Appendix XVI B (Vet) 5. Avian Live Virus Vaccines: Tests for Extraneous Agents in Batches of Finished Product

(Ph. Eur. method 2.6.25)

GENERAL provisions

a) In the following tests, chickens and/or chicken material such as eggs and cell cultures shall be derived from chicken flocks free from specified pathogens (SPF) (5.2.2).
b) Cell cultures for the testing of extraneous agents comply with the requirements for the master cell seed of general chapter 5.2.4. Cell cultures for the production of veterinary vaccines, with the exception of the karyotype test and the tumorigenicity test, which do not have to be carried out.
c) In tests using cell cultures, precise specifications are given for the number of replicates, monolayer surface areas and minimum survival rate of the cultures. Alternative numbers of replicates and cell surface areas are possible as well, provided that a minimum of 2 replicates are used, the total surface area and the total volume of vaccine test applied are not less than that prescribed here and the survival rate requirements are adapted accordingly.
d) In these tests, use the liquid vaccine or reconstitute a quantity of the freeze-dried preparation to be tested with the liquid stated on the label or another suitable diluent such as water for injections. Unless otherwise stated or justified, the test substance contains the equivalent of 10 doses in 0.1 mL of inoculum.
e) If the vaccine virus would interfere with the conduct and sensitivity of the test, neutralise the virus in the preparation with a monospecific antiserum.
f) Monospecific antiserum and serum of avian origin used for cell culture and any other purpose, in any of these tests, shall be free of antibodies against and free from inhibitory effects on the organisms listed under 7 Antibody specifications for sera used in extraneous agents testing (2.6.24).
g) Where specified in a monograph or otherwise justified, if neutralisation of the vaccine virus is required but difficult to achieve, the tests described below are adapted, as required, to provide the necessary guarantees of freedom from contamination with an extraneous agent.

If the vaccine virus cannot be completely neutralised – using monospecific antiserum – for the yolk sac inoculation test, the following tests may be performed:

— for enveloped viruses such as Newcastle disease virus: perform the test for extraneous agents using embryonated hens’ eggs by the yolk sac route with prior inactivation of enveloped vaccine viruses with lipid solvents,
— for avian encephalomyelitis virus and avian nephritis viruses: perform appropriate tests to detect these viruses. For avian nephritis virus, the test in chicken kidney cells described in section 2 of general chapter 2.6.24. Avian viral vaccines: tests for extraneous agents in seed lots may be used.

If the vaccine virus cannot be completely neutralised – using monospecific antiserum – for the other tests below, alternatively or in addition to in vitro tests conducted on the batch, a test for extraneous agents may be conducted on chick sera obtained from testing the batch of vaccine, as described under 6 Test for extraneous agents using chicks in general chapter 2.6.24. Avian viral vaccines: tests for extraneous agents in seed lots.

h) Other types of tests than those indicated may be used, provided they are at least as sensitive as those indicated and of appropriate specificity. Nucleic acid amplification techniques (2.6.21) give specific detection for many agents and can be used after validation for sensitivity and specificity.

1 TEST FOR EXTRANEOUS AGENTS USING EMBRYONATED HENS’ EGGS

Prepare the test vaccine, diluted if necessary, to contain neutralised virus equivalent to 10 doses of vaccine in 0.2 mL of inoculum. Suitable antibiotics may be added. Inoculate the test vaccine into 3 groups of 10 embryonated hens′ eggs as follows:

— group 1: 0.2 mL into the allantoic cavity of each 9- to 11-day-old embryonated egg,
— group 2: 0.2 mL onto the chorio-allantoic membrane of each 9- to 11-day-old embryonated egg,
— group 3: 0.2 mL into the yolk sac of each 5- to 6-day-old embryonated egg.

Candle the eggs in groups 1 and 2 daily for 7 days and the eggs in group 3 for 12 days. Discard embryos that die during the first 24 h as non-specific deaths; the test is not valid unless at least 6 embryos in each group survive beyond the first 24 h after inoculation. Examine macroscopically for abnormalities all embryos which die more than 24 h after inoculation, or which survive the incubation period. Examine also the chorio-allantoic membranes of these eggs for any abnormality and test the allantoic fluids for the presence of haemagglutinating agents.

Carry out a further embryo passage. Pool separately material from live and from the dead and abnormal embryos. Inoculate each pool into 10 eggs for each route as described above, chorio-allantoic membrane material being inoculated onto chorio-allantoic membranes, allantoic fluids into the allantoic cavity and embryo material into the yolk sac. For eggs inoculated by the allantoic and chorio-allantoic routes, candle the eggs daily for 7 days, proceeding and examining the material as described above. For eggs inoculated by the yolk sac route, candle the eggs daily for 12 days, proceeding and examining the material as described above.

