March 30, 2001 

VETERINARY SERVICES MEMORANDUM NO. 800.81 

Subject: Bursal Disease Vaccine of Chicken Bursa Origin 

To: Biologics Licensees, Permittees, and Applicants 

Directors, Center for Veterinary Biologics 

I. PURPOSE 

This memorandum gives guidance to licensees, permittees, and applicants concerning the 

production of Bursal Disease Vaccine, Killed Virus. The Center for Veterinary Biologics 

(CVB) considers license applications for this vaccine produced from any variant or 

standard strain(s) of infectious bursal disease virus (IBDV) propagated in chicken bursas. 

This memorandum specifies production recommendations and test procedures to ensure 

the purity of products which contain IBDV propagated in chicken bursas. 

II. CANCELLATION 

This memorandum cancels Veterinary Biologics Memorandum No. 800.81 dated 

January 18, 2000. 

III. BACKGROUND 

APHIS has published the Standard Requirement for Bursal Disease Vaccine, Killed 

Virus, in 9 CFR 113.212. This regulation requires that the vaccine “shall be prepared 

from virus-bearing cell culture fluids or embryonated chicken eggs.” APHIS has granted 

an exemption to this requirement under 9 CFR 113.4(a) because some firms encountered 

difficulties in producing vaccines with some isolates by the standard methods. 

This exemption permits the preparation of bursal disease vaccines by propagating an 

isolate of IBDV in chickens and then harvesting the bursal tissue from such infected 

chickens for the vaccine. However, this production method places these products at an 

increased risk of contamination with extraneous agents which may infect the chicken 

prior to or during production. APHIS has granted this exemption for the production of 

either variant or standard strains of IBDV virus in chicken bursas provided firms address 

this concern by establishing production procedures that ensure the purity of their product. 

This is under 9 CFR 113.4(b). 

The January 18, 2000 version of this memorandum is being replaced after it was brought 

to the attention of the CVB that source flocks serologically positive for Chicken Anemia 

Virus (CAV) are frequently being used in the production of bursal origin IBDV due to 

the demand for large numbers of birds for the production of this product. The 

requirement that source flocks remain seronegative to antibodies to CAV for 3 weeks 
Veterinary Services Memorandum No. 800.81 2 

following the selection of the chickens for use in vaccine production has been dropped. 

Currently, the CVB is reviewing the purity regulations for poultry products. While this 

review is not complete, the CAV status of the source flock and CAV testing of final 

product will be addressed in a future Proposed Rule. Input from the biologics industry on 

the most feasible means for such testing would be welcomed. 

IV. PRODUCTION RECOMMENDATIONS 

Since current Standard Requirements for virus products (9 CFR 113.200 and 113.300) are 

insufficient to detect chicken anemia virus (CAV) contamination and the production of 

bursal disease vaccine in chicken bursa presents a significant risk of such contamination, 

firms should use the following production procedures to ensure the purity of this vaccine 

when produced in chickens. 

A. Master Seed Virus

Until a standard assay can be established, firms should test all avian Master Seed Viruses 

for extraneous CAV according to one of the optional test procedures described in 

Veterinary Services Memorandum No. 800.89, section IV. A. Firms may obtain test 

methods and training on the PCR test for CAV from the Center for Veterinary BiologicsLaboratory upon request. 

B. Chickens

Firms should use only infectious bursal disease susceptible chickens derived from a 

specific-pathogen-free (SPF) source flock (as defined in Veterinary Services 

Memorandum No. 800.65) for vaccine production. Prior to their use in production, firms 

should house and handle chickens in isolation in order to maintain their pathogen-free 

status. Firms should also prevent exposure to avian pathogens through feed or transport. 

Since all SPF flocks are not necessarily free of CAV, firms should document that their 

IBDV inactivation procedure will inactivate extraneous CAV, or alternately, the firm 

should test the bulks prior to inactivation for the presence of CAV by a test acceptable to 

APHIS (specifics outlined below). 

C. Validation of Inactivation Procedures

Because the risk of contamination with CAV is high and the virus can replicate in bursal 

tissue, each firm should also provide documentation that the inactivation procedure 

described in their filed Outline of Production is capable of inactivating CAV as follows: 

1. Develop a Protocol - Each firm should develop a protocol for 

validation testing that is appropriate for the specific inactivation procedure 

defined in its Outline of Production according to the following guidelines: 
Veterinary Services Memorandum No. 800.81 3 

a. Inoculate SPF chicks from CAV-seronegative flocks of the age 

specified in the Outline of Production for virus propagation with a dose of 

CAV sufficient to meet the validity requirements in section e below. 

(1) Collect and pool bursa from these birds at 7 to 9 days 

post inoculation (CAV-infected bursal pool). 

(2) Collect and pool bursa from uninoculated control birds 

of the same age and hatch (uninfected bursal pool). 

