Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples

EPA Science Inventory

There are numerous PCR-based methods available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in method performance. Laboratory comparisons ...

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers

EPA Science Inventory

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined using sp...

Comparing Microbiome Sampling Methods in a Wild Mammal: Fecal and Intestinal Samples Record Different Signals of Host Ecology, Evolution

PubMed Central

Ingala, Melissa R.; Simmons, Nancy B.; Wultsch, Claudia; Krampis, Konstantinos; Speer, Kelly A.; Perkins, Susan L.

2018-01-01

The gut microbiome is a community of host-associated symbiotic microbes that fulfills multiple key roles in host metabolism, immune function, and tissue development. Given the ability of the microbiome to impact host fitness, there is increasing interest in studying the microbiome of wild animals to better understand these communities in the context of host ecology and evolution. Human microbiome research protocols are well established, but wildlife microbiome research is still a developing field. Currently, there is no standardized set of best practices guiding the collection of microbiome samples from wildlife. Gut microflora are typically sampled either by fecal collection, rectal swabbing, or by destructively sampling the intestinal contents of the host animal. Studies rarely include more than one sampling technique and no comparison of these methods currently exists for a wild mammal. Although some studies have hypothesized that the fecal microbiome is a nested subset of the intestinal microbiome, this hypothesis has not been formally tested. To address these issues, we examined guano (feces) and distal intestinal mucosa from 19 species of free-ranging bats from Lamanai, Belize, using 16S rRNA amplicon sequencing to compare microbial communities across sample types. We found that the diversity and composition of intestine and guano samples differed substantially. In addition, we conclude that signatures of host evolution are retained by studying gut microbiomes based on mucosal tissue samples, but not fecal samples. Conversely, fecal samples retained more signal of host diet than intestinal samples. These results suggest that fecal and intestinal sampling methods are not interchangeable, and that these two microbiotas record different information about the host from which they are isolated. PMID:29765359

Comparing Microbiome Sampling Methods in a Wild Mammal: Fecal and Intestinal Samples Record Different Signals of Host Ecology, Evolution.

PubMed

Ingala, Melissa R; Simmons, Nancy B; Wultsch, Claudia; Krampis, Konstantinos; Speer, Kelly A; Perkins, Susan L

2018-01-01

The gut microbiome is a community of host-associated symbiotic microbes that fulfills multiple key roles in host metabolism, immune function, and tissue development. Given the ability of the microbiome to impact host fitness, there is increasing interest in studying the microbiome of wild animals to better understand these communities in the context of host ecology and evolution. Human microbiome research protocols are well established, but wildlife microbiome research is still a developing field. Currently, there is no standardized set of best practices guiding the collection of microbiome samples from wildlife. Gut microflora are typically sampled either by fecal collection, rectal swabbing, or by destructively sampling the intestinal contents of the host animal. Studies rarely include more than one sampling technique and no comparison of these methods currently exists for a wild mammal. Although some studies have hypothesized that the fecal microbiome is a nested subset of the intestinal microbiome, this hypothesis has not been formally tested. To address these issues, we examined guano (feces) and distal intestinal mucosa from 19 species of free-ranging bats from Lamanai, Belize, using 16S rRNA amplicon sequencing to compare microbial communities across sample types. We found that the diversity and composition of intestine and guano samples differed substantially. In addition, we conclude that signatures of host evolution are retained by studying gut microbiomes based on mucosal tissue samples, but not fecal samples. Conversely, fecal samples retained more signal of host diet than intestinal samples. These results suggest that fecal and intestinal sampling methods are not interchangeable, and that these two microbiotas record different information about the host from which they are isolated.

Combining Land Use Information and Small Stream Sampling with PCR-Based Methods for Better Characterization of Diffuse Sources of Human Fecal Pollution

EPA Science Inventory

Diffuse sources of human fecal pollution allow for the direct discharge of waste into receiving waters with minimal or no treatment. Traditional culture-based methods are commonly used to characterize fecal pollution in ambient waters, however these methods do not discern between...

Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8 weeks.

PubMed

Hale, Vanessa L; Tan, Chia L; Knight, Rob; Amato, Katherine R

2015-06-01

Studies of the gut microbiome have become increasingly common with recent technological advances. Gut microbes play an important role in human and animal health, and gut microbiome analysis holds great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, many common fecal preservation protocols (e.g. freezing at -80 °C) are not suitable for field conditions, or have not been tested for long-term (greater than 2 weeks) storage. In this study, we collected fresh fecal samples from captive spider monkeys (Ateles geoffroyi) at the Columbian Park Zoo (Lafayette, IN, USA). The samples were pooled, homogenized, and preserved for up to 8 weeks prior to DNA extraction and sequencing. Preservation methods included: freezing at -20 °C, freezing at -80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater. At 0 (fresh), 1, 2, 4, and 8 weeks from fecal collection, DNA was extracted and microbial DNA was amplified and sequenced. DNA concentration, purity, microbial diversity, and microbial composition were compared across all methods and time points. DNA concentration and purity did not correlate with microbial diversity or composition. Microbial composition of frozen and ethanol samples were most similar to fresh samples. FTA card and RNAlater-preserved samples had the least similar microbial composition and abundance compared to fresh samples. Microbial composition and diversity were relatively stable over time within each preservation method. Based on these results, if freezers are not available, we recommend preserving fecal samples in ethanol (for up to 8weeks) prior to microbial extraction and analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

[Isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris in Yunnan Safari Park].

