AbstractBrucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (10⁸ and 10⁵ CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (10⁵ CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 10⁵ CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.