December 22, 1999 

VETERINARY SERVICES MEMORANDUM NO. 800.89 

Subject: Chicken Anemia Virus 

To: Biologics Licensees, Permittees, and Applicants 

Directors, Center for Veterinary Biologics 

I. PURPOSE 

This memorandum provides guidance for licensees, permittees, and applicants interested 

in using chicken anemia virus (CAV) in licensed facilities for product testing or for the 

preparation of live or killed CAV vaccines according to 9 CFR 103.1 and 102.5. 

II. CANCELLATION 

This memorandum cancels Veterinary Services Memorandum No. 800.89 dated 

February 24, 1997. 

III. BACKGROUND 

Chicken anemia virus is a non-enveloped, single-stranded, circular DNA virus classified 

in the genus Circovirus (F. Murphy et al., Virus Taxonomy, 1995). It can cause anemia, 

immunosuppression, and mortality in young chickens. CAV is widespread throughout the 

U.S. poultry industry and is extremely stable in the environment. The Center for 

Veterinary Biologics (CVB) is interested in preventing biological product contamination 

with CAV and encouraging CAV vaccine development. 

A. Product Contamination

Title 9 CFR 102.3(b) requires that applicants establish the purity of a biological product 

prior to issuance of a U.S. Veterinary Biological Product License. However, the tests 

found in the current Standard Requirements for viral products (9 CFR 113.200 and 

113.300) are insufficient to detect CAV contamination. Therefore, the CVB-Laboratory 

(CVB-L) now performs a polymerase chain reaction (PCR) test for CAV contamination 

when conducting confirmatory testing of new poultry Master Seed Viruses (MSVs). CVB 

is also reviewing the purity regulations for poultry products. While this review is not 

complete, we plan to include CAV testing in a future Proposed Rule; and we will need 

input from the biologics industry on the most feasible means for such testing. In 

anticipation of that proposal, we encourage firms to begin CAV testing of all new poultry 

MSVs to assist in the development of a mutually agreeable Standard Requirement. While 

it is not mandatory until a Standard Requirement is in place, prospective licensees should 

test new poultry MSVs for CAV contamination prior to the submission of MSV samples 

to CVB-L for confirmatory testing. Furthermore, we encourage firms to develop a 
Veterinary Services Memorandum No. 800.89 2 

program for testing previously approved poultry MSVs for CAV contamination. 

B. Vaccine Development

On November 25, 1991, CVB issued a Veterinary Biologics Notice entitled “Chicken 

Anemia Virus Vaccines.” Because the ecology of the virus was incompletely understood 

at that time, we indicated that we would consider license applications for killed CAV 

vaccines. Since then, more has been learned about this virus; and we now accept 

applications for licensure of live or killed CAV vaccines. 

IV. GUIDELINES 

A. Poultry MSV Testing

Until a standard assay can be established, CVB suggests the following options for CAV 

testing of poultry MSVs. Each firm must develop the specifics of these tests. CVB will 

also consider other test methods developed by individual firms. 

1. PCR Test - Conduct this test directly on the MSV sample. Upon 

request, CVB-L can make available the method they use to perform their PCR 

test. Alternatively, individual firms could develop similar tests1,2. 

2. Virus Isolation 

a. Firms may evaluate the MSV sample directly for the presence of 

live CAV by inoculation of MDCC-MSB1 cells. Isolation procedures 

should consist of 8-12 passages (every 48-72 hours) over a 3-week period. 

Firms may score cultures as positive or negative based on the presence or 

absence of cytopathogenic effect (CPE). However, firms should confirm 

the induction of CPE by CAV on a representative number of cultures 

using a technique which employs a CAV-specific antibody reagent (e.g., 

virus neutralization or fluorescent antibody techniques)3,4,5. 

b. Firms should report the results of tests conducted on new MSVs 

to CVB on the APHIS Form 2008 they submit when requesting 

confirmatory testing of the MSV. Individual firms should submit the test 

method used, along with data defining the sensitivity and specificity of the 

method described, with the data package accompanying the APHIS Form 

2008. CVB also encourages firms to test previously approved MSVs 

for CAV and to immediately report any positive results to CVB-Inspection 

and Compliance (CVB-IC). 

B. Facility Recommendations

Because CAV is environmentally stable, the licensee should make every effort to prevent 

CAV contamination of biological products within an establishment. 
Veterinary Services Memorandum No. 800.89 3 

1. CAV in Laboratory Facilities - When CAV is present as an agent in a 

firm’s quality assurance testing laboratory, the firm must manage the laboratory 

facilities, personnel, equipment, supplies, and test animals in such a manner as to 

prevent contamination of the production facility with this virus. 