PubMed

Cao, Yanru; Jiang, Yi; Li, Youlong; Chen, Xiu; Jin, Rongxian; He, Wenxiang

2012-07-04

We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches. Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park. Pretreatment of the samples, isolation media and inhibitors were tested for actinobacteria isolation. 16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis. The abundance of culturable actinobacteria was 1.10 x 10(8) cfu/g colony forming units (CFU) per gram of feces (wet weight). We obtained 110 purified cultural actinobacterium strains. The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least, and most of them were non-filamentous, such as Arthrobacter, Dietzia, Kocuria, Corynebacterium and Microbacterium. Streptomyces was the mainly classical filamentous actinobacteria, and up to 64% of total. Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria. Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria. The culturable actinobacteria are abundant in Panthera tigris tigris feces. Our study found an effective method to isolate animals' fecal actinobacteria and it's useful for studying and exploiting animals' fecal actinobacteria.

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

PubMed Central

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J.; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

Background In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Methodology Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Principal Findings Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). Conclusions The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments PMID:26360049

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

PubMed

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification.

PubMed

McMahan, Lanakila; Grunden, Amy M; Devine, Anthony A; Sobsey, Mark D

2012-04-15

The sensitivity and specificity of the H(2)S test to detect fecal bacteria in water has been variable and uncertain in previous studies, partly due to its presence-absence results. Furthermore, in groundwater samples false-positive results have been reported, with H(2)S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H(2)S-negative samples found to contain E. coli. Using biochemical and molecular methods and a novel quantitative test format, this research identified the types and numbers of microbial community members present in natural water samples, including fecal indicators and pathogens as well as other bacteria. Representative water sources tested in this study included cistern rainwater, a protected lake, and wells in agricultural and forest settings. Samples from quantitative H(2)S tests of water were further cultured for fecal bacteria by spread plating onto the selective media for detection and isolation of Aeromonas spp., E. coli, Clostridium spp., H(2)S-producers, and species of Salmonella and Shigella. Isolates were then tested for H(2)S production, and identified to the genus and species level using biochemical methods. Terminal Restriction Fragment Length Polymorphisms (TRFLP) was the molecular method employed to quantitatively characterize microbial community diversity. Overall, it was shown that water samples testing positive for H(2)S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H(2)S bacteria. Of the microorganisms isolated from natural water, greater than 70 percent were identified using TRFLP analysis to reveal a relatively stable group of organisms whose community composition differed with water source and over time. These results further document the validity of the H(2)S test for detecting and quantifying fecal contamination of water. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis

PubMed Central

Gao, Anli; Odumeru, Joseph; Raymond, Melinda; Hendrick, Steven; Duffield, Todd; Mutharia, Lucy

2009-01-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used. PMID:19337397

Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data.

PubMed

Blekhman, Ran; Tang, Karen; Archie, Elizabeth A; Barreiro, Luis B; Johnson, Zachary P; Wilson, Mark E; Kohn, Jordan; Yuan, Michael L; Gesquiere, Laurence; Grieneisen, Laura E; Tung, Jenny

2016-08-16

Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples-unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. Here, we extend previous work on storage methods for gut microbiome samples by comparing immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: lyophilization, storage in ethanol, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts.

Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿

PubMed Central

Wells, Scott J.; Collins, Michael T.; Faaberg, Kay S.; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R.; Collins, James E.; Cernicchiaro, Natalia; Whitlock, Robert H.

2006-01-01

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle. PMID:16928884

Presumptive Sources of Fecal Contamination in Four Tributaries to the New River Gorge National River, West Virginia, 2004

USGS Publications Warehouse

Mathes, Melvin V.; O'Brien, Tara L.; Strickler, Kriston M.; Hardy, Joshua J.; Schill, William B.; Lukasik, Jerzy; Scott, Troy M.; Bailey, David E.; Fenger, Terry L.