The batch of vaccine complies with the test if no test embryo shows macroscopic abnormalities or dies from causes attributable to the vaccine and if examination of the chorio-allantoic membranes and testing of the allantoic fluids show no evidence of the presence of extraneous agents.

As previously mentioned under General provisions (paragraph g), if neutralisation of the virus is not possible and as a consequence the yolk sac inoculation cannot be evaluated, suitable tests other than those indicated may be carried out to provide the necessary guarantees of freedom from contamination with an extraneous agent; in particular other tests to detect avian encephalomyelitis and avian nephritis viruses may be carried out. In this case, justification to use other tests must be provided, and the vaccine complies if there is no evidence of the presence of avian encephalomyelitis virus or avian nephritis virus.

2 TEST IN CHICKEN embryo fibroblast cells

Prepare 7 monolayers of primary or secondary chicken embryo fibroblasts, from the tissues of 9- to 11-day-old embryos, each monolayer having an area of about 25 cm2. Maintain 2 monolayers as negative controls and treat these in the same way as the 5 monolayers inoculated with the test vaccine, as described below. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers. Allow adsorption for 1 h and add culture medium. Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made with pooled cells and fluids from all 5 monolayers after carrying out a freeze-thaw cycle. Inoculate 0.1 mL of pooled material onto each of 5 recently prepared monolayers of chicken embryo fibroblast cells, each monolayer having an area of about 25 cm2 each as before. For the last passage, grow the cells also on a suitable substrate so as to obtain an area of about 10 cm2 of cells from each of the monolayers, for test A. The test is not valid if less than 80 per cent of the test monolayers, or neither of the 2 negative control monolayers survive after any passage.

Examine microscopically all the cell cultures frequently throughout the entire incubation period for any signs of cytopathic effect or other evidence of the presence of contaminating agents in the test vaccine. At the end of the total incubation period, carry out the following procedures.

A. Fix and stain (with Giemsa or haematoxylin and eosin) about 10 cm2 of confluent cells from each of the 5 original monolayers. Examine the cells microscopically for any cytopathic effect, inclusion bodies, syncytial formation, or any other evidence of the presence of a contaminating agent from the test vaccine.
B. Drain and wash about 25 cm2 of cells from each of the 5 monolayers. Cover these cells with a 0.5 per cent suspension of washed chicken red blood cells (using at least 1 mL of suspension for each 5 cm2 of cells). Incubate the cells at 4 °C for 20 min and then wash gently in phosphate buffered saline pH 7.4. Examine the cells microscopically for haemadsorption attributable to the presence of a haemadsorbing agent in the test vaccine.
C. Test individually samples of the fluid from each cell culture using chicken red blood cells for haemagglutination attributable to the presence of a haemagglutinating agent in the test vaccine.

The test is not valid if there are any signs of extraneous agents in the negative control cultures. The batch of vaccine complies with the test if there is no evidence of the presence of any extraneous agent.

3 TEST FOR EGG DROP SYNDROME VIRUS

Prepare 11 monolayers of chicken embryo liver cells, from the tissues of 14- to 16-day-old embryos, each monolayer having an area of about 25 cm2. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers (test monolayers). Allow adsorption for 1 h, add culture medium. Inoculate 4 of the monolayers with a suitable strain of egg drop syndrome virus (not more than 10 CCID50 in 0.1 mL) to serve as positive control monolayers. Maintain 2 non-inoculated monolayers as negative control monolayers.

Incubate the cells for a total of at least 21 days, subculturing every 4-5 days. Each passage is made as follows: carry out a freeze-thaw cycle; prepare separate pools of the cells plus fluid from the test monolayers, from the positive control monolayers and from the negative control monolayers; inoculate 0.1 mL of the pooled material onto each of 5, 4 and 2 recently prepared monolayers of chicken embryo liver cells, each monolayer having an area of about 25 cm2 as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative control monolayers survive after any passage.

Examine microscopically all the cell cultures at frequent intervals throughout the entire incubation period for any signs of cytopathic effect or other evidence of the presence of a contaminating agent in the test vaccine. At the end of the total incubation period, carry out the following procedure: test separately, cell culture fluid from the test monolayers, positive control monolayers and negative control monolayers, using chicken red blood cells, for haemagglutination attributable to the presence of haemagglutinating agents.

The test is not valid if egg drop syndrome virus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive. If the results for more than 1 of the test monolayers are inconclusive then further subcultures of reserved portions of the monolayers shall be made and tested until an unequivocal result is obtained.

The batch of vaccine complies with the test if there is no evidence of the presence of egg drop syndrome virus or any other extraneous agent.