2. CAV in Production Facilities 

a. When CAV is present as an MSV in the production facility, the 

most direct method of preventing product cross-contamination with this 

virus is for the firm to establish CAV production facilities which are 

physically separate and apart from the production of all other products. 

CVB-IC personnel will determine the adequacy of this separation upon 

review and inspection. 

b. If the firm does not establish separate and apart CAV production 

facilities, then it should identify, evaluate, and minimize all the potential 

means of facility contamination throughout the production process. In 

addition, the firm should test all other poultry products prepared in that 

facility for CAV contamination. Such firms would not need to test killed 

products, provided they show the inactivation procedure for the killed 

product is capable of inactivating CAV as described in Section IV. B. of 

3. Disinfection - In any facility (laboratory or production) in which CAV 

is present, the firm should propose and validate, with appropriate studies, the 

cleaning and disinfection methods used in the facility6. 

C. CAV Vaccine Recommendations

For licensure, a killed or live CAV vaccine should meet the requirements in 9 CFR 

113.200 and 113.300, guidelines provided in CVB General Licensing Considerations 

Nos. 800.200 and 800.201, and the following specific licensing considerations: 

1. CAV Vaccine Substrates - Because of the fastidious growth 

requirements of this virus, CVB will consider any of the documented growth 

systems (MDCC-MSB1 cell culture, chicken embryos, and chicken tissues, e.g., 

liver) as potential substrates for a killed or live CAV vaccine. However, firms 

should give due consideration to the potential for contamination with extraneous 

agents inherent to the substrate chosen. 

a. Firms should derive embryos or chicks used in production from 

specific pathogen free (SPF) flocks, as defined in VS Memorandum 

No. 800.65. Since SPF flocks are not necessarily CAV-free, firms should 

monitor source flocks to ensure they remain seronegative for antibodies to 

CAV for 3 weeks following the collection of the eggs or chicks used in 

vaccine production. 
Veterinary Services Memorandum No. 800.89 4 

b. Chicks used as a substrate should be reared, inoculated, and 

harvested in a facility dedicated to this activity. If not, the producer should 

conduct an additional final product test to demonstrate the absence of 

concurrent infection of production chickens by other organisms used in the 

production facility, as described in the supplemental test found in Section 

V of VS Memorandum No. 800.81. 

2. CAV Vaccine Efficacy - CAV causes clinical disease only in young 

chicks. Chicks can become infected either through vertical or horizontal 

transmission. Therefore, the label claim for any CAV vaccine (live or killed) 

should be for the prevention of clinical signs in chicks. This claim should be 

supported by a challenge study conducted on progeny derived from vaccinated 

dams. 

3. CAV Vaccine Safety - The principal safety concern, with either a live or 

a killed CAV vaccine, is the possible transmission of live CAV vertically to the 

offspring of vaccinated dams. 

a. Firms should evaluate live CAV vaccines for their ability to shed 

from vaccinated hens (both in feces and in eggs) and to spread to contact 

chickens. Firms should use these data to establish an upper age limit for 

vaccination so that vaccination does not result in the shedding of vaccine 

virus in eggs. 

b. Killed CAV vaccine production must include an adequate test to 

demonstrate complete inactivation of the virus. 

/s/ Thomas E. Walton for 

Alfonso Torres 

Deputy Administrator 

Veterinary Services 
Veterinary Services Memorandum No. 800.89 5 

REFERENCES 

1Noteborn, M., C. Verschueren, D. Van Roozelaar, S. Veldkamp, A. Van Der Eb, 

and G. De Boer. Detection of chicken anemia virus by DNA hybridization and 

polymerase chain reaction. Avian Pathol. 21:107-118. 1992. 

2Tham, K., and W. Stanislawek. Polymerase chain reaction amplification for 

direct detection of chicken anemia virus DNA in tissues and sera. Avian Dis. 36:1000-

1006. 1992. 

3Goryo, M., T. Suwa, S. Matsumoto, T. Umemura, and C. Itakura. Serial 

propagation and purification of chicken anemia agent in MDCC-MSB1 cell line. Avian 

Pathol. 16:149-163. 1987. 

4Todd, D., J. Creelan, D. Mackie, G. Rixon, and M. McNulty. Purification and 

biochemical characterization of chicken anemia agent. J. Gen. Virol. 71:819-823. 1990. 

5McNulty, M., T. Conner, F. McNeilly, K. Kirkpatrick, and J. McFerran. A 

serological survey of domestic poultry in the United Kingdom for antibody to chicken 

anemia agent. Avian Pathol. 17:315-324. 1988. 

6Yuasa, N., (1992). Effect of chemicals on the infectivity of chicken anemia virus. 

Avian Pathol., 21:315-319.