2007-01-01

Several methods were used to determine the sources of fecal contamination in water samples collected during September and October 2004 from four tributaries to the New River Gorge National River -- Arbuckle Creek, Dunloup Creek, Keeney Creek, and Wolf Creek. All four tributaries historically have had elevated levels of fecal coliform bacteria. The source-tracking methods used yielded various results, possibly because one or more methods failed. Sourcing methods used in this study included the detection of several human-specific and animal-specific biological or molecular markers, and library-dependent pulsed-field gel electrophoresis analysis that attempted to associate Escherichia coli bacteria obtained from water samples with animal sources by matching DNA-fragment banding patterns. Evaluation of the results of quality-control analysis indicated that pulsed-field gel electrophoresis analysis was unable to identify known-source bacteria isolates. Increasing the size of the known-source library did not improve the results for quality-control samples. A number of emerging methods, using markers in Enterococcus, human urine, Bacteroidetes, and host mitochondrial DNA, demonstrated some potential in associating fecal contamination with human or animal sources in a limited analysis of quality-control samples. All four of the human-specific markers were detected in water samples from Keeney Creek, a watershed with no centralized municipal wastewater-treatment facilities, thus indicating human sources of fecal contamination. The human-specific Bacteroidetes and host mitochondrial DNA markers were detected in water samples from Dunloup Creek, Wolf Creek, and to a lesser degree Arbuckle Creek. Results of analysis for wastewater compounds indicate that the September 27 sample from Arbuckle Creek contained numerous human tracer compounds likely from sewage. Dog, horse, chicken, and pig host mitochondrial DNA were detected in some of the water samples with the exception of the October 5 sample from Dunloup Creek. Cow, white-tailed deer, and Canada goose DNA were not detected in any of the samples collected from the four tributaries, despite the presence of these animals in the watersheds. Future studies with more rigorous quality-control analyses are needed to investigate the potential applicability and use of these emerging methods. Because many of the detections for the various methods could vary over time and with flow conditions, repeated sampling during both base flow and storm events would be necessary to more definitively determine the sources of fecal contamination for each watershed.

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds.

PubMed

Salgado, Miguel; Kruze, Juan; Collins, Michael T

2007-01-01

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.

Bacteria holding times for fecal coliform by mFC agar method and total coliform and Escherichia coli by Colilert®-18 Quanti-Tray® method

USGS Publications Warehouse

Aulenbach, Brent T.

2010-01-01

Bacteria holding-time experiments of up to 62 h were performed on five surface-water samples from four urban stream sites in the vicinity of Atlanta, GA, USA that had relatively high densities of coliform bacteria (Escherichia coli densities were all well above the US Environmental Protection Agency criterion of 126 colonies (100 ml) − 1 for recreational waters). Holding-time experiments were done for fecal coliform using the membrane filtration modified fecal coliform (mFC) agar method and for total coliform and E. coli using the Colilert®-18 Quanti-Tray® method. The precisions of these analytical methods were quantified. Precisions determined for fecal coliform indicated that the upper bound of the ideal range of counts could reasonably be extended upward and would improve precision. For the Colilert®-18 method, analytical precisions were similar to the theoretical precisions for this method. Fecal and total coliform densities did not change significantly with holding times up to about 27 h. Limited information indicated that fecal coliform densities might be stable for holding times of up to 62 h, whereas total coliform densities might not be stable for holding times greater than about 27 h. E. coli densities were stable for holding times of up to 18 h—a shorter period than indicated from a previous studies. These results should be applicable to non-regulatory monitoring sampling designs for similar urban surface-water sample types.

Diversity, abundance, and possible sources of fecal bacteria in the Yangtze River.

PubMed

Sun, Haohao; He, Xiwei; Ye, Lin; Zhang, Xu-Xiang; Wu, Bing; Ren, Hongqiang

2017-03-01

The fecal bacteria in natural waters may pose serious risks on human health. Although many source tracking methods have been developed and used to determine the possible sources of the fecal pollution, little is known about the overall diversity and abundance of fecal bacterial community in natural waters. In this study, a method based on fecal bacterial sequence library was introduced to evaluate the fecal bacterial profile in the Yangtze River (Nanjing section). Our results suggested that the Yangtze River water harbors diverse fecal bacteria. Fifty-eight fecal operational taxonomic units (97% identity level) were detected in the Yangtze River water samples and the relative abundance of fecal bacteria in these samples ranged from 0.1 to 8%. It was also found that the relative abundances of the fecal bacteria in locations near to the downstream of wastewater treatment plants were obviously higher than those in other locations. However, the high abundance of fecal bacteria could decrease to the normal level in 2~4 km in the river due to degradation or dilution, and the overall fecal bacteria level changed little when the Yangtze River flew through the Nanjing City. Moreover, the fecal bacteria in the Yangtze River water were found to be highly associated (Spearman rho = 0.804, P < 0.001) with the potential pathogenic bacteria. Collectively, the findings in this study reveal the diversity, abundance, and possible sources of fecal bacteria in the Yangtze River and advance our understandings of the fecal bacteria community in the natural waters.

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method

EPA Science Inventory

A quantitative polymerase chain reaction (qPCR) method for the detection of entercocci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of wat...

Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl.

