Analysis of human clinical and environmental Leptospira to elucidate the eco-epidemiology of leptospirosis in Yaeyama, subtropical Japan

PLoS Negl Trop Dis. 2022 Mar 31;16(3):e0010234. doi: 10.1371/journal.pntd.0010234. eCollection 2022 Mar.


Background: Leptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama region in Okinawa, subtropical Japan. Species of the P1 subclade "virulent" group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region.

Findings: Polymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade "virulent" subgroup and 11 environmental soil isolates of the P1subclade "low virulent" subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal.

Conclusion: Our study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Humans
  • Japan / epidemiology
  • Leptospira*
  • Leptospirosis* / epidemiology
  • Leptospirosis* / microbiology
  • Rats
  • Serogroup
  • Zoonoses / microbiology

Grant support

This study was supported by JSPS KAKENHI 20K12258 to Y.S. and 21H02732 to C.T.; and JSPS Core-to-Core Program JPJSCCB20200007 ( and TBRC Joint Usage Project Grant 2019-2020 to T. Kajita; (JSPS: Japan Society for the Promotion of Science, The Spatiotemporal Genomics Project promoted by the University of the Ryukyus to Y.S., H.I., H.N., R.K., and C.T.; a research grant from the Disaster Prevention Research Center for Island Region, University of the Ryukyus (2021) to C.T; and a grant for research promotion from the Okinawa Medical Science Research Foundation to I.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.