August 23, 1999

VETERINARY SERVICES MEMORANDUM NO. 800.88

Subject: Testing for Reticuloendotheliosis Virus Contamination

To: Biologics Licensees, Permittees, and Applicants

Directors, Center for Veterinary Biologics

I. PURPOSE

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

testing Master Seed Viruses (MSVs) of poultry biological products for

reticuloendotheliosis virus (REV) contamination.

II. CANCELLATION

This memorandum cancels Veterinary Biologics Memorandum No. 800.88 dated June 12,

1996.

III. BACKGROUND

REV is a retrovirus of poultry which can cause neoplasia and immunosuppression in

infected birds. The MSV of a licensed Fowl Pox Vaccine was found to be contaminated

with REV. The regulations in 9 CFR 102.3(b) require that the purity of a biological

product be established prior to issuance of a U.S. Veterinary Biological Product License.

However, the tests found in the current Standard Requirements for viral products in 

9 CFR 113.200 and 113.300 are insufficient to detect REV contamination.

In order to prevent a recurrence of this problem, Center for Veterinary BiologicsLaboratory (CVB-L) now performs a polymerase chain reaction (PCR) test for REV

contamination when conducting confirmatory testing of new poultry MSVs. Additionally,

the use of specific pathogen free (SPF) substrates in the production of poultry biologics, as

specified in Veterinary Services Memorandum No. 800.65, provides assurance that REV

contamination will not occur during the production process. 

The Center for Veterinary Biologics (CVB) is currently reviewing the purity regulations

for poultry products. While this review is not yet complete, we have determined that a

future Proposed Rule should include a Standard Requirement test for contamination of

poultry MSVs with REV. In anticipation of that proposal, we encourage firms to begin

REV testing now so that they may assist in the development of a mutually agreeable

Standard Requirement.
Veterinary Services Memorandum No. 800.88 2

Therefore, this memorandum describes procedures for testing poultry MSVs for REV

contamination. While not mandatory until a Standard Requirement is in place, prospective

licensees should test new poultry MSVs for REV contamination prior to the submission of

MSV samples to CVB-L for confirmatory testing. Furthermore, we encourage firms to

develop a program for testing previously approved poultry MSVs for REV contamination.

IV. TEST PROCEDURES

Until a standard assay can be established, any one of the following three options is

acceptable for REV testing of MSVs. This section, however, serves only as a general

guideline; the specifics of the tests must be developed by each individual firm. 

A. PCR Test

Conduct this test directly on the MSV sample. Upon request, the CVB-L can make

available the method they use to perform their PCR test, or individual firms may develop

similar tests.

1,2

B. In-vivo Virus Amplification 

Inoculate 1-day-old SPF chicks with the MSV; 2 weeks later evaluate blood samples for

the presence of REV using an enzyme immunoassay (ELISA), fluorescent antibody (FA), or

PCR test.3,4

C. Serology

Inoculate SPF chicks twice (at 2 and 4 weeks of age) with the MSV; 3 to 4 weeks later

evaluate blood samples for the presence of REV-specific antibodies using ELISA or FA

tests.4,5

[General comment: Do not filter the MSV sample (e.g., to remove pox virus) prior to

conducting tests for REV. Filtering could reduce the titer of any potentially contaminating

REV.]
Veterinary Services Memorandum No. 800.88 3

 

V. TEST REPORTING

A. New MSVs

Report the results of tests conducted on new MSVs to CVB on the APHIS Form 2008

submitted when requesting confirmatory testing of the MSV. Submit the test method(s)

used by an individual firm, along with data defining the sensitivity and specificity of the

method described, with the data package accompanying the APHIS Form 2008.

B. Previously Approved MSVs

Again, we encourage firms to also test previously approved MSVs for REV. Firms

conducting such tests should immediately report any positive results to CVB-Inspection and

Compliance.

 /s/

Alfonso Torres

Deputy Administrator

Veterinary Services

Veterinary Services Memorandum No. 800.88 4

REFERENCES

1Aly M, Smith E, Fadly A. Detection of reticuloendotheliosis virus infection using

the polymerase chain reaction. Avian Pathol., 22:543-554. 1993.

2Davidson I, Borovskaya A, Perl S, Malkinson M. Use of the polymerase chain

reaction for the diagnosis of natural infection of chickens and turkeys with Marek’s disease

virus and reticuloendotheliosis virus. Avian Pathol., 24:69-94. 1995.

3Fadly A, Witter R. Comparative evaluation of three methods for detection of

reticuloendotheliosis virus as a contaminant in a live-virus vaccine of poultry (abstract).

Proceedings of the 132nd Annual Meeting of the AVMA. 1995.

4Witter R. Reticuloendotheliosis. In: A laboratory manual for the isolation and

identification of avian pathogens, 3rd ed. Purchase HG, Arp LH, Domermuth CH, Pearson

JE, eds. Kendall/Hunt Publishing Co., Dubuque, IA. pp. 143-148. 1989.

5Melchior F. Oral presentation, Poultry section, 6th USDA Veterinary Biologics

Public Meeting, 1995.