Published guidelines for the production of veterinary vaccines will include rigorous 'quality control' to ensure a very high standard of vaccine is produced. Vaccine production laboratories that decide to produce the I-2 thermostable Newcastle disease vaccine will need to prepare their own in-house protocol for 'quality control' based on these guidelines.
There is always a risk of a vaccine becoming contaminated with extraneous microorganisms. Before the production of the I-2 Newcastle disease vaccine commences, an assessment of these risks should be carried out. A strategy to minimize the potential for contamination can then be developed. The I-2 vaccine has been developed for use in village chickens and must be of low cost. The capacity of the vaccine production laboratory to carry out a large number of quality control tests should be considered. There will inevitably be a compromise between the certainty with which the vaccine can be considered free of contaminants and the need for a cheap, locally produced thermostable Newcastle disease vaccine. Such a vaccine must be suitable for use in programmes that aim to control Newcastle disease in village chickens.
Testing for gross bacterial contamination of the vaccine should take place as soon as the allantoic fluid is harvested. This will involve inoculation and incubation of a suitable broth with the vaccine. If the broth is free of gross bacterial contamination after overnight incubation, the vaccine can then be divided into aliquots and stored as a wet vaccine or it can be freeze dried. Further testing of the stored vaccine can then be carried out before the vaccine is distributed for use in village chickens. As the level at which testing is carried out in each vaccine production laboratory will vary, this manual will deal with the basic tests used on batches of experimental vaccine. This section has been prepared after discussions with veterinarians involved in the production of the I-2 vaccine in Mozambique, Bhutan and Myanmar.
The following publications have been used as references and are recommended reading:
ASEAN (1998) Manual of ASEAN Standards for Animal Vaccines, Livestock Publication Series No 2A.
Allan, W.H. and J.E. Lancaster (1978) Newcastle Disease Vaccines, Their Production and Use, FAO Animal Production and Health Series No 10, 1978.
Office International des Epizooties (OIE) Manual of Standards for Diagnostic Tests and Vaccines, 2000.
Mowat, N. and M. Rweyemamu (1997) Vaccine Manual: The Production and Quality Control of Veterinary Vaccines for Use in Developing Countries, FAO Animal Health and Production Series No 35.
Sources of contamination
There are two possible sources of contamination with microorganisms:
1. Environmental contamination during the production of the vaccine. This risk is minimized by the use of aseptic technique, sterilized materials where appropriate, rigorous cleaning and disinfecting of all surfaces and equipment.
2. Vertical transmission through the eggs. By using embryonated eggs from a healthy flock, this risk is minimized. See Section 4.
Testing for bacterial contamination
Correct storage and preparation of media is essential for all the following tests.
Testing for aerobic contaminants
Use a general purpose broth culture medium that supports the growth of most likely contaminants for example Tryptic soy broth or nutrient broth.
1. Inoculate 1 mL of the wet vaccine or reconstituted freeze dried vaccine into 9 mL of broth.
2. Incubate at 30°C to 37°C for 24 hours.
3. Record results
Gross contamination after 24 hours, retest in twice the number of cultures. If contamination is evident after the second culture, discard the vaccine
Testing for anaerobic contaminants
Many anaerobic bacteria have fastidious growth requirements. Using aseptic technique will minimize environmental contamination by anaerobes during the harvesting of the allantoic fluid. Protocols published by FAO, ASEAN and the OIE detail procedures for testing Newcastle disease vaccine for the presence of anaerobic bacteria.
Testing for extraneous viruses
There is a range of tests available for detecting the presence of extraneous viruses. Deciding which pathogens to test for and which test to use is beyond the scope of this manual. Testing for extraneous viruses is also likely to be beyond the capacity of many vaccine production centres. In these centres, the risk of including extraneous viruses in vaccine is minimized by using embryonated eggs from healthy flocks free of viral infections.
The OIE Manual of Standards for Diagnostic Tests and Vaccines, 2000 recommends reading the purity test procedures for avian vaccines published in the European Pharmacopoeia.
