The role of the Yersinia pestis virulence-associated plasmid, pYV019, in the expression of Ca++ dependence, virulence, and the production of the V antigen was investigated. Derivatives of bacteriophage P1 were used to deliver the transposon Tn5 into Y pestis strain EV76. Ca++-independent mutants in which transposon Tn5 had been inserted into plasmid pYV019 were isolated, the resulting plasmids--pYV019::Tn5--were transformed into an Escherichia coli K12 derivative, and the site of insertion of transposon Tn5 was mapped with restriction endonucleases. The plasmids were then transduced by bacteriophage P1 into avirulent strain 195-P1 of Y pestis. The transductants were analyzed for expression of Ca++ dependence, virulence in mice, and the expression of the V antigen. Introduction of plasmid pYV019 with insertions outside of the Ca++ dependence loci restored strain 195-P1 to full virulence, while disruption of plasmid genes associated with Ca++ dependence led to loss of virulence. Using Western blotting analysis and E coli minicells, it was shown that genes specific for the V antigen are plasmid encoded.

A rapid and sensitive radioimmunoassay for the detection of soluble Yersinia pestis fraction I antigen using antibody-coated beads and radiolabeled immunoglobulin was applied to spleen-liver extracts, rendered noninfectious by ether treatment or filtration, from rodents dead of plague. The procedure was capable of detecting nanogram amounts of soluble plague antigen.

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When equal volumes of 6% lactose and a broth culture of Yersinia pestis were mixed before freezing, approximately 50% of the cells survived lyophilization and reconstitution on the following day. Concomitantly, the number of viable cells per 50% lethal dose increased from about 16 to 125 organisms. On subsequent storage of the lyophilized cells under vacuum in glass ampoules at 4 degrees C for 25 years, more than 25% of the cells remained viable. When stored cultures were assayed immediately after reconstitution, virulence for mice was significantly reduced (as many as 4,000 cells/50% lethal dose), but the virulence was fully restored when reconstituted cultures were held for 24 h at room temperature, or when a subculture was prepared in fresh medium.