A vaccine prepared from a non-pathogenic H7N7 virus isolated from natural reservoir conferred protective immunity against the challenge with lethal dose of highly pathogenic avian influenza virus in chickens.

During 2001-2004, 41 H7 influenza viruses (2 H7N1 and 39 H7N7 strains) were isolated from fecal samples of migratory ducks that flew from Siberia in the autumn of each year to Japan and Mongolia. A phylogenetic analysis of the hemagglutinin (HA) genes of the nine representative isolates revealed that they belonged to the Eurasian lineage and the deduced amino acid sequence at the cleavage site of the HAs represented apathogenic profiles. One of the H7 isolates A/duck/Mongolia/736/02 (H7N7) was chosen from these H7 isolates for the preparation of the test vaccine. To improve the growth potential of A/duck/Mongolia/736/02 (H7N7) in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7) was generated by genetic reassortment between A/duck/Mongolia/736/02 (H7N7) as the donor of the PB2, PB1, PA, HA, NA, and NS genes and A/duck/Hokkaido/49/98 (H9N2) as that of NP and M genes. The test vaccine was prepared as follows; A/duck/Hokkaido/Vac-2/04 (H7N7) was propagated in chicken embryos and the virus in the allantoic fluid was inactivated and adjuvanted to form an oil-in-water emulsion. The test vaccine conferred immunity to chickens, completely protecting the manifestation of clinical signs against the challenge with lethal dose of H7 highly pathogenic avian influenza virus. These results indicate that influenza viruses isolated from natural reservoirs are useful for vaccine strains.

Increase of antibody titer against Leucocytozoon caulleryi by oral administration of recombinant R7 antigen.

In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.

Method for estimation of toxic endotoxin in inactivated salmonella vaccine in D-galactosamine-sensitized mice.

We developed a method to estimate the content of the toxic endotoxin in inactivated Salmonella vaccine in D-galactosamine-sensitized mice. Ten-fold serially diluted vaccines were injected intraperitoneally into D-galactosamine-sensitized mice. Lethality in the mice was judged 3 days after the injection. The best result was obtained when C3H/HeN mice were used for the test. Correlation was observed between the endotoxin content measured by Limulus amoebocyte lysate assay and the LD50 in the mouse safety test (r=0.81). These results suggested that this test could be applied to the estimation of endotoxin content in inactivated vaccines of Salmonella.

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.