenized sample to an appropriate tube and centrifuge.
- Filter
the supernatant through a 0.45 mm filter if available.
- Candle,
mark and disinfect the inoculation site of 10 ten-day old embryonated eggs. Use
five eggs per sample. See Section 5.
- Inoculate
the allantoic cavity of the embryonated eggs with 0.1 mL of the supernatant.
See Section 6.
- Place
the inoculated eggs in the incubator.
- 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.
- 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
- Use
aseptic technique to harvest allantoic fluid from those eggs with positive HA.
Dispense fluid into centrifuge tubes.
- Centrifuge
the harvested allantoic fluid, pool and then prepare aliquots of the
supernatant. Label clearly and store at -20°C.
- 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.
- 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
- 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.
- 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.
- 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.
- Do not infect the remainder of the birds. Their
response will indicate whether contact spread between the birds occurs.
- 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.
- 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!
Source:
FAO (http://www.fao.org/docrep/005/ac802e/ac802e0i.htm)
