Attenuated S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α were constructed, as described elsewhere (17). Attenuated Nutlin-3a molecular weight S. enterica serovar Typhimurium χ8501 (hisG Δcrp-28 ΔasdA16) (21) was used as the host bacteria for the delivery of swIL-18 and swIFN-α and grown at 37°C in Lennox broth, Luria-Bertani (LB) broth, or on LB agar. Diaminopimelic acid (DAP; Sigma-Aldrich, St. Louis, MO, USA) was added (50 μg/mL) to induce the growth of Asd-negative bacteria (22). PBS

(pH 7.4) containing 0.01% gelatin (BSG) was used for the resuspension of Salmonella vaccines that were concentrated by centrifugation at 7000 g at 4°C for 5 min. A total of 30 seronegative crossbred F1 (Large white-Landrace × Duroc) piglets (3–4 weeks old) were housed separately in six groups (n= 5/group). The first group (control) was a negative control orally administered PBS containing 0.01% gelatin without S. enterica

serovar Typhimurium expressing swIL-18 and swIFN-α. The second group (vehicle) was orally administered S. enterica serovar Typhimurium harboring pYA3560 vector (1011 cfu/piglet) as a control for the empty pYA series vectors. The third group (alum) was vaccinated with Alum-absorbed inactivated PrV vaccine (equivalent to 2 × 1010 plaque-forming unit [pfu]/piglet). Alum-absorbed inactivated PrV vaccine was made by agitating alum (Sigma-Aldrich, 10 mg/piglet) with thymidine kinase-deleted p38 MAPK signaling PrV inactivated with 0.5% formalin. The fourth (swIL-18) and fifth (swIFN-α) groups were orally administered with S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α (1011 cfu/piglet), respectively. The sixth group (swIL-18 + swIFN-α) was orally co-administered Mannose-binding protein-associated serine protease with S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α after mixing the two constructs together (each 1011 cfu/piglet). Oral administration

of Salmonella bacteria was performed by depositing resuspended bacteria (10 mL/piglet) into the stomach using a flexible gavage feeding needle (Fine Science Tools, British Columbia, Canada) after starvation for 2 h. The groups that received attenuated S. enterica serovar Typhimurium were immunized with formalin-inactivated PrV vaccine (equivalent to 2 × 1010 pfu/piglet) via the intramuscular (i.m.) route 3 days after Salmonella administration (d0). Primarily vaccinated piglets were boosted with inactivated PrV vaccine by the same protocol 2 weeks later (d14). Three weeks after boosting (d35), piglets were intranasally (i.n.) challenged with the virulent PrV YS strain (108 pfu/piglet). After challenge with the virulent PrV, progression of clinical symptoms in piglets such as depression, anorexia, respiratory distress (cough/sneeze), and trembling started 3–5 days post-challenge.