Look up http://pharmacos.eudra.org/F2/eudralex/vol-7/B/7Blm3a.pdf.
Testing for Salmonella species
Brilliant Green agar
Desoxycholate citrate agar
Detailed information about preparing and using these media is available on the Oxoid website at http://www.oxoid.com/uk/index.asp
1. Inoculate 9 mL of one of the liquid media with 1 mL of vaccine
2. Incubate at 37°C for 18 to 24 hours
3. Inoculate a loopful of incubated broth onto one of the solid media.
4. Incubate 18 to 24 hours. Observe. If no Salmonella growth is detected, incubate again for 18 to 24 hours.
5. Discard the vaccine, if Salmonella are detected.
Testing for Mycoplasma species
Several Mycoplasma species are transmitted through the egg. Embryonated eggs used to produce Newcastle disease vaccine should ideally be collected from a flock free of pathogenic Mycoplasma species. If the flock cannot be verified free of Mycoplasma, the vaccine should be tested for their presence. They are fastidious organisms and require specialized expertise to culture and identify. A commonly used media and protocol should be used to determine whether the vaccine is contaminated with Mycoplasma.
Testing for contamination with fungi
Sabouraud dextrose agar is used to cultivate fungi. Chloramphenicol at 0.05 g/L can be added to inhibit bacteria. Inoculate the agar with 100 mL of vaccine and spread over the plate. Inoculate a control plate with diluent only. Incubate at 25°C to 30°C for one week. Observe daily and record results. Growth on the control plate invalidates the test, which should then be repeated. Vaccine should be free of fungal contamination.
Further information about bacteriological media
Difco Laboratories and Oxoid are international manufacturers and suppliers of culture media.
Visit the Oxoid website at http://www.oxoid.com/uk/index.asp
This website contains detailed product information including formulas, techniques and material safety data sheets and is very informative.
Difco Laboratories have combined with BD Biosciences.
Visit their website at http://www.bd.com/industrail/difco/nmce.html
See Appendix 3 for recipes for TSB and Sabouraud agar
Testing virus content of vaccine
The virus content is a measure of the amount of infective Newcastle disease virus in the vaccine. This is important to know to ensure that vaccinated chickens receive enough virus to induce a protective immunity.
To measure the infectivity titre of wet vaccine or reconstituted freeze dried vaccine, follow the procedure described in Section 12.
Summary of eye drop size and vaccine dose
The recommended route of inoculation is eye drop.
The standard dose for the I-2 Newcastle disease vaccine is 106.0 EID50 per chicken.
The size and number of eye-drops delivered may vary.
The drop size is usually in the range of 10 mL to 50 mL.
For a 10 mL eye-drop to contain one standard vaccine dose, the undiluted vaccine must contain ³ 109.0 EID50/mL.
For a 50 mL eye-drop to contain one standard vaccine dose, the undiluted vaccine must contain ³ 108.3 EID50/mL.
Safety test in young chicks
This test involves giving susceptible chickens ten doses of vaccine. The formal test described by OIE uses one-day-old SPF chicks. In many circumstances, it will be necessary to use commercial chickens and they may be older than one day old. In this case collect serum and test for HI titres to make sure they do not have maternal antibodies to Newcastle disease virus.
Use two groups of chickens, a minimum of ten chickens per group.
Take a serum sample from the chickens.
Use the HI test to test for antibodies to determine susceptibility.
Use only chickens that test negative for Newcastle disease antibodies, preferably SPF chickens.
Administer ten doses (107.0 EID50) by eye-drop of the I-2 vaccine to each chick in the treatment group.
Administer diluent only by eye-drop to each chick in the control group.
Observe daily for 21 days for clinical signs.
If any serious clinical signs or deaths attributable to the vaccine are observed, discard the vaccine.
Efficacy test of vaccine
This test involves vaccination of susceptible chickens and measuring their antibody response to the vaccine. As in the safety test a source of susceptible chickens must be found and bled. Test the serum samples for the presence of antibodies to Newcastle disease virus. Suitable chickens will have HI titres of 21 or less.
