Influence of Limnological Conditions on Clostridium Botulinum Type E Presence in Eastern Lake Erie Sediments (Great Lakes, USA)

Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol-chloroform-isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest rate of recovery (geometric mean of 11%, with a geometric standard deviation of 0.02; n ‫؍‬ 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest rate of PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River, Miyagi, Japan, and its estuary from November 2007 to April 2009, and the genomic RNAs of enterovirus and human norovirus in these samples were quantified by reverse transcription (RT)-quantitative PCR (qPCR). The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 ؋ 10 2 and 2.4 ؋ 10 2 copies/g (wet weight), respectively. This is the first report to obtain quantitative data for a human pathogenic virus in a river and in estuarine sediments using RT-qPCR. [6,14], and rotavirus [6]) pathogens have been detected in environmental sediments. Whittington et al. reported previously that M. avium in sediments from a dam lake survived 12 to 26 weeks longer than it did in a water column (29). Moreover, the persistence of viral pathogens in environmental sediments has been reported. Smith et al. demonstrated previously that poliovirus 1 (PV1), coxsackieviruses B3 and A9, and echovirus 1 survived significantly longer when associated with marine sediments (23). A 3-log reduction in the infectivity of PV1 was observed in 14 days in seawater having marine sediments, whereas such a reduction was observed in 4 days in seawater without sediments (23). These results suggest that environmental sediments have a protective effect on pathogens (1, 20), and the association of pathogens with environmental sediments cannot be ignored when considering the fate of pathogens in water environments (22). Bacterial (Mycobacterium aviumWhen storms, tides, or strong winds cause sediment resuspension, pathogens in sediments are also resuspended, resulting in high pathogen levels in the water column. Dorner et al. indicated previously the importance of the resuspension of microorganisms from stream sediments rather than land-based sources according to the hydrological simulation of Escherichia coli during storm events (4). The quantitative detection of pathogens in environmental sediments is thus crucial for ...

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confidence: 99%

Development of an Effective Method for Recovery of Viral Genomic RNA from Environmental Silty Sediments for Quantitative Molecular Detection

Miura

Masago

Sano

et al. 2011

“…BoNT/E outbreaks in the Great Lakes region typically occur between June and December and correlate with low mean annual water levels and increased surface water temperatures (25). It has been hypothesized that BoNT/E is mobilized through Great Lakes aquatic food webs consisting of exotic species (19,25,32,33), providing a potential link between sediment-inhabiting clostridia and the intoxication of fish-eating birds. Using the BoTest Matrix E assay to conduct enhanced epidemiological investigations of avian BoNT/E outbreaks, including the analysis of aquatic food web components, would aid in our understanding of toxin mobilization pathways and provide critical insights for the management and conservation of bird species in the Great Lakes region.…”

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confidence: 99%

“…This is supported by the isolation of Clostridium botulinum from fish muscle and alimentary canal tissues as well as detection of the BoNT/E gene in fish liver and intestine samples collected within the Great Lakes Basin (8,16,47). Additional studies have identified the BoNT/E gene in sediment samples collected from Lake Erie, suggesting that sediment ingestion is a route for bacterial infection or toxin accumulation in bottom-feeding fish or bottom-dwelling invertebrates such as filter-feeding dreissenid mussels (32,33).…”

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confidence: 99%

“…PCR-based assays have been used to identify BoNT types C and E genes in fish and in lake-bed sediment samples (16,30,32), suggesting the presence of C. botulinum, but PCR does not indicate whether catalytically active BoNT is present. Other in vitro assays for detection of BoNTs use fluorescent substrates or mass spectrometry coupled with immunological techniques (5,9,13,15,17,20,23,35).…”

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confidence: 99%

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In Vitro Detection and Quantification of Botulinum Neurotoxin Type E Activity in Avian Blood

Piazza

Blehert

Dunning

et al. 2011

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n ‫؍‬ 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.

“…Increased water temperatures may enhance the type E detection in sediment, potentially indicating bacterial growth under anoxic conditions (9,19,26). In addition, other physicochemical parameters such as redox potential, pH, salinity, available phosphorus, and turbidity may contribute to C. botulinum type E growth (19,27,28). Relationships among these parameters are complex; a multivariate regression model developed using various sediment and water variables indicated that type C outbreak risk increased with 7.5 to 9.0 pH, negative redox potential, Ͼ20°C water temperature, and Ͻ2.0 ppm salinity (29).…”

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confidence: 99%

Spatial, Temporal, and Matrix Variability of Clostridium botulinum Type E Toxin Gene Distribution at Great Lakes Beaches

Wijesinghe

Oster

Haack

et al. 2015

c Clostridium botulinum type E toxin is responsible for extensive mortality of birds and fish in the Great Lakes. The C. botulinum bontE gene that produces the type E toxin was amplified with quantitative PCR from 150 sloughed algal samples (primarily Cladophora species) collected during summer 2012 from 10 Great Lakes beaches in five states; concurrently, 74 sediment and 37 water samples from four sites were also analyzed. The bontE gene concentration in algae was significantly higher than in water and sediment (P < 0.05), suggesting that algal mats provide a better microenvironment for C. botulinum. The bontE gene was detected most frequently in algae at Jeorse Park and Portage Lake Front beaches (Lake Michigan) and Bay City State Recreation Area beach on Saginaw Bay (Lake Huron), where 77, 100, and 83% of these algal samples contained the bontE gene, respectively. The highest concentration of bontE was detected at Bay City (1.98 ؋ 10 5 gene copies/ml of algae or 5.21 ؋ 10 6 g [dry weight]). This study revealed that the bontE gene is abundant in the Great Lakes but that it has spatial, temporal, and matrix variability. Further, embayed beaches, low wave height, low wind velocity, and greater average water temperature enhance the bontE occurrence.