Edition: BP 2025 (Ph. Eur. 11.6 update)
1 DEFINITION
Avian infectious bursal disease vaccine (live) [Gumboro disease vaccine (live)] is a preparation of a suitable strain of infectious bursal disease virus type 1. This monograph applies to vaccines intended for administration to chickens for active immunisation; it applies to vaccines containing strains of low virulence but not to those containing strains of higher virulence that may be needed for disease control in certain epidemiological situations.
2 PRODUCTION
2-1 PREPARATION OF THE VACCINE
The vaccine virus is grown in embryonated hens’ eggs or in cell cultures.
2-2 SUBSTRATE FOR VIRUS PROPAGATION
2-2-1 Embryonated hens’ eggs
If the vaccine virus is grown in embryonated hens’ eggs, they are obtained from flocks free from specified pathogens (SPF) (5.2.2).
2-2-2 Cell cultures
If the vaccine virus is grown in cell cultures, they comply with the requirements for cell cultures for the production of vaccines for veterinary use (5.2.4).
2-3 CHOICE OF VACCINE VIRUS
The vaccine virus shall be shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) for the chickens for which it is intended.
The following tests for safety (section 2-3-1), damage to the bursa of Fabricius (section 2-3-2), immunosuppression (section 2-3-3), increase in virulence (section 2-3-4) and immunogenicity (section 2-3-5) may be used during the demonstration of safety and efficacy.
2-3-1 Safety
Carry out the test for each route and method of administration to be recommended for vaccination using in each case chickens not older than the minimum age to be recommended for vaccination and from an SPF flock (5.2.2). Use vaccine virus at the least attenuated passage level that will be present in a batch of the vaccine.
For each test performed in chickens younger than 3 weeks of age, use not fewer than 10 chickens. For each test performed in chickens older than 3 weeks of age, use not fewer than 8 chickens. Administer to each chicken a quantity of the vaccine virus equivalent to not less than 10 times the maximum virus titre likely to be contained in 1 dose of the vaccine. Observe the chickens at least daily for at least 14 days.
The test is not valid if more than 10 per cent of the chickens younger than 3 weeks of age show abnormal signs of disease or die from causes not attributable to the vaccine. For chickens older than 3 weeks of age, the test in not valid if non- specific mortality occurs.
The vaccine virus complies with the test if no chicken shows abnormal signs of disease or dies from causes attributable to the vaccine virus.
2-3-2 Damage to the bursa of Fabricius
Carry out the test for the route to be recommended for vaccination likely to be the least safe using chickens not older than the minimum age to be recommended for vaccination. Use virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. Use not fewer than 20 chickens from an SPF flock (5.2.2). Administer to each chicken a quantity of the vaccine virus equivalent to 10 times the maximum titre likely to be contained in a dose of the vaccine. On each of days 7, 14, 21 and 28 after administration of the vaccine virus, euthanise not fewer than 5 chickens and prepare a section from the site with the greatest diameters of the bursa of Fabricius of each chicken. Carry out histological examination of the section and score the degree of bursal damage using the following scale.
| 0 | No lesion, normal bursa. |
| 1 | 1 per cent to 25 per cent of the follicles show lymphoid depletion (i.e., less than 50 per cent depletion in 1 affected follicle); influx of heterophils in lesions. |
| 2 | 26 per cent to 50 per cent of the follicles show nearly complete lymphoid depletion (i.e., more than 75 per cent depletion in 1 affected follicle), affected follicles show necrosis and severe influx of heterophils may be detected. |
| 3 | 51 per cent to 75 per cent of the follicles show lymphoid depletion; affected follicles show necrosis and severe influx of heterophils is detected. |
| 4 | 76 per cent to 100 per cent of the follicles show nearly complete lymphoid depletion, hyperplasia and cyst structures are detected; affected follicles show necrosis and severe influx of heterophils is detected. |
| 5 | 100 per cent of the follicles show nearly complete lymphoid depletion; complete loss of follicular structure, thickened and folded epithelium, fibrosis of bursal tissue. |
Calculate the average score for each group of chickens. The vaccine virus complies with the test if:
— no chicken shows notable clinical signs of disease or dies from causes attributable to the vaccine virus;
— the average score for bursal damage 21 days after administration of the vaccine virus is less than or equal to 2.0 and 28 days after administration is less than or equal to 0.6;
— during the 21 days after administration a notable repopulation of the bursae by lymphocytes has taken place.
2-3-3 Immunosuppression
Carry out the tests for the route to be recommended for vaccination likely to be the least safe using chickens not older than the minimum age to be recommended for vaccination. Use vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. Use not fewer than 30 chickens from an SPF flock (5.2.2). Divide them randomly into 3 groups each of not fewer than 10 and maintain the groups separately. Administer to each chicken of 1 group a quantity of the vaccine virus equivalent to not less than the maximum titre likely to be contained in 1 dose of the vaccine. At the time after administration when maximal bursal damage is likely to be present, as judged from the results obtained in the test for damage to the bursa of Fabricius (section 2-3-2), administer by eye-drop to each vaccinated chicken and to each chicken of another group 1 dose of Hitchner B1 strain Newcastle disease vaccine (live).
