BALB/c mice were infected orally with Salmonella dublin strains Lane and LD842, an isogenic derivative of the former that is avirulent because it was cured of its 80-kilobase-pair virulence plasmid pSDL2. Both strains colonized the intestine and invaded Peyer's patches with equivalent efficiency. However, the parent strain multiplied in mesenteric nodes and in the spleen; the plasmid-cured strain reached these organs, but the infection was low grade and remained relatively static until the mice developed active immunity and cured themselves. The histological response to plasmid-free strain LD842 was mononuclear, whereas the virulent parent strain produced abscesses. Sublethal irradiation of the mice before infection with strain LD842 prevented the mononuclear infiltrate in the liver and made the animals susceptible. Thus the virulence plasmid of S. dublin allows multiplication within the reticuloendothelial system and does not have any effect on the organism's ability to colonize the intestine or invade Peyer's patches.

Plasmids of -80 kb in size are found in nearly all clinical isolates of Salmonella dublin and are believed to be essential for virulence. We have shown previously that the 80-kb plasmid pSDL2 is required for the S. dublin Lane strain to establish a lethal systemic infection in BALB/c mice after oral or intraperitoneal inoculation. We now present a physical and genetic characterization of pSDL2. We have established a complete restriction endonuclease cleavage map of pSDL2 for five enzymes: Xba I, Bam HI, Xho I, Sal I, and Hind III. The region specifying autonomous replication has been localized to a 10.5-kb region of the Sal I A fragment by subcloning on the vector pBR322. Using transposon insertion mutagenesis with Tn5-oriT, a region encoding the virulence phenotype has been mapped within a 6.4-kb portion of the Sal I B fragment. Deletions generated by partial Eco RI restriction digestion demonstrate that at least 50 kb of the plasmid DNA are not required for replication or virulence functions, confirming the map location of these phenotypes. Plasmids of different sizes and restriction patterns were found in mouse virulent strains of S.dublin Vi+, S. enteritidis, and S. choleraesuis. By Southern hybridization, these putative virulence plasmids share a common 4-kb Eco RI fragment with the virulence region of pSDL2, and the plasmids from S. dublin Vi+ and S. enteritidis were shown to express mouse virulence comparable to pSDL2.

Salmonella dublin, a serotype which causes invasive disease in cattle and humans, carries a characteristic 80-kilobase plasmid (pSDL2). We were able to cure the plasmid from a strain of S. dublin. The cured strain was avirulent for mice by either the oral or intraperitoneal route of infection. A derivative of Tn5 which contains the transfer origin of the broad-host-range plasmid RK2 (TnS-ori7) was transposed onto pSDL2, allowing mobilization of the plasmid by an RK2 helper plasmid. Reintroduction of the pSDL2 derivative plasmid into the cured strain restored virulence, demonstrating that the plasmid is necessary for virulence. These studies also demonstrate the usefulness of the TnS-oriT construct for genetic manipulations.

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