THE PRODUCTION OF AN AUXOTROPHIC MARKED, PLASMID-CURED SALMONELLA SER. TYPHIMURIUM AS A LIVE ATTENUATED VACCINE

A number of amino acid requiring auxotrophic strains of Salmonella Typ himurium were produced by chemical mutagenesis. One of them, strain 81 , was cured of the virulence plasmid and attenuated for mice. This str ain had an auxotrophic requirement for serine, which could be used as a marker for the differentiation of the vaccine strain from other isol ates in the field. The strain still contained the smooth form of the O -antigen, was resistant to Complement-mediated killing of serum and pr oduced type 1 fimbriae. Of the six auxotrophic mutants only this mutan t differed in its outer membrane protein profile from that of the pare nt strain in that an outer membrane protein of about 30 kDa was absent . With the use of the polymerase chain reaction, using total DNA of th e cell as template, and with primers targeted to the virulence plasmid , it was shown that the virulence plasmid of Salmonella Typhimurium wa s completely cured from this strain. This strain also had a LD50-value of 4 log units lower for mice than the parent strain. The plasmid-cur ed strain gave a very high degree of protection to mice after systemic immunization, but not after oral vaccination. Compared to the parent, strain 81 also had a lower multiplication rate in the liver and splee n after intraperitoneal inoculation, characteristics that could be att ributed to plasmid-loss, and it could also not be recovered from the s pleen and liver of orally inoculated mice.