Test vaccines comprised of inactivated water-in-oil emulsions containing various antigen levels were prepared using a non-pathogenic H5N1 avian influenza (AI) virus, A/duck/Hokkaido/Vac-1/04 (H5N1). The potencies of these test vaccines were evaluated by two experiments. In the first experiment, the triangular relationship among the antigen levels of test vaccines, the hemagglutination inhibition (HI) antibody response, and the protective effect against challenge with a highly pathogenic avian influenza (HPAI) virus, A/chicken/Yamaguchi/7/04 (H5N1), was confirmed. Then lasting immunity of chickens after a single-shot vaccination was confirmed in the second experiment. As a result, complete protection after the challenge was observed in chickens immunized by test vaccines with an antigen level of 160 HA units/dose or higher. Thus, it was ascertained that the minimum antigen level in the AI vaccine was 160 HA units/dose, and the minimum HI antibody titer that could protect chickens from HPAI virus infection-related death was considered to be 1:16. Dose-dependent HI antibody responses were observed in chickens after the vaccination. Thus, 640 HA units/dose were thought to be similar to the optimal antigen level. Alternatively, the HI antibody titers of chickens, injected with the vaccine containing 640 HA units/dose, were maintained at 1:181 or higher for 100 weeks after the single-shot vaccination.

The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV1, such as CVI988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV1, suggesting hat MDV1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV1. Both meq and L-meq were detected in CD4(+) and CD8(+) T cells infected with strain Md5 or CVI988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8(+) T cell subset from Md5- infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8(+) T cell subset from CVI988/R6-infected chickens. These results suggest that a dominant subpopulation of MDV1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8(+) T cells which play a role in the clearance of pathogenic agents.

Forty-seven Newcastle disease virus (NDV) strains isolated from fecal samples of waterfowls in Alaska and Siberia from 1991 to 1996 were analyzed for their virulence. None of the viruses formed plaques on MDBK cells in the absence of trypsin. Of these, 29 strains showed virulent character by the mean death time with the minimum lethal dose in chicken embryos comparable to velogenic NDV strains. Of the 29 strains, 11 were sequenced for their fusion protein (F) gene. The results showed that 5 of them contained a pair of dibasic amino acids at the cleavage site of the F, which is of a virulent type. The present results suggest that potentially virulent strains of NDV are maintained in migratory waterfowl populations in nature, and that some of those may be transmitted to domestic poultry and acquire pathogenicity during passages in chicken population