ABSTRACT
Salmonella enterica serovar Typhimurium ATCC 13311 is virulent at a dose as low as 10(2) colony-forming units when administered intraperitoneally to BALB/c mice. In order to develop highly attenuated mutant strain through the combination of 2 phenotypically attenuated markers, we constructed a number of amino acid requiring auxotrophic strains of S. enterica serovar Typhimurium by means of UV-induced mutations. One of them, strain NDMC-B1, was highly attenuated for mice, with an LD50-value of 6 and 3 log units lower for mice than the wild-type strain and S. enterica serovar Typhimurium aroA strain, respectively. This strain still contained the Salmonella O- and H-antigens but had a requirement for cysteine and was unable to utilize citrate as its sole carbon source. NDMC-B1 colonized the gut-associated lymphoid tissue more efficiently than the wild-type strain, but its capacities to colonize spleen and liver were significantly reduced. Mice intraperitoneally or orally vaccinated with NDMC-B1 were highly protected against either an intraperitoneal challenge with 10(6) colony-forming units or an oral challenge with 10(9) colony-forming units of the wild-type strain. Taken together, the results illustrate that through the combination of 2 independently phenotypical attenuating markers, the requirement for cysteine and the inability to use citrate, we have successfully constructed a highly attenuated, stable, and immunogenic S. enterica serovar Typhimurium vaccine strain which can induce protective immunity in a mouse model against lethal challenge of wild-type strain.