A number of amino acid requiring auxotrophic strains of SalmonellaTyphimurium were produced by
chemical mutagenesis. One of them, strain 81, was cured of the virulence plasmid and attenuated
for mice. This strain had an auxotrophic requirement for serine, which could be used as a marker for
the differentiation of the vaccine strain from other isolates in the field. The strain still contained the
smooth form of the 0-antigen, was resistant to Complement-mediated killing of serum and produced
type 1 fimbriae. Of the six auxotrophic mutants only this mutant differed in its outer membrane protein
profile from that of the parent 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 the cell as template, and with primers
targeted to the virulence plasmid, it was shown that the virulence plasmid of SalmonellaTyphimurium
was completely cured from this strain. This strain also had a LD<sub>50</sub>-value of 4 log units lower for mice
than the parent strain. The plasmid-cured 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 spleen after intraperitoneal inoculation, characteristics that
could be attributed to plasmid-loss, and it could also not be recovered from the spleen and liver of
orally inoculated mice.