PubMed

Wasser, Samuel K; Hunt, Kathleen E

2005-06-01

There is an urgent need for noninvasive methods to study reproduction and environmental stress in at-risk species such as the northern spotted owl (Strix occidentalis caurina). Two related owl species (barred owl and great horned owl) were used as surrogates to validate hormone assays for fecal metabolites of progesterone, 17beta-estradiol, testosterone, and corticosterone. Infusions of radiolabeled hormones showed that the owls excreted most hormone within 6 h. Feces and urine contained roughly equal amounts of hormone, and most fecal hormone metabolites were quite polar. The testosterone and corticosterone assays in this study bound to the major excreted metabolites of these hormones, but two progesterone assays did not appreciably bind to the major progesterone metabolites. All assays showed excellent parallelism with hydrolyzed and unhydrolyzed samples and with previously dried or undried fecal samples. Thus, samples do not require hydrolysis or prior drying. Samples from a female barred owl had significantly higher fecal estrogen, lower fecal testosterone, and higher fecal estrogen/testosterone ratio than samples from two male barred owls. The fecal estrogen/testosterone ratio was the most accurate predictor of owl gender, particularly if two or more samples are available from the same individual. Fecal corticosterone metabolites also demonstrated considerable utility for wild northern spotted owls. Fecal glucocorticoid levels varied by gender and breeding stage, being highest in male northern spotted owls early in the breeding season and highest in females when nestlings were fledging. Collectively, these studies show that noninvasive fecal hormone measurements show great promise for noninvasive assessment of reproduction and stress in wild owls.

Bacteriological quality of bottled drinking water versus municipal tap water in Dharan municipality, Nepal.

PubMed

Pant, Narayan Dutt; Poudyal, Nimesh; Bhattacharya, Shyamal Kumar

2016-06-07

Water-related diseases are of great concern in developing countries like Nepal. Every year, there are countless morbidity and mortality due to the consumption of unsafe drinking water. Recently, there have been increased uses of bottled drinking water in an assumption that the bottled water is safer than the tap water and its use will help to protect from water-related diseases. So, the main objective of this study was to analyze the bacteriological quality of bottled drinking water and that of municipal tap water. A total of 100 samples (76 tap water and 24 bottled water) were analyzed for bacteriological quality and pH. The methods used were spread plate method for total plate count (TPC) and membrane filter method for total coliform count (TCC), fecal coliform count (FCC), and fecal streptococcal count (FSC). pH meter was used for measuring pH. One hundred percent of the tap water samples and 87.5 % of the bottled water samples were found to be contaminated with heterotrophic bacteria. Of the tap water samples, 55.3 % were positive for total coliforms, compared with 25 % of the bottled water. No bottled water samples were positive for fecal coliforms and fecal streptococci, in contrast to 21.1 % and 14.5 % of the tap water samples being contaminated with fecal coliforms and fecal streptococci, respectively. One hundred percent of the tap water samples and 54.2 % of the bottled water samples had pH in the acceptable range. All of the municipal tap water samples and most of the bottled drinking water samples distributed in Dharan municipality were found to be contaminated with one or more than one type of indicator organisms. On the basis of our findings, we may conclude that comparatively, the bottled drinking water may have been safer (than tap water) to drink.

Comparison of individual, pooled, and composite fecal sampling methods for detection of Salmonella on U.S. dairy operations

USDA-ARS?s Scientific Manuscript database

The objectives of this study were to estimate the prevalence of Salmonella for individual, pooled, and composite fecal samples and to compare culture results from each sample type for determining herd Salmonella infection status and identifying Salmonella serotype(s). The USDA’s National Animal Hea...

Selection and Evaluation of Chemical Indicators for Waste Stream Identification

NASA Astrophysics Data System (ADS)

DeVita, W. M.; Hall, J.

2015-12-01

Human and animal wastes pose a threat to the quality of groundwater, surface water and drinking water. This is especially of concern for private and public water supplies in agricultural areas of Wisconsin where land spreading of livestock waste occurs on thin soils overlaying fractured bedrock. Current microbial source tracking (MST) methods for source identification requires the use of polymerase chain reaction (PCR) techniques. Due to cost, these tests are often not an option for homeowners, municipalities or state agencies with limited resources. The Water and Environmental Analysis Laboratory sought to develop chemical methods to provide lower cost processes to determine sources of fecal waste using fecal sterols, pharmaceuticals (human and veterinary) and human care/use products in ground and surface waters using solid phase extraction combined with triple quadrupole mass spectrometry. The two separate techniques allow for the detection of fecal sterol and other chemical markers in the sub part per billion-range. Fecal sterol ratios from published sources were used to evaluate drinking water samples and wastewater from onsite waste treatment systems and municipal wastewater treatment plants. Pharmaceuticals and personal care products indicative of human waste included: acetaminophen, caffeine, carbamazepine, cotinine, paraxanthine, sulfamethoxazole, and the artificial sweeteners; acesulfame, saccharin, and sucralose. The bovine antibiotic sulfamethazine was also targeted. Well water samples with suspected fecal contamination were analyzed for fecal sterols and PPCPs. Results were compared to traditional MST results from the Wisconsin State Laboratory of Hygiene. Chemical indicators were found in 6 of 11 drinking water samples, and 5 of 11 were in support of MST results. Lack of detection of chemical indicators in samples contaminated with fecal waste supports the need for confirmatory methods and advancement of chemical indicator detection technologies.

Chapter A7. Section 7.2. Fecal Indicator Viruses

USGS Publications Warehouse

Bushon, Rebecca N.

2003-01-01

More than 100 types of human pathogenic viruses may be present in fecal-contaminated waters. Coliphages are used as indicators of virus-related fecal contamination and of the microbiological quality of waters. This report provides information on the equipment, sampling protocols, and laboratory methods that are in standard use by U.S. Geological Survey (USGS) personnel for the collection of data on fecal indicator viruses.

A PILOT STUDY TO COMPARE MICROBIAL AND CHEMICAL INDICATORS OF HUMAN FECAL CONTAMINATION IN WATER

EPA Science Inventory

Limitations exist in applying traditional microbial methods for the detection of human fecal contamination of water. A pilot study was undertaken to compare the microbial and chemical indicators of human fecal contamination of water. Sixty-four water samples were collected in O...

Extraction of 3,4,4'-Trichlorocarbanilide from Rat Fecal Samples for Determination by High Pressure Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Kennedy, Rebekah C; Fling, Russell R; Terry, Paul D; Menn, Fu-Min; Chen, Jiangang; Borman, Christopher J

2015-07-15

Triclocarban (3,4,4'-Trichlorocarbanilide; TCC) in the environment has been well documented. Methods have been developed to monitor TCC levels from various matrices including water, sediment, biosolids, plants, blood and urine; however, no method has been developed to document the concentration of TCC in fecal content after oral exposure in animal studies. In the present study, we developed and validated a method that uses liquid extraction coupled with HPLC-MS/MS determination to measure TCC in feces. The limit of detection and limit of quantitation in control rats without TCC exposure was 69.0 ng/g and 92.9 ng/g of feces, respectively. The base levels of TCC in feces were lower than LOD. At 12 days of treatment, the fecal TCC concentration increased to 2220 µg/g among 0.2% w/w exposed animals. The concentration in fecal samples decreased over the washout period in 0.2% w/w treated animals to 0.399 µ/g feces after exposure was removed for 28 days. This method required a small amount of sample (0.1 g) with simple sample preparation. Given its sensitivity and efficiency, this method may be useful for monitoring TCC exposure in toxicological studies of animals.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

PubMed

Paar, Jack; Doolittle, Mark M; Varma, Manju; Siefring, Shawn; Oshima, Kevin; Haugland, Richard A

2015-05-01

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination. Published by Elsevier B.V.

Usefulness of detection of clarithromycin-resistant Helicobacter pylori from fecal specimens for young adults treated with eradication therapy.

PubMed

Osaki, Takako; Mabe, Katsuhiro; Zaman, Cynthia; Yonezawa, Hideo; Okuda, Masumi; Amagai, Kenji; Fujieda, Shinji; Goto, Mitsuhide; Shibata, Wataru; Kato, Mototsugu; Kamiya, Shigeru

2017-10-01

To prevent Helicobacter pylori infection in the younger generation, it is necessary to investigate the prevalence of antibiotic-resistant H. pylori. The aim of this study was to evaluate the method of PCR-based sequencing to detect clarithromycin (CAM) resistance-associated mutations using fecal samples as a noninvasive method. DNA extracted from fecal specimens and isolates from gastric biopsy specimens were collected from patients with H. pylori infection. Antibiotic resistance to CAM was analyzed by molecular and culture methods. The detection rates of CAM resistance-associated mutations (A2142C or A2143G) were compared before and after eradication therapy. With CAM resistance of H. pylori evaluated by antibiotic susceptibility test as a gold standard, the sensitivity and the specificity of gene mutation detection from fecal DNA were 80% and 84.8%, respectively. In contrast, using DNA of isolated strains, the sensitivity and the specificity were 80% and 100%. Of the seven cases in which eradication was unsuccessful by triple therapy including CAM, CAM-resistant H. pylori, and resistance-associated mutations were detected in three cases, CAM-resistant H. pylori without the mutation was detected in two patients, and resistance-associated mutation was only detected in one patient. PCR-based sequencing to detect CAM resistance-associated mutations using isolates or fecal samples was useful for finding antibiotic-resistant H. pylori infection. Although the specificity of the detection from fecal samples compared with antibiotic susceptibility testing was lower than that from isolates, this fecal detection method is suitable especially for asymptomatic subjects including children. Further improvement is needed before clinical application. © 2017 John Wiley & Sons Ltd.

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study.

PubMed

Harwood, Valerie J; Boehm, Alexandria B; Sassoubre, Lauren M; Vijayavel, Kannappan; Stewart, Jill R; Fong, Theng-Theng; Caprais, Marie-Paule; Converse, Reagan R; Diston, David; Ebdon, James; Fuhrman, Jed A; Gourmelon, Michele; Gentry-Shields, Jennifer; Griffith, John F; Kashian, Donna R; Noble, Rachel T; Taylor, Huw; Wicki, Melanie

2013-11-15

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Detection of cytomegalovirus DNA in fecal samples as a method for CMV enterocolitis diagnosis after allogeneic stem cell transplantation.

