Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.

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.

Potency of an inactivated influenza vaccine prepared from a non-pathogenic H5N1 virus against a challenge with antigenically drifted highly pathogenic avian influenza viruses in chickens.

Antigenic variants of H5N1 highly pathogenic avian influenza virus (HPAIV) have selected and are prevailing in poultry populations in Asia. In the present study, the potency of inactivated influenza vaccine prepared from a non-pathogenic H5N1 avian influenza virus, A/duck/Hokkaido/Vac-3/2007 (H5N1), was assessed by challenging with H5N1 HPAIV variants, A/muscovy duck/Vietnam/OIE-559/2011 (H5N1), A/whooper swan/Hokkaido/4/2011 (H5N1), and A/peregrine falcon/Hong Kong/810/2009 (H5N1) belonging to clades 1, 2.3.2.1, and 2.3.4, respectively. All chickens immunized with the Vac-3 vaccine survived without showing any clinical signs after intranasal challenge either with A/whooper swan/Hokkaido/4/2011 (H5N1) or A/muscovy duck/Vietnam/OIE-559/2011 (H5N1). After challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1), 10 out of 12 vaccinated chickens survived and the other 2 died on 4 or 7 post-challenge days. The Vac-3 vaccine of 2.4-fold antigen concentration conferred complete protective immunity in chickens against challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1).

Detection of highly pathogenic avian influenza virus infection in vaccinated chicken flocks by monitoring antibodies against non-structural protein 1 (NS1).

H5 and H7 highly pathogenic avian influenza virus (HPAIV) represent a major global concern in poultries and human health. Avian influenza (AI) vaccines are available but not preferred for field applications, primarily because vaccination interferes with sero-surveillances of AIV infection. To overcome the problem, ELISA systems using non-structural protein 1 (NS1) of AIV as antigens (NS1-ELISA) have been developed to measure anti-NS1 antibodies that are raised in AIV-infected but not in vaccinated chickens. However, some AI-vaccinated chickens having a weak anti-virus immune response may subsequently be infected with AIV and spread the virus. This raises a concern for the validity of NS1-ELISA to detect AIV infection in previously vaccinated chickens. In this study, we developed NS1-ELISA and assessed its feasibility to detect HPAIV infection in chickens previously immunized with H5 or H7 AI vaccines. The results indicated that the NS1-ELISA could identify HPAIV infection in both unvaccinated and vaccinated chickens at 1 week after infection in correlation with results from time-consuming virus isolation tests. Taken together, the NS1-ELISA system would be valuable tool to define HPAIV infection when AI vaccine program is in place.

Long lasting immunity in chickens induced by a single shot of influenza vaccine prepared from inactivated non-pathogenic H5N1 virus particles against challenge with a highly pathogenic avian influenza virus.

An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octadecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs.