The complement-fixation test for detection of avian lymphoid leukosis provided in this section shall be conducted on all biological products containing virus which has been propagated in substrates of chicken origin: Provided, An inactivated viral product shall be exempt from this requirement if the licensee can demonstrate to Animal and Plant Health Inspection Service that the agent used to inactivate the vaccine virus would also inactivate lymphoid leukosis virus.

(a) Propagation of contaminating lymphoid leukosis viruses, if present, shall be done in chick embryo cell cultures.

(1) Each vaccine virus, cytopathic to chick embryo fibroblast cells, shall be effectively neutralized, inactivated, or separated so that minimal amounts of lymphoid leukosis virus can be propagated on cell culture during the 21-day growth period. If a vaccine virus cannot be effectively neutralized, inactivated, or separated, a sample of another vaccine prepared the same week from material harvested from each source flock (or other sampling procedure acceptable to Animal and Plant Health Inspection Service) used for the preparation of the questionable vaccine virus that cannot be neutralized, inactivated, or separated shall be tested each week during the preparation of such questionable vaccine.

(2) When cell cultures are tested, 5 ml of the final cell suspension as prepared for seeding of production cell cultures shall be used as inoculum. When vaccines are tested, the equivalent of 200 doses of Newcastle disease vaccine or 500 doses of other vaccines for use in poultry, or one dose of vaccine for use in other animals shall be used as inoculum. Control cultures shall be prepared from the same cell suspension as the cultures for testing the vaccine.

(3) Uninoculated chick embryo fibroblast cell cultures shall act as negative controls. One set of chick fibroblast cultures inoculated with subgroup A virus and another set inoculated with subgroup B virus shall act as positive controls, A and B respectively.

(4) The cell cultures shall be propagated at 35-37 °C for at least 21 days. They shall be passed when necessary to maintain viability and samples harvested from each passage shall be tested for group specific antigen.

(b) The microtiter complement-fixation test shall be performed using either the 50 percent or the 100 percent hemolytic end point technique to determine complement unitage. Five 50 percent hemolytic units or two 100 percent hemolytic units of complement shall be used for each test.

(1) All test materials, including positive and negative controls, shall be stored at −60 °C or colder until used in the test. Before use, each sample shall be thawed and frozen three times to disrupt intact cells and release the group specific antigen.

(2) The antiserum used in the microtiter complement-fixation test shall be a standard reagent supplied or approved by the Animal and Plant Health Inspection Service. Four units of antiserum shall be used for each test.

(3) Presence of complement-fixing activity in the harvested samples (from passages) at the 1:4 or higher dilution, in the absence of anticomplementary activity, shall be considered a positive test unless the activity can definitely be established to be caused by something other than lymphoid leukosis virus, subgroups A and/or B. Activity at the 1:2 dilution shall be considered suspicious and the sample further subcultured to determine presence or absence of the group specific antigen.

(4) Biological products or primary cells which are found contaminated with lymphoid leukosis viruses are unsatisfactory. Source flocks from which contaminated material was obtained are also unsatisfactory.