PubMed

Zavrelova, Alzbeta; Radocha, Jakub; Pliskova, Lenka; Paterova, Pavla; Vejrazkova, Eva; Cyrany, Jiri; Gabalec, Filip; Podhola, Miroslav; Zak, Pavel

2018-05-16

Cytomegalovirus enterocolitis is a rare but potentially life threatening complication after allogeneic stem cell transplantation. Its early diagnosis and treatment are essential for a successful outcome. To determine the potential benefit of fecal CMV DNA detection in the diagnosis of CMV colitis among stem cell transplant recipients. Biopsies from the lower gastrointestinal tract, taken during 69 episodes of diarrhea, were compared with fecal samples previously examined for CMV DNA in 45 patients after allogeneic stem cell transplantation. Six confirmed cases of CMV colitis were observed, with 16 out of 69 (23%) fecal samples proving positive for CMV DNA. Only one positive sample correlated with histologically confirmed CMV colitis, and 15 samples were evaluated as false positive. These results provide a 16.7% sensitivity and 76.2% specificity in the diagnosis of CMV enterocolitis. The examination of fecal samples for the presence of CMV DNA has very low potential in the diagnosis of CMV enterocolitis after allogeneic stem cell transplantation; therefore, a biopsy of the gastrointestinal mucosa is still warranted for correct diagnosis.

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

PubMed Central

Eriksson, Per; Mourkas, Evangelos; González-Acuna, Daniel; Olsen, Björn; Ellström, Patrik

2017-01-01

ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples. PMID:29152162

Surface Sampling Collection and Culture Methods for Escherichia coli in Household Environments with High Fecal Contamination

PubMed Central

Kosek, Margaret N.; Schwab, Kellogg J.

2017-01-01

Empiric quantification of environmental fecal contamination is an important step toward understanding the impact that water, sanitation, and hygiene interventions have on reducing enteric infections. There is a need to standardize the methods used for surface sampling in field studies that examine fecal contamination in low-income settings. The dry cloth method presented in this manuscript improves upon the more commonly used swabbing technique that has been shown in the literature to have a low sampling efficiency. The recovery efficiency of a dry electrostatic cloth sampling method was evaluated using Escherichia coli and then applied to household surfaces in Iquitos, Peru, where there is high fecal contamination and enteric infection. Side-by-side measurements were taken from various floor locations within a household at the same time over a three-month period to compare for consistency of quantification of E. coli bacteria. The dry cloth sampling method in the laboratory setting showed 105% (95% Confidence Interval: 98%, 113%) E. coli recovery efficiency off of the cloths. The field application demonstrated strong agreement of side-by-side results (Pearson correlation coefficient for dirt surfaces was 0.83 (p < 0.0001) and 0.91 (p < 0.0001) for cement surfaces) and moderate agreement for results between entrance and kitchen samples (Pearson (0.53, p < 0.0001) and weighted Kappa statistic (0.54, p < 0.0001)). Our findings suggest that this method can be utilized in households with high bacterial loads using either continuous (quantitative) or categorical (semi-quantitative) data. The standardization of this low-cost, dry electrostatic cloth sampling method can be used to measure differences between households in intervention and non-intervention arms of randomized trials. PMID:28829392

Surface Sampling Collection and Culture Methods for Escherichia coli in Household Environments with High Fecal Contamination.

PubMed

Exum, Natalie G; Kosek, Margaret N; Davis, Meghan F; Schwab, Kellogg J

2017-08-22

Empiric quantification of environmental fecal contamination is an important step toward understanding the impact that water, sanitation, and hygiene interventions have on reducing enteric infections. There is a need to standardize the methods used for surface sampling in field studies that examine fecal contamination in low-income settings. The dry cloth method presented in this manuscript improves upon the more commonly used swabbing technique that has been shown in the literature to have a low sampling efficiency. The recovery efficiency of a dry electrostatic cloth sampling method was evaluated using Escherichia coli and then applied to household surfaces in Iquitos, Peru, where there is high fecal contamination and enteric infection. Side-by-side measurements were taken from various floor locations within a household at the same time over a three-month period to compare for consistency of quantification of E. coli bacteria. The dry cloth sampling method in the laboratory setting showed 105% (95% Confidence Interval: 98%, 113%) E. coli recovery efficiency off of the cloths. The field application demonstrated strong agreement of side-by-side results (Pearson correlation coefficient for dirt surfaces was 0.83 ( p < 0.0001) and 0.91 ( p < 0.0001) for cement surfaces) and moderate agreement for results between entrance and kitchen samples (Pearson (0.53, p < 0.0001) and weighted Kappa statistic (0.54, p < 0.0001)). Our findings suggest that this method can be utilized in households with high bacterial loads using either continuous (quantitative) or categorical (semi-quantitative) data. The standardization of this low-cost, dry electrostatic cloth sampling method can be used to measure differences between households in intervention and non-intervention arms of randomized trials.

Excreta Sampling as an Alternative to In Vivo Measurements at the Hanford Site.

PubMed

Carbaugh, Eugene H; Antonio, Cheryl L; Lynch, Timothy P

2015-08-01

The capabilities of indirect radiobioassay by urine and fecal sample analysis were compared with the direct radiobioassay methods of whole body counting and lung counting for the most common radionuclides and inhalation exposure scenarios encountered by Hanford workers. Radionuclides addressed by in vivo measurement included 137Cs, 60Co, 154Eu, and 241Am as an indicator for plutonium mixtures. The same radionuclides were addressed using gamma energy analysis of urine samples, augmented by radiochemistry and alpha spectrometry methods for plutonium in urine and fecal samples. It was concluded that in vivo whole body counting and lung counting capability should be maintained at the Hanford Site for the foreseeable future, however, urine and fecal sample analysis could provide adequate, though degraded, monitoring capability for workers as a short-term alternative, should in vivo capability be lost due to planned or unplanned circumstances.

