Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/17792
Title: An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples
Authors: Riediger, Irina Nastassja
Hoffmaster, Alex R
Casanovas-Massana, Arnau
Biondo, Alexander Welker
Ko, Albert Icksang
Stoddard, Robyn A
Affilliation: Central Laboratory of the State of Paraná. Curitiba, PR, Brasil
National Center for Emerging and Zoonotic Infectious Diseases. Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Yale University. School of Public Health. Department of Epidemiology of Microbial Diseases. New Haven, Connecticut, USA
Federal University of the State of Paraná. Department of Veterinary Medicine. Curitiba, PR, Brazil
Yale University. School of Public Health. Department of Epidemiology of Microbial Diseases. New Haven, Connecticut, USA / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
National Center for Emerging and Zoonotic Infectious Diseases. Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Abstract: Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.
Keywords: Leptospira
Leptospirosis
Environment
Water
Animals
keywords: Leptospira
Leptospirose
Meio ambiente
Água
Animais
Issue Date: 2016
Publisher: Public Library of Science
Citation: RIEDIGER, I. N. et al. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples. Plos One, v. 11, n. 8, p. e0160523, 2016.
DOI: 10.1371/journal.pone.0160523
ISSN: 1932-6203
Copyright: open access
Appears in Collections:BA - IGM - Artigos de Periódicos

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