A basic laboratory manual for the small-scale production and testing of I-2 ...

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A basic laboratory manual for the small-scale production and testing of I-2 ...
Grimes, S.E.
FAO Document Repository
13. The production of I-2 Newcastle disease vaccine
The seedlot system
Preparation of vaccines by a seedlot system reduces the chance of genetic alterations occurring during continuous passages in eggs. With each passage, there is the possibility of genetic alterations to the virus. These alterations may affect virulence, antigenicity and the yield of vaccine. A selected isolate is used to produce a volume of virus that comprises the master seed, which is divided into aliquots and stored. The master seed is usually freeze dried. An aliquot of master seed is used to produce a volume of working seed, which is again divided into aliquots and stored. An aliquot of the working seed is then used to produce a batch of vaccine. All batches of vaccine are then only two passages removed from the master seed.
Preparation of the I-2 Newcastle disease vaccine master seed
The I-2 Newcastle disease vaccine master seed was prepared at the John Francis Virology Laboratory, School of Veterinary Science, University of Queensland. The I-2 strain is an avirulent Australian Newcastle disease isolate. It was chosen for its antigenicity and thermostability. The master seed stock of virus was derived from parent stock that had survived at 56?C for thirty minutes.
The I-2 master seed was propagated in embryonated chicken eggs obtained from a minimal disease flock. Rigorous testing of the master seed revealed no evidence of contamination with aerobic or anaerobic bacteria, mycoplasmas, fungi or viruses other than Newcastle disease virus. The allantoic fluid was diluted 1:1 with a 10 percent skim milk solution and freeze dried in glass ampoules.
Experimental I-2 vaccine produced from the I-2 master seed was further tested for thermostability. The vaccine's safety and immunogenicity in chickens were tested. It was used to vaccinate a simulated village chicken flock. As clutches of chicks were produced by the flock, they were vaccinated. The flock maintained adequate antibody levels for 9 months with HI titres apparently boosted by the vaccine virus excreted by the chicks.
The I-2 vaccine administered by eye drop protected village chickens from a local velogenic challenge strain in both laboratory and village trails in several countries including Vietnam.
The I-2 Newcastle disease vaccine is now being manufactured in many countries for use in controlling Newcastle disease in village chickens.
References:
Tu, Phuc, et al., 1998.
Bensink, Z. and P. Spradbrow, 1999.
Supply and transport of the I-2 master seed
The I-2 Newcastle disease master seed virus is stored at the School of Veterinary Science at the University of Queensland. It is supplied to Vaccine Production Laboratories in developing countries to produce vaccine for use in programmes to control Newcastle disease virus in village chickens.
For more information contact the Head of the School of Veterinary Science at hosvetsci@uq.edu.au
To keep the cost of transport at a reasonable level, the master seed is transported by non refrigerated airfreight, usually with DHL World-wide Express. Three ampoules of freeze dried master seed are packed into a HAZPAK packaging system that has been specially designed for transport of biological materials. Dry ice or chiller packs are added to keep the vaccine cool for as long as possible. However the vaccine usually reaches ambient temperature before delivery. It is anticipated that during transport the infectivity titre of the master seed may be reduced. It is therefore necessary to titrate the vaccine on arrival to establish the infectivity titre.
An information sheet is supplied with the master seed. The sheet includes instructions for reconstitution, titration and storage of the master seed.
Note:
The ampoules of master seed are best stored at -70?C.
Use aseptic technique to open one vial of the freeze dried I-2 master seed and resuspend the contents in 1 mL sterile water.
Further dilute with phosphate buffered saline or antibiotic solution (PSG) to 20 mL.
Prepare one mL aliquots of the reconstituted master seed.
Titrate one ampoule (see Section 12), label and store the remaining aliquots at -70?C.
Production of the I-2 Newcastle disease vaccine working seed
1. Calculate the volume of diluted master seed required to inoculate the eggs with 0.1 mL per egg.
2. Remove and thaw an aliquot of reconstituted master seed from the -70?C freezer. Further dilute the thawed vaccine to ensure each egg receives at least 102.0 EID50 preferably 103.0 EID50 per 0.1 mL of inoculum. Label unused reconstituted master seed with the date of thawing and return to freezer for future use. Remember thawing and freezing can reduce the infectivity titre. Retitration of a sample returned to the freezer will reveal any loss of infectivity titre during the freeze/thaw.