Fecal-indicator bacteria in surface waters of the Santee River Basin and coastal drainages, North and South Carolina, 1995-98

USGS Publications Warehouse

Wilhelm, Lance J.; Maluk, Terry L.

1998-01-01

IntroductionHigh levels of fecal-indicator bacteria in rivers and streams can indicate the possible presence of pathogenic (disease-causing) microorganisms. Cholera, typhoid fever, bacterial dysentery, infectious hepatitis, and cryptosporidiosis are some of the well known waterborne diseases that spread through water contaminated and fecal matter. Eye, ear, nose, and throat infections also can result from contact with contaminated water. In general, methods are not routinely used to detect pathogens in water. Instead, bacteria such as total coliforms, fecal coliforms, fecal streptococci, Escherichia coli (E coli), and enterococci are used as indicators of sanitary water quality, because they are present in high numbers in fecal material and have been shown to be associated with some waterborne disease-causing organisms. Indicator bacteria usually are harmless, more plentiful, and easier to detect than pathogens. The concentration of bacteria in a sample of water is usually expressed as the number of bacterial colonies per 100 milliliters of water sample.As part of the U.S. Geological Survey National Water-Quality Assessment Program, 145 samples were collected and analyzed for selected water-quality constituents, fecal coliforms, and fecal streptococci at 17 sites in North and South Carolina from October 1995 through September 1996. Of the original 17 sites, 4 in South Carolina were sampled for E. coli and total coliforms from April through September 1997. At two sites, this sampling continued from October 1997 through April 1998.

Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

EPA Science Inventory

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds.

PubMed

Yoshida, Hiromi; Sakoda, Yoshihiro; Endo, Mayumi; Motoshima, Masayuki; Yoshino, Fumi; Yamamoto, Naoki; Okamatsu, Masatoshi; Soejima, Takahiro; Senba, Syouhei; Kanda, Hidetoshi; Kida, Hiroshi

2011-06-01

Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.

Rapid regrowth and detection of microbial contaminants in equine fecal microbiome samples

PubMed Central

Beckers, Kalie F.; Childers, Gary W.

2017-01-01

Advances have been made to standardize 16S rRNA gene amplicon based studies for inter-study comparisons, yet there are many opportunities for systematic error that may render these comparisons improper and misleading. The fecal microbiome of horses has been examined previously, however, no universal horse fecal collection method and sample processing procedure has been established. This study was initialized in large part to ensure that samples collected by different individuals from different geographical areas (i.e., crowdsourced) were not contaminated due to less than optimal sampling or holding conditions. In this study, we examined the effect of sampling the surface of fecal pellets compared to homogenized fecal pellets, and also the effect of time of sampling after defecation on ‘bloom’ taxa (bloom taxa refers to microbial taxa that can grow rapidly in horse feces post-defecation) using v4 16S rRNA amplicon libraries. A total of 1,440,171 sequences were recovered from 65 horse fecal samples yielding a total of 3,422 OTUs at 97% similarity. Sampling from either surface or homogenized feces had no effect on diversity and little effect on microbial composition. Sampling at various time points (0, 2, 4, 6, 12 h) had a significant effect on both diversity and community composition of fecal samples. Alpha diversity (Shannon index) initially increased with time as regrowth taxa were detected in the amplicon libraries, but by 12 h the diversity sharply decreased as the community composition became dominated by a few bloom families, including Bacillaceae, Planococcaeae, and Enterococcaceae, and other families to a lesser extent. The results show that immediate sampling of horse feces must be done in order to ensure accurate representation of horse fecal samples. Also, several of the bloom taxa found in this study are known to occur in human and cattle feces post defecation. The dominance of these taxa in feces shortly after defecation suggests that the feces is an important habitat for these organisms, and horse fecal samples that were improperly stored can be identified by presence of bloom taxa. PMID:29091944

Combining watershed attributes with culture- and PCR-based methods for improved characterization and management of fecal pollution

EPA Science Inventory

Culture- and PCR-based methods for characterization of fecal pollution were evaluated in relation to physiographic, biotic, and chemical indicators of stream condition. Stream water samples (n = 235) were collected monthly over a two year period from ten channels draining subwat...

[Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

PubMed

Ruan, Jia; Ren, Dong-xia; Yang, Dan-ni; Long, Pin-pin; Zhao, Hong-yue; Wang, Yi-qi; Li, Yong-xin

2015-07-01

To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.

False positive fecal coliform in biosolid samples assayed using A-1 medium.

