In order to prepare the recombinants (p3D-NT19/ S. cho, pLacZ/S. cho and p3D-NT56/S. cho), the attenuated S. typhimurium LB5010, an intermediate host cell, was first transformed with plasmids (pLacZ, p3D-NT56 and p3D-NT19) using a standard CaCl 2 method. Then, the plasmids were extracted and transferred to a terminal host cell-attenuated S. choleraesuis C500 by electroporation. The preparation of cells for electroporation was as follows: C500 cells were cultured in 3 mL LB medium at 200 rpm overnight at 37°C, and then were inoculated into 25 mL liquid LB medium at 1% (v/v) at 37°C, again with shaking at 200 rpm. Bacteria were grown to an optical density (OD) of 0.6-0.7, chilled and then collected by centrifugation at 4 000 rpm for 10 min at 4°C. The cells were then washed with 10% glycerol three times. The cells were re-suspended in 10% glycerol at a concentration of 3 · 10 10 cells/mL, frozen in dry ice, and stored at À70°C. A 200 lL aliquot of the cells was thawed and used for transformation. One microgram DNA (the control setup with no DNA) was mixed with cold cell suspension and then transferred to a chilled cuvette. Transformation was carried out with a Bio-Rad Gene Pulser at 2.0 kV, 25 lF, 200 X at 0-4°C. Transformed competent cells were transferred to a tube, and 400 lL of media were added prior to incubating the cultures for 1.5 h at 50 rpm at 37°C. Then, 80 lL of the cultures were transferred onto solid LB medium containing 100 lL/mL ampicillin. Recombinant bacteria were identified via PCR. The presence of the recombinant plasmid was confirmed by digestion with XhoI. Serological characteristics of recombinant S. choleraesuis (rS. cho) were tested using a congregation reaction on a glass plate to confirm its immunogenicity.