Virulence markers of LCR plasmid in Indian isolates of Yersinia pestis

Presence of 10 important yop genes in Yersinia pestis isolates (18 in number) of Indian origin from 1994 plague outbreak regions of Maharashtra (6 Rattus rattus & Tetera indica rodents) and Gujarat (11 from human patients, 1 from R. rattus) and from plague endemic regions of the Deccan plateau (8 from T. indica) was located by PCR and specific enzyme immunoassay. PCRs were standardized for six effector yops (YopE, YopH, YopJ, YopM, YopO and YopT), three translocator yops (YopB, YopD and YopK) and a regulator LcrV gene. Amplification of all the 10 yop genes was observed in isolates recovered from pneumonic patients and in 5 of 7 rodents from outbreak regions. Among these, amplification of the yopD gene was absent in all eight isolates, and that of yopM in all except one (10R). One of the isolates from rodents of the Deccan plateau (24H) was consistently negative for all the yops. Cloning and expression of truncated yopM (780 bp), yopB (700 bp) and lcrV (796 bp) genes in pQE vectors with SG13009 host cells yielded recombinant proteins for generation of monoclonal antibodies for further use in enzyme immunoassay. Ten stable reactive clones for YopB, nine for YopM and six for LcrV were obtained, all of them exhibiting specific reactions only to Y. pestis. Testing of 26 Y. pestis isolates by monoclonal antibody dot-ELISA and Western blotting provided results identical to PCR, suggesting that the isolates that failed to show PCR amplification also had no expression of their respective proteins. The Y. pestis isolates of outbreak regions had their virulence factors intact in the LCR plasmid. Yersinia pestis isolates recovered from rodents of the Deccan plateau were relatively heterogeneous. It appears that a long residency of Y. pestis of nearly 100 years in the enzootic plague foci has resulted in shedding of virulence genes in the LCR plasmid region in a fairly large proportion of the organisms, possibly due to natural recombination.