scholarly journals Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

2010 ◽  
Vol 76 (5) ◽  
pp. 1359-1366 ◽  
Author(s):  
Orin C. Shanks ◽  
Karen White ◽  
Catherine A. Kelty ◽  
Sam Hayes ◽  
Mano Sivaganesan ◽  
...  
Keyword(s):  
Genetic Markers ◽  
A Priori ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
End Point ◽  
Assay Performance ◽  
Different Populations

ABSTRACT There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

scholarly journals Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

2007 ◽  
Vol 74 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Orin C. Shanks ◽  
Emina Atikovic ◽  
A. Denene Blackwood ◽  
Jingrang Lu ◽  
Rachel T. Noble ◽  
...  
Keyword(s):  
Genetic Marker ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Broad Distribution ◽  
Assay Performance ◽  
Target Dna ◽  
Bovine Feces

ABSTRACTAccurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing totalBacteroidetes,Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.

scholarly journals Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

2013 ◽  
Vol 80 (2) ◽  
pp. 612-617 ◽  
Author(s):  
Kruti Ravaliya ◽  
Jennifer Gentry-Shields ◽  
Santos Garcia ◽  
Norma Heredia ◽  
Anna Fabiszewski de Aceituno ◽  
...  
Keyword(s):  
Fresh Produce ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rrna Gene ◽  
Specific Marker ◽  
Production Environment ◽  
Content Type ◽  
Food Borne

ABSTRACTIn recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based onBacteroidales16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universalBacteroidalesmarker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation betweenBacteroidalesand genericEscherichia coliacross all samples. This study provides evidence thatBacteroidalesmarkers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.

scholarly journals Quantitative PCR for Genetic Markers of Human Fecal Pollution

2009 ◽  
Vol 75 (17) ◽  
pp. 5507-5513 ◽  
Author(s):  
Orin C. Shanks ◽  
Catherine A. Kelty ◽  
Mano Sivaganesan ◽  
Manju Varma ◽  
Richard A. Haugland
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Quantitative Methods ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Target Dna ◽  
Pcr Assays ◽  
Wastewater Samples ◽  
Human Specific

ABSTRACT Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 � 106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

Fecal pollution source tracking in waters intended for human supply based on archaeal and bacterial genetic markers

2015 ◽  
Vol 13 (4) ◽  
pp. 985-995 ◽  
Author(s):  
Kayo Bianco ◽  
Camila Barreto ◽  
Samara Sant'Anna Oliveira ◽  
Leonardo Henriques Pinto ◽  
Rodolpho Mattos Albano ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Pollution Source ◽  
Fecal Pollution ◽  
Source Tracking ◽  
The Novel ◽  
Tropical Water ◽  
Novel Host ◽  
Bacterial Genetic

The determination of fecal pollution sources in aquatic ecosystems is essential to estimate associated health risks. In this study, we evaluate eight microbial source tracking (MST) markers including host-specific Bacteroidales and Methanobrevibacter spp. for discrimination between human, bovine, equine, and swine fecal contamination in waters intended for human supply. Overall, the novel host-specific archaeal and bacterial primers proposed in this study demonstrated high sensitivity and specificity. Markers for the Archaea domain were more prevalent in the fecal and water samples studied. We conclude that the investigations regarding the sources of fecal pollution in public water supplies can contribute to improve the quality of human health. To our knowledge, this is the first analysis using both archaeal and bacterial fecal MST markers on tropical water bodies of Rio de Janeiro city, Brazil.

scholarly journals Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups

2015 ◽  
Vol 81 (20) ◽  
pp. 7023-7033 ◽  
Author(s):  
Jenny C. Fisher ◽  
A. Murat Eren ◽  
Hyatt C. Green ◽  
Orin C. Shanks ◽  
Hilary G. Morrison ◽  
...  
Keyword(s):  
Fecal Pollution ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Source Tracking ◽  
Rrna Gene ◽  
Content Type ◽  
Fundamental Relationship ◽  
The Family ◽  
Scale Population ◽  
Taxonomic Groups

ABSTRACTMost DNA-based microbial source tracking (MST) approaches target host-associated organisms within the orderBacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the familyLachnospiraceaeto provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

Host-specific 16S rRNA gene markers of Bacteroidales for source tracking of fecal pollution in the subtropical coastal seawater of Hong Kong

2010 ◽  
Vol 44 (20) ◽  
pp. 6164-6174 ◽  
Author(s):  
Rulong Liu ◽  
Miranda H.Y. Chiang ◽  
Clare H.I. Lun ◽  
Pei-Yuan Qian ◽  
Stanley C.K. Lau
Keyword(s):  
Hong Kong ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Gene Markers ◽  
Coastal Seawater

In-treatment monitoring and use of a novel topical therapy shorten the duration to eliminate Taylorella asinigenitalis in donkey stallions

2020 ◽  
Vol 8 (2) ◽  
pp. e001082
Author(s):  
Catherine May ◽  
Alan John Guthrie ◽  
Martin Lance Schulman
Keyword(s):  
Topical Therapy ◽  
Treatment Monitoring ◽  
Off Label ◽  
Real Time Quantitative Pcr ◽  
The Third ◽  
End Point ◽  
Multiple Sampling ◽  
Equus Asinus ◽  
Quarantine Station

Postarrival quarantine testing of a consignment of imported miniature donkeys (Equus asinus) with a duplex Taylorella equigenitalis/asinigenitalis real-time quantitative PCR (qPCR) assay and bacteriological culture identified T asinigenitalis in one donkey stallion from the third set of samples collected at weekly intervals. Following transportation to an approved quarantine station, further testing showed the second companion donkey stallion to be additionally positive for T asinigenitalis. Daily topical treatment included the off-label application of a bovine intramammary antimicrobial preparation to the predilection sites with concurrent in-treatment sampling for qPCR to determine treatment end point. Treatment successfully eliminated colonisation with the organism in both donkey stallions within seven days. This report strongly supported multiple sampling opportunities and the inclusion of a duplex qPCR during postimportation screening. The modified treatment regimen appeared to facilitate the rapid interval to elimination of the organism and the prompt determination of treatment end point.

scholarly journals Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

2008 ◽  
Vol 74 (22) ◽  
pp. 6839-6847 ◽  
Author(s):  
Yong-Jin Lee ◽  
Marirosa Molina ◽  
Jorge W. Santo Domingo ◽  
Jonathan D. Willis ◽  
Michael Cyterski ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Gene Markers ◽  
Specific Pcr ◽  
Pcr Assays ◽  
The Impact

ABSTRACT Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

scholarly journals Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

2012 ◽  
Vol 78 (12) ◽  
pp. 4338-4345 ◽  
Author(s):  
Hodon Ryu ◽  
Jingrang Lu ◽  
Jason Vogel ◽  
Michael Elk ◽  
Felipe Chávez-Ramírez ◽  
...  
Keyword(s):  
Host Specificity ◽  
Quantitative Pcr ◽  
Water Samples ◽  
False Negative ◽  
Environmental Water ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Content Type ◽  
Sandhill Crane

ABSTRACTWhile the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous toBacteroidetesandClostridia. The Crane1 marker targeted a dominant clade of unclassifiedLactobacillalessequences closely related toCatellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e.,n= 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n= 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n= 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.

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