3. Candle eggs and mark the inoculation site according to the method in Section 5.
4. Swab the inoculation site with 70 percent alcohol and inoculate 0.1 mL of the diluted I-2 master seed into the allantoic cavity of each embryonated egg according to the method described in Section 6.
5. Place eggs in incubator. Check the temperature and humidity.
6. After 24 hours of incubation candle the eggs. Discard any dead embryos.
7. After 4 days of incubation, remove the eggs from the incubator and chill for at least 2 hours, preferably overnight.
8. Test each egg for haemagglutinin activity. See Section 10.
9. Discard embryos that do not test positive for the virus or are visibly contaminated.
10. Harvest the allantoic fluid from the eggs into sterile containers preferably that can be centrifuged. See Section 9.
11. Test for gross bacterial contamination. See Section 14.
12. Centrifuge or stand overnight at 4?C to clarify the allantoic fluid.
13. Pool allantoic fluid to ensure homogeneity.
14. Prepare 1 mL aliquots in sterile containers.
15. Clearly label the aliquots with contents and date.
16. Store at -70?C.
17. Titrate the working seed to establish infectivity titre. See Section 12.
Production of I-2 Newcastle disease vaccine
As with the production of the working seed, a reliable source of healthy embryonated eggs is necessary. Estimates of the quantity of vaccine required will determine the number of eggs to be inoculated with the working seed. Large eggs will yield up to 10 mL of allantoic fluid. Each mL should contain in excess of 109.0 EID50 (about 1 000 doses of vaccine).
It is recommended that each egg is inoculated with at least 104.5 EID50 of the working seed in a volume of 0.1 mL (Allan, et al. 1978). This is a dilution of 1 in 10 000 of working seed containing ? 109.5 EID50 of virus per mL. Thus a 1 mL aliquot of working seed will be adequate for inoculating 100 000 eggs.
1. Remove an aliquot of I-2 working seed and thaw.
2. Dilute with antibiotic solution or PBS to give an adequate volume to inoculate each egg with 0.1 mL.
3. Swab inoculation site with 70 percent alcohol and proceed with egg inoculation according to Section 6.
4. Place the eggs in an incubator for 4 days. See Section 4.
5. Candle the eggs after one day. See Section 5. Discard any dead eggs.
6. After four days, remove the eggs from the incubator, chill and harvest the allantoic fluid according to the method in Section 9.
Quality control of the I-2 Newcastle disease vaccine
See Section 15.
Protective diluents for wet vaccine
Dilution in either a 2 percent gelatin solution or 4 percent skim milk is recommended. (Bensink and Spradbrow, 1999.)
At the John Francis Virology Laboratory, the standard wet I-2 Newcastle disease vaccine has been diluted as follows:
Mix one part allantoic fluid with one part 2 percent gelatin solution and two parts antibiotic solution. This gives a 0.5 percent solution of gelatin in which the infectivity titre of the virus remains high after storage at 4?C for eight weeks. (Unpublished results of experiments at the John Francis Virology Laboratory.)
Both the gelatin and skim milk solutions can be prepared in distilled water or phosphate buffered saline and sterilized by autoclaving.
Freeze drying of the I-2 Newcastle disease vaccine
It is beyond the scope of this manual to describe the freeze drying process. Vaccine production centres with freeze drying facilities will have detailed instructions on how to manage and operate the facility. Staff trained to maintain and use the freeze drying equipment will be required to supervise this operation.
Labeling individual vials of vaccine
Labels designed to fit even very small vials can be prepared on a computer using a basic word processing programme. The label should include the following information:
I-2 thermostable Newcastle disease vaccine
Place of production of vaccine
Number of doses
Batch number and expiry date
Recommended storage conditions
For veterinary use only
Preparing a leaflet to distribute with the vaccine
After the experimental I-2 vaccine has been tested in the laboratory and in the field, commercial vaccine will be produced for distribution and sale to farmers. A leaflet describing the vaccine and instructions for its use should be prepared. As well as repeating the information on the labels, the leaflet would include the following additional information:
A description of the composition of the vaccine.
Properties of the I-2 Newcastle disease virus strain.
More details of dose and administration of the vaccine.
More details about the production of the vaccine.
Note: The manufacturer determines the expiry date of vaccine after a consideration of the viral content of the vaccine, and the rate of heat inactivation under local conditions.
More information about labels and leaflets
Refer to the Principles of Veterinary Production section of the OIE website for guidelines. http://www.oie.int/eng/normes/MMANUAL/A_00017.htm
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