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Brucella  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 (Bs2Δ wbkF ) or only producing internal O-chain precursors (Bs2Δ wzm ) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2Δ wadB  and Bs2Δ wadD ). We also investigated mutants in the pyruvate phosphate dikinase (Bs2Δ ppdK ) and phosphoenolpyruvate carboxykinase (Bs2Δ pckA ) 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 (10 8  and 10 5  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 Bs2Δ wbkF , Bs2Δ wzm,  Bs2Δ wadB  and the Rev1 control (10 5  CFU). As described before for  B. abortus ,  B. suis  biovar 2 carried a disabled  pckA  so that a double mutant Bs2Δ ppdK Δ pckA  had the same metabolic phenotype as Bs2Δ ppdK  and  ppdK  mutation was enough to generate attenuation. At 10 5  CFU, Bs2Δ ppdK  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 Bs2Δ wbkF , the latter seems a better R vaccine candidate than Bs2Δ wzm . However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2Δ wbkF  is advantageous over Bs2Δ wadB  or Bs2Δ ppdK  requires experiments in the natural host.

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background