Definitions.

Only stock seed virus which has been established as pure, safe and immunogenic shall be used. Each jot of stock seed virus shall be tested for other pathogens by chicken embryo inoculation tests as follows: - A lot of seed virus shall be mixed with 9 volumes of sterile heat inactivated specific anti-serum to neutralize and the vaccine virus serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos of 9-11 days old (0.1 ml on CAM and 0.1 ml in the allantoic sac). Eggs are candled daily for 7 days. Deaths occurring during first 24 hours shall be discarded but at least 18 viable embryos shall survive 24 hours post inoculation for a valid test. All embryo and CAMS from embryos shall be examined which die after 24 hours. If necessary embryo subcultures shall be made to determine the cause of death. The test shall be concluded on the 7th day post inoculation and surviving embryos including the CAM shall be examined. If death and or abnormality attributable to the stock seed virus occur, the seed lot is unsatisfactory. Each lot of stock seed virus shall be tested for immunogenicity as below: - Bronchitis susceptible chickens of the same age and source shall be used. For each method of administration recommended on the label and for each serotype against which protection is claimed, 20 chicks shall be used as vaccinates. Ten additional chickens for each serotypes against which protection is claimed shall be held as unvaccinated controls. 21 to 28 days post vaccination all vaccinates and controls shall be challenged by eye drops with virulent Bronchitis virus. A separate set of vaccinates and controls shall be used for each serotype against which protection is claimed. The challenge virus shall have a titre of at least 104.6 EID50 per ml. Trachea swabs shall be taken once 5 days post challenge from each vaccinated and controls. Each swab shall be placed in test tube containing 3 ml of tryptose phosphate broth and antibiotics. The tubes and swabs shall be swirled thoroughly and stored at minus 40o C pending egg inoculation. For each chicken swabs at least 5 chicken embryos, 9-11 days old shall be inoculated in the allantoic cavity with 0.2 ml of broth from each tube. All the embryos surviving 3rd day post inoculation shall be used in evaluation. A tracheal swab shall be positive for virus recovery when any of the embryos show typical infections bronchitis virus lesions such as stunting, curling, kidney urates, clubbed down or death during 4-7 days post inoculation period. 90 percent of the controls should prove positive for virus recovery. If less than 90 per cent of the controls are negative for virus recovery, the stock seed is unsatisfactory. The stock seed virus should be tested for immunogenicity once in 5 years provided it is maintained under standard conditions of the bronchitis virus storage.