Tenosynovitis Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs.

Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300, except (a)(3)(ii) and (c), and the special requirements in this section.

(b) Each lot of Master Seed shall be tested for:

(1) Pathogens by the chicken inoculation test prescribed in §113.36.

(2) Lymphoid leukosis virus contamination as follows:

(i) Each of at least 10 3-week-old or older lymphoid leukosis free chickens from the same source and hatch shall be injected intra-muscularly with an amount of Master Seed equal to 100 label doses of vaccine. At least 15 chickens of the same source and hatch shall be used as controls; 5 or more shall be unvaccinated and serve as negative controls; 5 or more shall be injected with subgroup A lymphoid leukosis virus; and 5 or more with subgroup B lymphoid leukosis virus. Each group of control chickens shall be held isolated from each other and from the vaccinates.

(ii) Twenty-one to 28 days postinoculation, blood samples shall be taken from each chicken and the serum separated using a technique conducive to virus preservation. These serums shall be used as inocula in the complement fixation for avian lymphoid leukosis (COFAL) test prescribed in §113.31.

(iii) Serums from the vaccinates shall be tested separately, but serums within each control group may be pooled. A valid test shall have positive COFAL reactions from each virus inoculated group and negative reactions from the uninoculated controls. If any of the chickens injected with the Master Seed have positive COFAL test reactions in a valid test, the Master Seed is unsatisfactory.

(3) Identity using the following agar gel immunodiffusion test. The undiluted Master Seed may be used as test antigen or the Master Seed may be inoculated onto the chorioallantoic membrane (CAM) of fully susceptible chicken embryos and the infected CAMs ground and used as antigen. A known tenosynovitis antiserum and a known tenosynovitis antigen shall be used in the test. A precipitin line shall form between the test antigen and the known antiserum in the center well which shows identity with the line formed between the antiserum and the known antigen, or the Master Seed is unsatisfactory.

(4) Safety using the following chicken test:

(i) For vaccines intended for use in chickens less than 14 days of age, Master Seed equal to 10 label doses shall be administered subcutaneously to each of 25 1-day-old tenosynovitis susceptible chickens.

(ii) For vaccines intended for use only in chickens 14 days of age or older, Master Seed equal to 10 label doses shall be administered subcutaneously to each of 25 4-week-old or older tenosynovitis susceptible chickens.

(iii) The vaccinates shall be observed each day for 21 days. If unfavorable reactions occur which are attributable to the vaccine, the Master Seed is unsatisfactory. If unfavorable reactions occur which are not attributable to the vaccine, the test is a No Test and may be repeated.

(c) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose shall be established as follows:

(1) Tenosynovitis susceptible chickens, of the same age and from the same source shall be used as test birds. Vaccines intended for use in very young chickens shall be administered to chickens of the youngest age for which the vaccine is recommended. Vaccines intended for use in older chickens shall be administered to 4-week-old or older chickens. Twenty or more vaccinates shall be used for each method of administration recommended on the label. Ten or more chickens shall be held as unvaccinated controls.

(2) A geometric mean titer of the vaccine produced at the highest passage from the Master Seed shall be established using a method acceptable to Animal and Plant Health Inspection Service before the immunogenicity test is conducted. A predetermined quantity of vaccine virus shall be administered to each vaccinate. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the dose.

(3) Twenty-one to 28 days postvaccination, each vaccinate and control shall be challenged by injecting virulent virus furnished or approved by Animal and Plant Health Inspection Service into one foot pad. The vaccinates and controls shall be observed each day for 14 days. If at least 90 percent of the controls do not develop swelling and discoloration in the phalangeal joint area of the injected foot pad typical of infection with tenosynovitis virus, the test is a No Test and may be repeated. If at least 19 of 20, 27 of 30, or 36 of 40 vaccinates do not remain free from these signs, disregarding transient swelling which subsides within 5 days postchallenge, the Master Seed is unsatisfactory.

(4) An Outline of Production change shall be made before authority for use of a new lot of Master Seed shall be granted by Animal and Plant Health Inspection Service.

(d) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300, except (c), and the requirements in this paragraph.

(1) Purity. Final container samples of completed product from each serial shall be tested for pathogens by the chicken inoculation test prescribed in §113.36.

(2) Safety.

(i) Final container samples of completed product from each serial shall be safety tested as follows:

(A) For vaccines intended for use in very young chickens, each of 25 1-day-old tenosynovitis susceptible chickens shall be vaccinated with the equivalent of 10 doses by one method recommended on the label.

(B) For vaccines intended for use in older chickens, each of 25 4-week-old or older tenosynovitis susceptible chickens shall be vaccinated with the equivalent of 10 doses by one method recommended on the label.

(ii) The vaccinates shall be observed each day for 21 days. If unfavorable reactions occur which are attributable to the product, the serial is unsatisfactory. If unfavorable reactions occur in more than two vaccinates which are not attributable to the product, the test is a No Test and may be repeated. If the test is not repeated, the serial is unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be titrated by the method used in paragraph (c)(2) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of the vaccine virus used in the immunogenicity test prescribed in paragraph (c) of this section to assure that, when tested at any time within the expiration period, each serial and subserial shall have a virus titer 100.7 times greater than that used in the immunogenicity test, but not less than 102.0 titration units (PFU or ID50) per dose.

(4) Identity. Bulk or final container samples of completed product from each serial shall be tested for identity as prescribed in paragraph (b)(3) of this section and shall meet the criteria stated therein.

[50 FR 438, Jan. 4, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, 1999; 72 FR 72564, Dec. 21, 2007]