Gaspak anaerobic system

The highly virulent S. suis serotype 2 (SS2) and the less prevalent S. suis serotype 14 (SS14), originally isolated from some diseased pigs were ATCC 700794 and 13730, respectively. The rarely reported S. suis serotypes, isolated from some healthy pigs, were S. suis serotype 18 (SS18) NT77 and S. suis serotype 19 (SS19) 42A, respectively. These four references S. suis serotypes were cultured on Columbia blood agar (Difco Laboratories, Detroit, MI, USA) with 5% (v/v) sheep’s blood at 37°C for 24 h. A GasPak Anaerobic System (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) was used to generate the anaerobic condition. The 16S rRNA gene was sequenced to confirm the S. suis serotype. The primers used for sequencing were F1 and R13 primers [30 (link)], the accession numbers of SS2, SS14, SS18, and SS19 are LS483418.1, AF009489.1, AF009493.1, and AF009494.1, respectively. Then, the bacterial colonies were cultured in a Todd–Hewitt broth (THB) (Difco Laboratories, Detroit, MI, USA) and preserved with 20% glycerol at −80°C for further study.

B. pilosicoli ATCC51139, Kyoto-C, and B. aalborgi NCTC11492, Yokosuka 24-d, B. ibaraki (one lineage of B. aalborgi) were used for the agglutination test. The organisms were grown anaerobically on 4% sheep blood agar containing 400 µg/ml spectinomycin using a GasPak Anaerobic System (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) as described previously.^{(13 (link))} Dark field agglutination tests were carried out as described previously.^{(11 (link))}B. pilosicoli was grown anaerobically for 7 days and B. aalborgi was grown anaerobically for 30 days. The cells grown on blood agar were harvested with physiological saline and after centrifugation, the precipitate was suspended in physiological saline. The cells were adjusted to MacFarland No.1. The sera collected from patients with diarrhea were diluted two-fold limiting dilution from 100 to 12,800. Into each well of the ceramic agglutination plate, an aliquot (0.05 µl) if the diluted serum pipetted and then 0.05 µl of the brachyspiral cell suspensions were dispensed into each well. After incubation for 60 min, the ceramic plates were left for 2 h. and then observed under the dark field microscope. Furthermore, the agglutination was observed again after 24 h and 48 h.
Informed consent was obtained from all subjects, and the experimental protocol was approved by the Ethics Committee of Tokyo Medical University Ibaraki Medical Center.

Six reference S. suis serotypes, originating from diseased pigs, were included in the present study. The virulent S. suis to humans were SS2 (ATCC 700794) and SS14 (13730) and the non-virulent S. suis to humans were SS7 (8074), SS9 (22083), SS25 (89-3576-3), and SS27 (89-5259). S. suis bacteria were cultured on Columbia blood agar (Difco Laboratories, Detroit, MI, USA) with 5% (v/v) sheep’s blood at 37 °C in anaerobic conditions using a GasPak Anaerobic System (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) for 24 h. Sequencing of the 16S rRNA gene was performed to validate their strains [10 (link)]. GenBank accession numbers of the 16S rRNA gene of SS2, SS14, SS7, SS9, SS25 and SS27 are LS483418.1, AF009489.1, AF009482.1, AF009484.1, AF009500.1 and AF009502.1, respectively. The bacterial colonies were then cultured in a Todd–Hewitt broth (THB) (Difco Laboratories, Detroit, MI, USA) and preserved with 20% glycerol at −80 °C for further study.