(3) Use a sufficient number of birds in each group to be 

representative of small scale production. 

b. Subject the infected and uninfected bursa pools to the physical 

processing procedures (homogenization, sonication, etc.) described in the 

Outline of Production. 

c. Subject one half of the processed CAV-infected bursal pool to 

the inactivation procedure described in the Outline of Production (test 

sample). Do not inactivate the other half of the processed CAV-positive 

pool (positive control sample). Subject the processed uninfected bursal 

pool to the inactivation procedure described in the Outline of Production 

(negative control sample). 

d. Evaluate the test sample, positive control sample, and the 

negative control sample for the presence of live CAV by titration in 

MDCC-MSB1 cells. 

(1) Subculture each dilution in the titration every 48-72 

hours over a 3-week period (8-12 passages each). 

 (2) Score each dilution as positive or negative based on the 

presence or absence of cytopathology. 

(3) Confirm any induction of CPE using a CAV-specific 

antibody reagent (virus neutralization or fluorescent antibody 

techniques) on a representative number of cultures. 

e. For a valid test, the positive control sample should have a CAV 

titer of at least 105 TCID50/ml, and the negative control sample should be 

negative for CAV. 

f. A satisfactory test should demonstrate complete inactivation of 

the CAV in the test sample. 
Veterinary Services Memorandum No. 800.81 4 

2. Submit Protocol - Firms should submit their protocols to the Center for 

Veterinary Biologics-Licensing and Policy Development for review and comment 

before initiating the study. 

3. Test Site - Firms may conduct studies to validate their inactivation 

procedures in the firm’s research facilities if they are separate and apart from 

production facilities or at an off-site research facility thereby preventing 

contamination of licensed production facilities with CAV. 

4. Changes in Inactivation Procedures - Should the results of the firm’s 

inactivation study indicate the need to change the method of inactivation for the 

product, CVB will accept changes in the method of inactivation in the Outline of 

Production based on data demonstrating the new procedure is adequate to 

inactivate CAV and successful potency assays on three consecutive production 

serials of the newly formulated product according to 9 CFR 113.212(d)(2). 

5. Changes to Outline - If data to accept the inactivation procedure are 

accepted, the firm should document the acceptance (including the date of 

acceptance) in section IV. A. of each affected Outline of Production. 

D. Serial Testing Option

If a firm is not able to demonstrate that its method of inactivation for the product is 

adequate to inactivate CAV as described in section IV. C. above or if it has not completed 

such testing, the firm may use the option of demonstrating the purity of its product by 

testing samples of each serial of vaccine prior to adding the killing agent by one of the 

CAV testprocedures described in Veterinary Services Memorandum No. 800.89, section 

IV. A. Firms using this option must designate specific test requirements in section V. A. 

of their Outlines of Production for such products and provide data demonstrating the 

validity and sensitivity of their test procedure. The acceptance of the test method studies 

along with the acceptance date should be documented in section V. A. along with the test. 

V. ADDITIONAL TESTING RECOMMENDATIONS 

A. Vaccines Produced in Facilities Producing Other Avian Vaccines

The production of Bursal Disease Vaccine in chickens in facilities that also produce other 

avian vaccines presents a risk of contamination of the final product due to the possible 

exposure of the production birds with other vaccine viruses in the production facilities. 

Firms should therefore test bulk or final containers of completed product produced under 

such conditions for purity according to the requirements found in 9 CFR 113.212 and by 

the following supplemental test: 

1. Inoculation of Birds - Inoculate bulk or final container samples of 

completed product from each serial into 10 SPF chickens, 21 to 28 days of age, by 

the recommended route, using twice the recommended dose. Three to four weeks 
Veterinary Services Memorandum No. 800.81 5 

later, inoculate each bird a second time, using twice the recommended dose. (Due 

to the doubled volumes of these inoculations, you may divide each inoculation 

into more than one site.) Hold five chickens of the same source and hatch as 

negative controls. 

2. Test for Antibodies to Extraneous Agents - Collect serum from each 

bird prior to the first inoculation and 2 to 3 weeks following the second 

inoculation. 

a. Test individual sera for the presence of antibodies to all the 

infectious agents of poultry authorized for use within the licensed 

production facility or within any facility housing the chickens. 

b. Use the serological test methods specified in the filed Outline of 

Production or a special outline. (Please note that ELISA assays may yield 

false positive results for some antigens in birds receiving oil adjuvants. 

Therefore, such assays may not be the test of choice for inclusion in 

section V of the outline.) 

c. A serial which induces a positive response to any of these 

agents, with the exception of the bursal vaccine virus or viruses, is 

unsatisfactory. 

B. Vaccines Produced in Isolation Facilities

If chickens used to produce Bursal Disease Vaccine are housed in isolation facilities and 

the production procedures used are found by the Center for Veterinary BiologicsInspection and Compliance to be satisfactory to prevent contamination of the product 

with other avian viruses, CVB will not require firms to conduct supplemental testing on 

each serial of product as described in section V. A. above. 

VI. OUTLINES OF PRODUCTION 

All Outlines of Production for Bursal Disease Vaccine, Killed Virus, of chicken bursa 

origin should conform with the above guidelines. 

/s/ Chester A. Gipson for 

Alfonso Torres 

Deputy Administrator 

Veterinary Services