4 TEST FOR MAREK’S DISEASE VIRUS

Prepare 11 monolayers of primary or secondary chick embryo fibroblasts from the tissues of 9- to 11-day-old embryos, each monolayer having an area of about 25 cm2. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers (test monolayers). Allow adsorption for 1 h, and add culture medium. Inoculate 4 of the monolayers with a suitable strain of Marek’s disease virus (not more than 10 CCID50 in 0.1 mL) to serve as positive controls. Maintain 2 non-inoculated monolayers as negative controls.

Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made as follows: trypsinise the cells, prepare separate pools of the cells from the test monolayers, from the positive control monolayers and from the negative control monolayers. Mix an appropriate quantity of each with a suspension of freshly prepared primary or secondary chick embryo fibroblasts and prepare 5, 4 and 2 monolayers, as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative control monolayers survive after any passage.

Examine microscopically all the cell cultures frequently throughout the entire incubation period for any signs of cytopathic effect or other evidence of the presence of a contaminating agent in the test vaccine.

For the last subculture, grow the cells on a suitable substrate so as to obtain an area of about 10 cm2 of confluent cells from each of the original 11 monolayers for the subsequent test: test about 10 cm2 of confluent cells derived from each of the original 11 monolayers by immunostaining for the presence of Marek’s disease virus. The test is not valid if Marek’s disease virus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive.

The batch of vaccine complies with the test if there is no evidence of the presence of Marek’s disease virus or any other extraneous agent.

5 TESTS FOR TURKEY RHINOTRACHEITIS VIRUS

A. In chicken embryo fibroblasts

NOTE: this test can be combined with Test 2 by using the same test monolayers and negative controls, for all stages up to the final specific test for turkey rhinotracheitis virus on cells prepared from the last subculture.

Prepare 11 monolayers of primary or secondary chick embryo fibroblasts from the tissues of 9- to 11-day-old embryos, each monolayer having an area of about 25 cm2. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers (test monolayers). Allow adsorption for 1 h, and add culture medium. Inoculate 4 of the monolayers with a suitable strain of turkey rhinotracheitis virus as positive controls (not more than 10 CCID50 in 0.1 mL). Maintain 2 non-inoculated monolayers as negative controls.

Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made as follows: carry out a freeze-thaw cycle; prepare separate pools of the cells plus fluid from the test monolayers, from the positive control monolayers and from the negative control monolayers; inoculate 0.1 mL of the pooled material onto each of 5, 4 and 2 recently prepared monolayers of chicken embryo fibroblasts cells, each monolayer having an area of about 25 cm2 as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative control monolayers survive after any passage.

For the last subculture, grow the cells on a suitable substrate so as to obtain an area of about 10 cm2 of confluent cells from each of the original 11 monolayers for the subsequent test: test about 10 cm2 of confluent cells derived from each of the original 11 monolayers by immunostaining for the presence of turkey rhinotracheitis virus. The test is not valid if turkey rhinotracheitis virus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive. If the results for both of the 2 test monolayers are inconclusive then further subcultures of reserved portions of the fibroblasts shall be made and tested until an unequivocal result is obtained.

The batch of vaccine complies with the test if there is no evidence of the presence of turkey rhinotracheitis virus or any other extraneous agent.

B. In Vero cells

Prepare 11 monolayers of Vero cells, each monolayer having an area of about 25 cm2. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers (test monolayers). Allow adsorption for 1 h, and add culture medium. Inoculate 4 of the monolayers with a suitable strain of turkey rhinotracheitis virus (not more than 10 CCID50 in 0.1 mL) to serve as positive controls. Maintain 2 non-inoculated monolayers as negative controls.

Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made as follows: carry out a freeze-thaw cycle. Prepare separate pools of the cells plus fluid from the test monolayers, from the positive control monolayers and from the negative control monolayers. Inoculate 0.1 mL of the pooled material onto each of 5, 4 and 2 recently prepared monolayers of Vero cells, each monolayer having an area of about 25 cm2 as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative controls survive after any passage.

For the last subculture, grow the cells on a suitable substrate so as to obtain an area of about 10 cm2 of confluent cells from each of the original 11 monolayers for the subsequent test: test about 10 cm2 of confluent cells derived from each of the original 11 monolayers by immunostaining for the presence of turkey rhinotracheitis virus. The test is not valid if turkey rhinotracheitis virus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive. If the results for more than 1 of the test monolayers are inconclusive then further subcultures of reserved portions of the monolayers shall be made and tested until an unequivocal result is obtained.

The batch of vaccine complies with the test if there is no evidence of the presence of turkey rhinotracheitis virus or any other extraneous agent.

