Research Laboratory Center, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan.
Center for Strategic Research Project, Organization for Research Promotion, University of the Ryukyus, Nishihara, Okinawa, Japan.
PLoS Negl Trop Dis. 2022 Mar 31;16(3):e0010234. doi: 10.1371/journal.pntd.0010234. eCollection 2022 Mar.
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.
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.
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.
钩端螺旋体病是一种由螺旋体属钩端螺旋体引起的人畜共患病，在日本冲绳的八重山地区流行。在钩端螺旋体属中，属于 P1 亚群“毒力”组的物种是冲绳钩端螺旋体病的主要病原体。然而，它们在环境中的持久性还不太清楚。本研究采用细菌分离和环境 DNA（eDNA）宏条形码方法相结合，了解该流行地区钩端螺旋体病的生态流行病学。
聚合酶链反应（PCR）鉴定了来自八重山地区的 12 个人类临床 L. interrogans 分离株，属于 P1 亚群“毒力”亚组和 11 株环境土壤分离株 P1 亚群“低毒力”亚组（与 L. kmetyi、n=1；L. alstonii、n=4；L. barantonii、n=6 遗传相关），针对四个毒力相关基因（lipL32、ligA、ligB 和 lpxD1）。临床分离株至少对 3 个靶基因呈 PCR 阳性，而所有环境分离株仅对 lipL32 呈阳性。对选定的临床和环境菌株感染的肾上皮细胞的分析表明，Hebdomadis 临床株血清群的细胞-细胞连接解体。冬季和夏季钩端螺旋体 eDNA 的比较确定了与土壤样本中分离的物种（L. kmetyi 和 L. barantonii）和其他未通过土壤培养检测到的 P2 亚群物种（L. licerasiae、L. wolffii 相关等）相对应的分类单元。夏季的总钩端螺旋体读数高于冬季，钩端螺旋体/动物 eDNA 关系的分析表明，Rattus spp.是一种潜在的储存动物。
本研究表明，八重山地区的环境钩端螺旋体多样性很高，特别是在夏季，大多数钩端螺旋体病病例都报告在这个季节。此外，还鉴定出了几种具有致病潜力的钩端螺旋体物种，这些物种尚未在八重山地区报告，但以前从该地区人类临床病例中分离出的 P1 亚群物种在环境中不存在，这表明需要进一步开发和监测方法。
