1. Does it matter if you harvest amniotic fluid as well as allantoic fluid?
No. Ideally, you should try to harvest only allantoic fluid. Amniotic fluid does not contain the I-2 Newcastle disease virus and will reduce the concentration of virus in the harvest. Take care not to rupture the amniotic membrane.
2. Does it matter if the fluid that is harvested contains red blood cells?
It is best to avoid contamination with red blood cells. Chill the eggs at 4°C for at least 2 hours or preferably overnight before harvesting to kill the embryos. Chilling also reduces the bleeding should the vessels rupture during harvesting of the allantoic fluid. Any red blood cells that are present should be removed by centrifugation and harvesting the supernatant using aseptic technique.
3. Can maternal antibodies in the embryos reduce the titre of the I-2 Newcastle disease vaccine harvest?
No. Avirulent strains of Newcastle disease virus such as V4 and I-2 inoculated into the allantoic cavity are confined in the cavity. They do not cross the allantoic membrane and do not enter the embryo. Maternal antibody is confined to the yolk sac until about 14 days of incubation and then enters the embryo. Virus harvested in allantoic fluid at 14 days will not have been exposed to antibody.
4. Is the haemagglutination test specific for Newcastle disease virus?
No. Many viruses will agglutinate chicken red blood cells. A specific identification requires a haemagglutination inhibition test with specific anti-Newcastle disease virus antiserum.
5. Can you give chickens too much I-2 Newcastle disease vaccine?
I-2 Newcastle disease vaccine is a safe, avirulent vaccine tolerated at high doses. If you have excess vaccine, it is better to increase the dose of vaccine than to discard a portion of the vaccine. The I-2 vaccine was shown to be safe when day old SPF chickens were given 100 times the standard dose by eye drop in a trial at the John Francis Virology Laboratory.
6. Can red blood cells from vaccinated donor chickens that have antibodies to Newcastle disease virus be used in the haemagglutination-inhibition test?
Yes. The cells will be washed free of antibody. It is recommended to pool red blood cells from at least three donor chickens to guard against individual birds whose cells may react in an unusual way. Remember to always use a red blood cell control when doing the haemagglutination inhibition test. See Section 11.
7. Can you use the same incubator for preparation of vaccine and isolation of Newcastle disease virus from field samples?
No. There would be too much risk of unrecognized contamination of vaccine with the field virus.
8. How do I clean the incubator?
Remove all solid debris with a damp cloth. Then swab all surfaces with a disinfectant or 70 percent alcohol. See Section 16 for instructions for fumigation with formaldehyde.
9. If an embryo is dead, do I collect the allantoic fluid?
The I-2 strain of Newcastle disease virus will rarely kill embryos when inoculated into the allantoic cavity. Dead embryos inoculated with I-2 are likely to have been killed by bacterial contamination and the embryo should be discarded. If you are working with more virulent strains of Newcastle disease virus, the virus will kill embryos. In this case allantoic fluid from these eggs will be collected for testing. See Section 15.
10. What do the standard positive and negative sera do?
They ensure that your tests are standard and producing similar results each time you perform them. They can also ensure standard results between different laboratories. See Section 11 for more details.
11. What does passaging the Newcastle disease virus through eggs achieve?
The virus is passaged through eggs by the inoculation of the allantoic cavity to increase the quantity of virus. A small amount of the I-2 Newcastle disease virus seed material produces a lot of vaccine. However, the number of passages must be kept as small as possible, or some characteristics of the vaccine may alter. This especially applies to thermostable Newcastle disease vaccines that have been selected for heat-resistance. This property of the virus may be lost during subsequent passaging in embryonated eggs without further heat selection.
12. Why is it necessary to change the micropipette tip when carrying out ten-fold serial dilutions but not when carrying out two-fold serial dilutions?
In this manual, two-fold serial dilutions are used in quantitative assays to indicate the concentration of Newcastle disease virus (HA test) or Newcastle disease virus antibodies (HI test) in each dilution. These data is then used to calculate a titre to the log base 2. In both assays, the small quantities of the antibodies or virus carried over on tips between the two-fold dilutions will not greatly influence the result of the titres.
In this manual, ten-fold dilutions are used in quantal assays to titrate viral infectivity. Each dilution is inoculated into eggs and a single viral particle is sufficient to infect the embryo. Infected embryos will give a positive result when the allantoic fluid is tested for the presence or absence of virus after incubation for four days. For this reason even a small number of infective virus particles that are carried over on the pipette tip will falsify the results. The infectivity titres calculated from these results will be erroneously high. By using clean tips for each dilution, this error will not be introduced into the assay.
