The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup 0:8, biotype lB isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.

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Forty-seven strains of Yersinia kristensenii from widely differing sources, representing all known 0 serogroups of this species, were investigated for virulence with a variety of animal and in vitro assays. Twenty-four (51%) of the isolates were lethal for mice pretreated with iron dextran. Mouse-lethal strains occurred predominantly within 0 serogroups 0:11, 0:12,25, and 0:16. Virulent Y. kristensenii strains generally did not express the virulence-associated phenotype (Ca2" dependence and binding of Congo red and crystal violet) which characterizes virulent strains of Y. enterocolitica, nor did they carry the Yersinia virulence plasmid. Although all strains hybridized with a DNA probe derived from the inv (invasin) gene of Y. enterocolitica, none was able to invade HEp-2 epithelial cell culture. Y. kristensenii strains were virulent only when inoculated parenterally into iron-loaded mice. Animals infected in this way succumbed rapidly to infection, generally within 24 h. This finding suggested that the pathogenicity of these bacteria may be attributable to a secreted toxin, but a search for such a substance and for other in vitro correlates of pathogenicity was unsuccessful. These observations indicate that some strains of Y. kristensenii kill mice by a mechanism not previously recognized in yersiniae.

The virulence plasmid (pYV) of Yersinia enterocolitica is necessary for production of conjunctivitis in guinea pigs and for mouse lethality. To identify the genes responsible for production of conjunctivitis in guinea pigs, we subcloned the BamHI and SalI restriction fragments of the virulence plasmid of Y. enterocolitica A2635 (serotype O:8) into derivatives of the broad-host-range plasmid pRK290 and introduced the constructions into plasmid-negative Y. enterocolitica strains. A mild, transient conjunctivitis was evident 24 h after inoculation with strains containing a 2.8-kilobase (kb) BamHI fragment of pYV. These strains were cytotoxic to HEp-2 cells but did not cause death in iron-loaded adult mice. When the 2.8- and adjacent 0.5-kb BamHI fragments were deleted from the virulence plasmid of a fully virulent Y. enterocolitica isolate, the resultant strain did not cause conjunctivitis in guinea pigs and was not cytotoxic to HEp-2 cells. However, the strain with the deletion appeared to be more virulent for mice, with more rapid dissemination after orogastric inoculation, compared with that of the parent strain. When the deletion was complemented by introduction of a plasmid containing the 2.8-kb BamHI fragment, the strain again caused conjunctivitis but had decreased virulence for mice.

concerning our recent publication on virulence testing of Yersinia species (1). We agree that the statement in our discussion to which Dr. Bhaduri has taken exception is ambiguous. What we wished to convey in this sentence was the fact that we were the first to evaluate the crystal violet binding and pyrazinamidase assays on a large and varied collection of Yersinia species using mouse virulence as the reference assay. It should be clear from the discussion in our paper that we did not intend to detract from Dr. Bhaduri's original and valuable observation on the usefulness of crystal violet binding as a means to detect virulent yersiniae. LITERATURE CITED