Safeguarding American Agriculture 

Federal Relay Service 

APHIS is an agency of USDA’s Marketing and Regulatory Programs (Voice/TTY/ASCII/Spanish)

An Equal Opportunity Provider and Employer 1-800-877-8339 

March 7, 2012 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-04 

TO: Biologics Licensees, Permittees, and Applicants 

 Directors, Center for Veterinary Biologics 

 Veterinary Services Leadership Team 

 

FROM: Richard E. Hill, Jr. /s/ Byron E. Rippke, for

 Director 

 Center for Veterinary Biologics 

SUBJECT: Appropriate Use of Controls for Chicken Anemia Virus Extraneous Agent 

 Testing, Availability of a PCR-based Testing Protocol, and Availability of 

 a New Testing Reagent 

I. PURPOSE 

This Notice provides guidance on the use of controls in the testing for extraneous chicken 

anemia virus (CAV) that will provide valid test results for interpretation. It also provides 

information on the availability of a new polymerase chain reaction (PCR) based protocol, 

discussed in Veterinary Services Memorandum 800.89, for the detection of extraneous 

CAV. The new version of the protocol contains a new reagent for use as a positive 

control. 

II. BACKGROUND 

Testing for extraneous CAV is recommended for firms in the case of Master Seed 

Viruses (MSV) prepared using poultry origin substrates. It is required for infectious 

bursal disease virus fractions propagated in chicken bursas and when outbreaks of CAV 

occur in CAV-negative specific pathogen free (SPF) flocks used in veterinary biologics 

preparation. 

The Center for Veterinary Biologics (CVB) laboratory routinely tests for extraneous 

CAV using the current version of our protocol, entitled “Polymerase Chain Reaction 

Assay for Detection and Identity of Extraneous Chicken Anemia Virus (CAV)”, and 

designated VIRPRO0118.04. A copy is attached to this Notice. This protocol has been 

posted to our web site with other selected protocols; future versions of this protocol will 

be posted there or available upon request. 

United States 

Department of 

Agriculture 

Animal and Plant 

Health Inspection 

Service 

Veterinary Services 

Center for Veterinary 

Biologics 

1920 Dayton Avenue 

PO Box 844 

Ames, IA 50010 

(515) 337- 6100 
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-04 

Page 2 

The current protocol makes use of internal and external positive controls to allow for the 

interpretation of negative test results. An internal control is added to the sample material 

being tested to ensure the sample material does not contain components that will inhibit 

or interfere with the amplification of CAV DNA. Inhibition of the PCR has been 

reported in the literature (Soine et al11) and observed in our laboratory (6/75 samples 

tested in Summer/Fall 2010). An external positive control is added to the PCR master 

mix to ensure amplification of the CAV DNA template by the reaction. 

Several recent CAV outbreaks in CAV-negative SPF flocks have resulted in use of the 

CVB PCR protocol and the CVB’s review of bench records provided by licensed firms. 

It was noted that specifics of the test method were not always on file with the CVB and 

positive controls were not always incorporated in the testing. 

III. POLICY 

When testing for extraneous CAV is conducted, the procedure used should be on file as a 

Special Outline and acceptable to the Animal and Plant Health Service (APHIS). A 

procedure acceptable to APHIS should include internal and external positive controls. In 

the case of a PCR-based test, the results of both the master mix (external control) and a 

spike for the material being tested (internal control) should be reported with test results 

relative to the presence or absence of CAV. If another test method is selected and 

acceptable to APHIS, the results of equivalent controls should be included in the Special 

Outline and reported. 

An example of a procedure acceptable to APHIS has been developed by the CVB 

laboratory and the current version is documented in VIRPRO0118. In connection with 

this, a 419 base pair (bp) fragment of CAV, which can be used as an internal or external 

positive control for the procedure described in VIRPRO0118.04, was developed. Its 

usefulness for other protocols should be evaluated on a case by case basis. This reagent 

will be available in limited quantites as it is being incorporated into the PCR protocols 

selected by firms. After the supply is exhausted, firms will be expected to prepare their 

own lots. The protocol used to prepare this reagent, entitled "Production of Chicken 

Anemia Virus (CAV) DNA Positive Control" and designated VIRNPP0001.01, is also 

attached to this Notice. This will be available on our web site or by request, as well. 

Updated versions of the protocol will be placed on the web site. If comparable positive 

controls have been prepared by firms, they should feel free to use them. 

                                                            

1 Soine, C., Watson, S. K., Rybicki, E., Lucio, B., Nordgren, R. M., Parrish, C. R., and Schat, K. A,

1993. Avian Diseases 37: 467‐476
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-04 

Page 3 

Outlines indicating the use of CAV extranaeous agent testing should reference a Special 

Outline that provides the details for conducting the test. The Special Outline should 

specifiy the positive and negative controls used, and provide details on the nature of the 

controls. If a firm chooses to use the CVB protocol, the details of our protocol may be 

incorporated into a Special Outline maintained by the firm. If another protocol is used, 

data equivalent to our protocol should be submitted to support it. 

IV. IMPLEMENTATION 

The use of CAV positive controls (internal and external) for the testing for extraneous 

CAV is considered a requirement for a valid negative test result for the material being 

tested. Firms should review their procedures to ensure that positive controls are included 

in the protocols. This should be in place within 30 days of the publication of this Notice. 

Bench records for CAV testing may be audited by CVB during inspections or as the 

result of an outbreak and should clearly indicate the controls that are used. It should be 

possible to determine the nature of such controls from firm documentation. 

Additionally, Outlines of Production should be revised and Special Outlines drafted to be 

consistent with these recommendations by the next annual review of the relative 

documents. Therefore, firms would be expected to be in compliance with this Notice 

within 18 months of publication of this Notice. Firms needing additional time to come 

into compliance with the filing of documents should contact their reviewer to discuss 

their particular situation.