Long-term persistence of virulent Yersinia pestis in soil

Plague is characterized by geographical foci from which it re-emerges after decades of silence, a fact currently explained by enzootic and epizootic cycles between plague-susceptible and plague-resistant rodents. To assess the potential role of soil in plague epidemiology, we experimentally investigated whether Yersinia pestis could persist alive and virulent in soil. Sterilized soil inoculated with virulent Y. pestis biotype Orientalis was regularly sampled for 40 weeks in duplicate. Each sample was observed by acridine orange staining and immunofluorescence using an anti-Y. pestis polyclonal antibody, and DNA was extracted for PCR amplification and sequencing of the Y. pestis ureD, caf1 and pla genes. All samples were inoculated onto selective agar, and samples from soil that had been incubated for 10, 60, 165, 210 and 280 days were also inoculated into each of two BALB/c female mice. The mouse experiment was performed in triplicate. Non-inoculated, sterilized soil samples were used as negative controls. Micro-organisms fluorescing orange and detected by immunofluorescence were identified as Y. pestis in all samples. They were recovered in pure agar cultures for up to 30 weeks but thereafter were contaminated with Pseudomonas spp. Soil that had been inoculated with Y. pestis proved to be fully virulent in mice, which died with Y. pestis septicaemia and multiple organ involvement. Negative control mice showed no signs of disease. These data indicate that Y. pestis biotype Orientalis can remain viable and fully virulent after 40 weeks in soil. This study is a first step on which to base further investigations of a potential telluric reservoir for Y. pestis, which could represent an alternative mechanism for the maintenance of plague foci.