Determine the seroresponse of each chicken of the 2 groups to the Newcastle disease virus 14 days after administration.
Challenge each chicken of the 3 groups by the intramuscular route with not less than 105 EID of virulent Newcastle disease virus and note the degree of protection in the 2 groups vaccinated with Hitchner B1 strain Newcastle vaccine compared with the non-vaccinated group.
The test is not valid if 1 or more of the non-vaccinated chickens does not die within 7 days of challenge. The degree of immunosuppression is estimated from the comparative seroresponses and protection rates of the 2 Hitchner B1 vaccinated groups.
The vaccine complies with the test if there is no significant difference between the 2 groups.
2-3-4 Increase in virulence
Carry out the test according to general chapter 5.2.6 using chickens from an SPF flock (5.2.2) and not older than the minimum age to be recommended for vaccination. If the properties of the vaccine virus allow sequential passage through 5 groups via natural spreading, this method may be used, otherwise passage as described below is carried out.
Administer to each chicken of the 1st group by eye-drop a quantity of the vaccine virus that will allow recovery of virus for the passages described below. Prepare 3 to 4 days after administration a suspension from the bursa of Fabricius of each chicken and pool these samples. Administer 0.05 mL of the pooled samples by eye-drop to each chicken of the next group. Carry out this passage operation not fewer than 4 times; verify the presence of the virus at each passage. If the virus is not found at a passage level, repeat the passage by administration to a group of 10 chickens.
Carry out the test for damage to the bursa of Fabricius (section 2-3-2) using the material used for the 1st passage and the virus at the final passage. Administer the virus by the route to be recommended for vaccination likely to be the least safe.
The vaccine virus complies with the test if no indication of increasing virulence of the virus recovered for the final passage compared with the material used for the 1st passage is observed. If virus is not recovered after an initial passage in 5 chickens and a subsequent repeat passage in 10 chickens, the vaccine virus also complies with the test.
2-3-5 Immunogenicity
A test is carried out for each route and method of administration to be recommended using in each case chickens not older than the minimum age to be recommended for vaccination. The quantity of vaccine virus administered to each chicken is not greater than the minimum virus titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of the vaccine. Use not fewer than 30 chickens of the same origin and from an SPF flock (5.2.2). Vaccinate by a route to be recommended not fewer than 20 chickens. Maintain not fewer than 10 chickens as controls.
Challenge each chicken after 14 days by eye-drop with a sufficient quantity of virulent avian infectious bursal disease virus. Observe the chickens at least daily for 10 days after challenge. Record the deaths due to infectious bursal disease and the surviving chickens that show clinical signs of disease. At the end of the observation period, euthanise all the surviving chickens and carry out histological examination for lesions of the bursa of Fabricius.
The test is not valid if one or more of the following applies:
— during the observation period following challenge, fewer than 50 per cent of the control chickens show characteristic signs of avian infectious bursal disease;
— 1 or more of the surviving control chickens does not show degree 3 lesions of the bursa of Fabricius;
— during the period between the vaccination and challenge more than 10 per cent of the vaccinated or control chickens show abnormal clinical signs or die from causes not attributable to the vaccine.
The vaccine virus complies with the test if during the observation period after challenge not fewer than 90 per cent of the vaccinated chickens survive and show no notable clinical signs of disease nor degree 3 lesions of the bursa of Fabricius.
3 BATCH TESTS
3-1 Identification
The vaccine, diluted if necessary, is identified using a suitable method. For example, when mixed with a monospecific infectious bursal disease virus type 1 antiserum, it is no longer able to infect embryonated hens’ eggs from an SPF flock (5.2.2) or susceptible cell cultures (5.2.4) into which it is inoculated.
3-2 Bacteria and fungi
Vaccines intended for administration by injection comply with the test for sterility prescribed in the general monograph Vaccines for veterinary use (0062).
Any diluent supplied for reconstitution of the vaccine complies with the test for sterility prescribed in the general monograph Vaccines for veterinary use (0062).
3-3 Mycoplasmas (2.6.7)
The vaccine complies with the test for mycoplasmas.
3-4 Extraneous agents (5.2.5)
The vaccine is free from extraneous agents.
3-5 Virus titre
Titrate the vaccine virus by inoculation into embryonated hens’ eggs from an SPF flock (5.2.2) or into suitable cell cultures (5.2.4). The vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label.
3-6 Potency
The vaccine complies with the requirements of the test prescribed under Immunogenicity (section 2-3-5) when administered by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label.