6 TEST FOR CHICKEN ANAEMIA VIRUS

Prepare eleven 20 mL suspensions of the MDCC-MSBI cell line or another cell line of equivalent sensitivity in 25 cm2 flasks containing about 5 × 105 cells/mL. Inoculate 0.1 mL of test vaccine into each of 5 of these flasks. Inoculate 4 other suspensions with 10 CCID50 chicken anaemia virus as positive controls. Maintain not fewer than 2 non-inoculated suspensions. Maintain all the cell cultures for a total of at least 24 days, subculturing 8 times at 3- to 4-day intervals. During the subculturing the presence of chicken anaemia virus may be indicated by a metabolic colour change in the infected cultures, the culture fluids becoming red in comparison with the control cultures. Examine the cells microscopically for cytopathic effect. At this time or at the end of the incubation period, centrifuge the cells from each flask at low speed, resuspend at about 106 cells per millilitre and place 25 µL in each of 10 wells of a multi-well slide. Examine the cells by immunostaining.

The test is not valid if chicken anaemia virus is detected in fewer than 3 of the 4 positive controls or in any of the non-inoculated controls. If the results for more than 1 of the test suspensions are inconclusive then further subcultures of reserved portions of the test suspensions shall be made and tested until an unequivocal result is obtained.

The batch of vaccine complies with the test if there is no evidence of the presence of chicken anaemia virus.

7 TEST FOR DUCK ENTERITIS VIRUS

This test is carried out for vaccines prepared on duck or goose substrates.

Prepare 11 monolayers of primary or secondary Muscovy duck embryo liver cells, from the tissues of 21- or 22-day-old embryos, each monolayer having an area of about 25 cm2. Remove the culture medium when the cells reach confluence. Inoculate 0.1 mL of test vaccine onto each of 5 of the monolayers (test monolayers). Allow adsorption for 1 h and add culture medium. Inoculate 4 of the monolayers with a suitable strain of duck enteritis virus (not more than 10 CCID50 in 0.1 mL) to serve as positive controls. Maintain 2 non-inoculated monolayers as negative controls.

Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made as follows: trypsinise the cells and prepare separate pools of the cells from the test monolayers, from the positive control monolayers and from the negative control monolayers. Mix a portion of each with a suspension of freshly prepared primary or secondary Muscovy duck embryo liver cells to prepare 5, 4 and 2 monolayers, as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative controls survive after any passage.

For the last subculture, grow the cells on a suitable substrate so as to obtain an area of about 10 cm2 of confluent cells from each of the original 11 monolayers for the subsequent test: test about 10 cm2 of confluent cells derived from each of the original 11 monolayers by immunostaining for the presence of duck enteritis virus. The test is not valid if duck enteritis virus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive. If the results for more than 1 of the test monolayers are inconclusive then further subcultures of reserved portions of the monolayers shall be made and tested until an unequivocal result is obtained.

The batch of vaccine complies with the test if there is no evidence of the presence of duck enteritis virus or any other extraneous agent.

8 TEST FOR DUCK AND GOOSE PARVOVIRUSES

This test is carried out for vaccines prepared on duck or goose substrates.

Prepare a suspension of sufficient primary or secondary Muscovy duck embryo fibroblasts from the tissues of 16- to 18-day-old embryos, to obtain not fewer than 11 monolayers, each having an area of about 25 cm2. Inoculate 0.5 mL of test vaccine into an aliquot of cells for 5 monolayers and seed into 5 replicate containers to form 5 test monolayers. Inoculate 0.4 mL of a suitable strain of duck parvovirus (not more than 10 CCID50 in 0.1 mL) into an aliquot of cells for 4 monolayers and seed into 4 replicate containers to form 4 positive control monolayers. Prepare 2 non-inoculated monolayers as negative controls.

Incubate the cultures for a total of at least 21 days, subculturing at 4- to 5-day intervals. Each passage is made as follows: carry out a freeze-thaw cycle. Prepare separate pools of the cells plus fluid from the test monolayers, from the positive control monolayers and from the negative control monolayers. Inoculate 0.5 mL, 0.4 mL and 0.2 mL of the pooled materials into aliquots of a fresh suspension of sufficient primary or secondary Muscovy duck embryo fibroblast cells to prepare 5, 4 and 2 monolayers, as before. The test is not valid if fewer than 4 of the 5 test monolayers or fewer than 3 of the 4 positive controls or neither of the 2 negative controls survive after any passage.

For the last subculture, grow the cells on a suitable substrate so as to obtain an area of about 10 cm2 of confluent cells from each of the original 11 monolayers for the subsequent test: test about 10 cm2 of confluent cells derived from each of the original 11 monolayers by immunostaining for the presence of duck or goose parvovirus. The test is not valid if duck parvovirus is detected in fewer than 3 of the 4 positive control monolayers or in any of the negative control monolayers, or if the results for both of the 2 negative control monolayers are inconclusive.

The batch of vaccine complies with the test if there is no evidence of the presence of duck (or goose) parvovirus or any other extraneous agent.