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Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan.

ABSTRACT: 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.

SUBMITTER: Sato Y

PROVIDER: S-EPMC6484013 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlthough previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.Methods and findingsA quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (approximately 10(3) leptospires/ml versus 0.5 x 10(2) leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.ConclusionsOur findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.

Project description:BackgroundLeptospirosis, 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.FindingsPolymerase 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.ConclusionOur 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.

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Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan.

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