Development of rapid canine fecal source identification PCR-based assays.

The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. We used genome fragment enrichment (GFE) to identify novel nonribosomal microbial genetic markers potentially useful for detecting dog fecal contamination with PCR-based methods in environmental samples. Of the 679 sequences obtained from GFE, we used 84 for the development of PCR assays targeting putative canine-associated genetic markers. Twelve genetic markers were shown to be prevalent among dog fecal samples and were rarely found in other animals. Three assays, DG3, DG37, and DG72, performed best in terms of specificity and sensitivity and were used for the development of SYBR Green and TaqMan quantitative PCR (qPCR) assays. qPCR analysis of 244 fecal samples collected from a wide geographic range indicated that marker concentrations were below limits of detection in noncanine hosts. As a proof-of-concept, these markers were detected in urban stormwater samples, suggesting a future application of newly developed methods for water quality monitoring.

Methane and Carbon Dioxide Emissions From Reservoirs: Controls and Upscaling.

Estimating carbon dioxide (CO2) and methane (CH4) emission rates from reservoirs is important for regional and national greenhouse gas inventories. A lack of methodologically consistent data sets for many parts of the world, including agriculturally intensive areas of the United States, poses a major challenge to the development of models for predicting emission rates. In this study, we used a systematic approach to measure CO2 and CH4 diffusive and ebullitive emission rates from 32 reservoirs distributed across an agricultural to forested land use gradient in the United States. We found that all reservoirs were a source of CH4 to the atmosphere, with ebullition being the dominant emission pathway in 75% of the systems. Ebullition was a negligible emission pathway for CO2, and 65% of sampled reservoirs were a net CO2 sink. Boosted regression trees (BRTs), a type of machine learning algorithm, identified reservoir morphology and watershed agricultural land use as important predictors of emission rates. We used the BRT to predict CH4 emission rates for reservoirs in the U.S. state of Ohio and estimate they are the fourth largest anthropogenic CH4 source in the state. Our work demonstrates that CH4 emission rates for reservoirs in our study region can be predicted from information in readily available national geodatabases. Expanded sampling campaigns could generate the data needed to train models for upscaling in other U.S. regions or nationally.

Effects of an Experimental Water-level Drawdown on Methane Emissions from a Eutrophic Reservoir.

Reservoirs are a globally significant source of methane (CH4) to the atmosphere. However, emission rate estimates may be biased low due to inadequate monitoring during brief periods of elevated emission rates (that is, hot moments). Here we investigate CH4 bubbling (that is, ebullition) during periods of falling water levels in a eutrophic reservoir in the Midwestern USA. We hypothesized that periods of water-level decline trigger the release of CH4-rich bubbles from the sediments and that these emissions constitute a substantial fraction of the annual CH4 flux. We explored this hypothesis by monitoring CH4 ebullition in a eutrophic reservoir over a 7-month period, which included an experimental water-level drawdown. We found that the ebullitive CH4 flux rate was among the highest ever reported for a reservoir (mean = 32.3 mg CH4 m-2 h-1). The already high ebullitive flux rates increased by factors of 1.4-77 across the nine monitoring sites during the 24-h experimental water-level drawdown, but these emissions constituted only 3% of the CH4 flux during the 7-month monitoring period due to the naturally high ebullitive CH4 flux rates that persist throughout the warm weather season. Although drawdown emissions were found to be a minor component of annual CH4 emissions in this reservoir, our findings demonstrate a link between water-level change and CH4 ebullition, suggesting that CH4 emissions may be mitigated through water-level management in some reservoirs.

Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.

Assessment of the effectiveness of low-pressure UV light for inactivation of Helicobacter pylori.

Three strains of Helicobacter pylori were exposed to UV light from a low-pressure source to determine log inactivation versus applied fluence. Results indicate that H. pylori is readily inactivated at UV fluences typically used in water treatment regimens. Greater than 4-log(10) inactivation was demonstrated on all three strains at fluences of less than 8 mJ cm(-2).