Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan
Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan
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DOI：
10.1038/s41598-019-42978-1
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发表时间：
2019-04-25
影响因子：
4.6
通讯作者：
Toma, Claudia
中科院分区：
文献类型：
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作者：
Sato, Yukuto;Mizuyama, Masaru;Toma, Claudia
钩端螺旋体，引起人畜共患病钩端螺旋体病，坚持在土壤和水环境。几个因素，包括降雨量，水库动物的存在，以及各种非生物和生物成分相互作用，影响钩端螺旋体的生存，持久性和致病性的环境。然而，这些因素如何调节感染的风险知之甚少。在这里，我们开发了一种方法，使用环境DNA（eDNA）metabarcoding检测微生物组，脊椎动物和病原性钩端螺旋体在水生样品。具体而言，我们组合了4组引物以生成PCR产物，用于通过下一代测序对多个扩增子进行高通量测序。使用我们的方法分析日本冲绳北方钩端螺旋体病流行区的eDNA，我们发现每条河流中的微生物群随着时间的推移而发生变化。对应于致病性L的操作分类单位。alstonii，L. kmetyi和L.在12种非致病性细菌中检测到问号。此外，其中11种的出现频率与降雨量相关。猪、狐蝠、钝尾蝾螈等10种脊椎动物与钩体eDNA检测结果高度相关。我们的eDNA元条形码方法是了解钩端螺旋体环境阶段和预测人类感染风险的有力工具。
Leptospires, which cause the zoonotic disease leptospirosis, persist in soil and aqueous environments. Several factors, including rainfall, the presence of reservoir animals, and various abiotic and biotic components interact to influence leptospiral survival, persistence, and pathogenicity in the environment. However, how these factors modulate the risk of infection is poorly understood. Here we developed an approach using environmental DNA (eDNA) metabarcoding for detecting the microbiome, vertebrates, and pathogenic Leptospira in aquatic samples. Specifically, we combined 4 sets of primers to generate PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. Using our method to analyze the eDNA of leptospirosis-endemic areas in northern Okinawa, Japan, we found that the microbiota in each river shifted over time. Operating taxonomic units corresponding to pathogenic L. alstonii, L. kmetyi, and L. interrogans were detected in association with 12 nonpathogenic bacterial species. In addition, the frequencies of 11 of these species correlated with the amount of rainfall. Furthermore, 10 vertebrate species, including Sus scrofa, Pteropus dasymallus, and Cynops ensicauda, showed high correlation with leptospiral eDNA detection. Our eDNA metabarcoding method is a powerful tool for understanding the environmental phase of Leptospira and predicting human infection risk.