Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

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Erysipelothrix rhusiopathiae causes swine erysipelas (SE). Sporadic SE outbreaks in Japan are mostly caused by the E. rhusiopathiae serovar 1a variant featured by methionine (M) and isoleucine (I) at amino acid positions 203 and 257 of the surface protective antigen (Spa) A protein (M203/I257 SpaA-type). To determine if current vaccines are effective against infection with this variant in pigs, one representative inactivated vaccine, SER-ME (containing E. rhusiopathiae serovar 2a), was evaluated. All vaccinated pigs survived without any apparent clinical signs after lethal challenge with the Fujisawa reference strain or the variant. This indicates that the SER-ME vaccine effectively protects pigs against the infection of E. rhusiopathiae M203/I257 SpaA-type variant. Current vaccines in Japan, including SER-ME, suggest that outbreaks in Japan are unlikely caused by vaccine failure.

Genotyping of avian infectious bronchitis virus (IBV) was performed on trachea and kidney samples of six chickens obtained from a single farm in Japan. Using two primer sets targeting the spike (S) protein gene, the S1 and S2 regions of DNA fragments were amplified. Sequences of amplified S1 fragments extracted from both organs were identical among the six chickens, showing a JP-I genotype. Sequences of amplified S2 fragments differed between trachea and kidney samples. The kidney profile showed a group IV genotype, whereas the trachea profile showed an unclassified group. This result showed that two different IBVs infected the six chickens. The first IBV infection induced poor protective immunity in this farm, permitting a second IBV infection to occur.

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