An adequate antibody response to vaccine can be measured in two ways:
1. Testing serum collected from vaccinated chickens.
2. Challenging vaccinated chickens with a virulent strain of Newcastle disease virus.
In 1974, Allan and Gough published trials that showed an HI titre of 23 indicated the birds would be protected against fatal Newcastle disease if challenged. At some laboratories including the John Francis Virology Laboratory, challenge with virulent Newcastle disease virus is not carried out and serological testing as described by Allan and Gough is regarded as sufficient for testing vaccine efficacy.
Most laboratories already producing other strains of Newcastle disease vaccine will have protocols in place for testing the vaccine according to international protocols. These protocols can be applied to testing I-2 Newcastle disease vaccine.
Serological testing of efficacy of I-2 Newcastle disease vaccine
Divide the chickens into two groups of approximately ten chickens per group. House the groups separately.
Vaccinate the chickens in one group with a standard vaccine dose of 106.0 EID50.
The other group is the unvaccinated control group.
Bleed the chickens in both groups two weeks after vaccination.
Test the serum for antibodies using the HI test.
HI titres of 23 and above are considered protective.
Reference: Allan, W.H. and R.E. Gough, 1974b.
See Section 7: The collection of blood from chickens.
See Section 11: Serology.
Further testing of the efficacy of the vaccine may be carried out by challenging both groups of chickens with virulent Newcastle disease virus.
Outline of a protocol for testing I-2 Newcastle disease vaccine efficacy by challenge with virulent Newcastle disease virus
Further reading of the references listed at the beginning of the section will be required before challenge trails can be designed and successfully carried out.
Following is an outline only, based on information contained in the references.
Select at least twenty chickens with antibody levels of 21 or less.
Divide the chickens into two groups, a control group and a treatment group.
House the two groups separately and arrange for two attendants to look after the chickens, one attendant for each group of chickens.
Implement reasonable biosecurity measures to minimize the risk of uncontrolled introduction of Newcastle disease virus to either of the groups.
The control group will not be vaccinated.
The treatment group will receive one vaccine dose per bird.
Bleed both groups at weekly intervals.
Test the serum to measure the HI antibody response of the chickens to vaccination and to indicate levels of protection.
Challenge the chickens with virulent Newcastle disease virus when HI titres of vaccinated birds are ³ 23 indicating a protective antibody response. This will be 2 to 3 weeks after vaccination. See Section 15 for a method for the isolation of virulent Newcastle disease virus.
Make daily observations of the chickens and record the results.
Collect serum from the surviving chickens after 2 weeks and determine HI antibody titres. See Section 11.
Challenging chickens with virulent Newcastle disease virus
The two groups of birds can be housed together for the challenge. The challenge virus can be introduced in more than one way.
Eye drop or intranasal infection of all the birds.
Eye drop or intranasal infection of a few susceptible birds that are then placed with the flock. This allows the virus to spread by contact.
OIE Manual of Standards for Diagnostic Tests and Vaccines 2000 recommends a challenge dose is 105.0 LD50.
Allan, W.H. and J.E. Lancaster (1978) Newcastle Disease Vaccines, Their Production and Use recommends a challenge dose of 106.0 LD50.
Interpretation of results
At least 90 percent of the vaccinated chickens should survive the challenge and show no clinical signs of the Newcastle disease.
All unvaccinated control chickens should die of Newcastle disease.
Notes on recording results of Newcastle disease vaccine efficacy trials
These trials can be laboratory or village based trials. Prepare tables to summarize the data collected. This can be done on a computer or tables can be hand drawn in the daybook.
Most of the data collected will be the results of serological haemagglutination inhibition (HI) tests. These results are usually expressed as HI titres to the log base 2.
Useful statistics calculated for each group of chickens are the geometric mean titre (GMT), number of chickens per group (n) and the percentage of the group having a protective HI titre. A protective titre is a titre that is greater than or equal to 23.
Chicken identification (usually numbers of wing tags or leg bands) must also be recorded.
See Appendix 6 for instructions for calculating GMT.