PubMed

Baker, Katherine H; Redmond, Brady; Herson, Diane S

2005-01-01

Two most probable number (MPN) methods-lauryl tryptose broth with Escherichia coli broth confirmation and direct A-1 broth incubation (A-1)--were compared for the enumeration of fecal coliform in lime-treated biosolid. Fecal coliform numbers were significantly higher using the A-1 method. Analysis of positive A-1 tubes, however, indicated that a high percentage of these were false positives. Therefore, the use of A-1 broth for 40 CFR Part 503 Pathogen Reduction (CFR, 1993) compliance testing is not recommended.

Assessment of Fecal Exposure Pathways in Low-Income Urban Neighborhoods in Accra, Ghana: Rationale, Design, Methods, and Key Findings of the SaniPath Study

PubMed Central

Robb, Katharine; Null, Clair; Teunis, Peter; Yakubu, Habib; Armah, George; Moe, Christine L.

2017-01-01

Abstract. Rapid urbanization has contributed to an urban sanitation crisis in low-income countries. Residents in low-income, urban neighborhoods often have poor sanitation infrastructure and services and may experience frequent exposure to fecal contamination through a range of pathways. There are little data to prioritize strategies to decrease exposure to fecal contamination in these complex and highly contaminated environments, and public health priorities are rarely considered when planning urban sanitation investments. The SaniPath Study addresses this need by characterizing pathways of exposure to fecal contamination. Over a 16 month period, an in-depth, interdisciplinary exposure assessment was conducted in both public and private domains of four neighborhoods in Accra, Ghana. Microbiological analyses of environmental samples and behavioral data collection techniques were used to quantify fecal contamination in the environment and characterize the behaviors of adults and children associated with exposure to fecal contamination. Environmental samples (n = 1,855) were collected and analyzed for fecal indicators and enteric pathogens. A household survey with 800 respondents and over 500 hours of structured observation of young children were conducted. Approximately 25% of environmental samples were collected in conjunction with structured observations (n = 441 samples). The results of the study highlight widespread and often high levels of fecal contamination in both public and private domains and the food supply. The dominant fecal exposure pathway for young children in the household was through consumption of uncooked produce. The SaniPath Study provides critical information on exposure to fecal contamination in low-income, urban environments and ultimately can inform investments and policies to reduce these public health risks. PMID:28722599

Determination of actinides in urine and fecal samples

DOEpatents

McKibbin, Terry T.

1993-01-01

A method of determining the radioactivity of specific actinides that are carried in urine or fecal sample material is disclosed. The samples are ashed in a muffle furnace, dissolved in an acid, and then treated in a series of steps of reduction, oxidation, dissolution, and precipitation, including a unique step of passing a solution through a chloride form anion exchange resin for separation of uranium and plutonium from americium.

Determination of actinides in urine and fecal samples

DOEpatents

McKibbin, T.T.

1993-03-02

A method of determining the radioactivity of specific actinides that are carried in urine or fecal sample material is disclosed. The samples are ashed in a muffle furnace, dissolved in an acid, and then treated in a series of steps of reduction, oxidation, dissolution, and precipitation, including a unique step of passing a solution through a chloride form anion exchange resin for separation of uranium and plutonium from americium.

Evaluation of PMS-PCR technology for detection of Mycobacterium avium subsp. paratuberculosis directly from bovine fecal specimens.

PubMed

Salgado, M; Steuer, P; Troncoso, E; Collins, M T

2013-12-27

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with <100 MAP per 2g feces (30.06) (p<0.05). PMS-PCR technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Environmental sampling to predict fecal prevalence of Salmonella in an intensively monitored dairy herd.

PubMed

Van Kessel, J S; Karns, J S; Wolfgang, D R; Hovingh, E; Jayarao, B M; Van Tassell, C P; Schukken, Y H

2008-10-01

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas-were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone.

A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

PubMed

Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

2018-01-01

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

[Investigation of Cryptosporidium sp. in workers of the Van municipality slaughterhouse and in slaughtered animals].

PubMed

Ciçek, Mutalip; Körkoca, Hanifi; Gül, Abdurrahman

2008-01-01

This study was carried out in order to investigate the prevalence of Cryptosporidium sp. in slaughtered animals and workers of the Van municipality slaughterhouse in Van. Animals slaughtered at different times and workers who had been working in different departments of the slaughter house were included in the study for three months. A total of 309 fecal specimens from animals including 167 sheep, 56 goats and 86 cattle and 87 fecal specimens from workers were examined for Cryptosporidium sp. oocysts. In slaughtered animals, the modified acid-fast staining method was used to determine the oocysts of Cryptosporidium sp. The fecal samples of slaughter workers were examined by using RIDA (R) Quick Cryptosporidium Strip Test (R-Biopharm, Germany) and the modified acid-fast staining method. Fecal samples found to be positive by stripe test were also confirmed with the ELISA method (R-Biopharm, Germany). Oocysts of Cryptosporidium sp. were found in fecal specimens of 22 sheep (13.17%), 6 goats (10.71%) and 7 cattle (8.13%). Intestinal parasites were observed in 34 fecal specimens of workers (39.08%). Cryptosporidium sp., Hymenolepis nana, Chilomastix mesnili, Endolimax nana, Iodamoeba bütschlii were found in the specimen of one worker (1.14%), Entamoeba coli in 4 workers (4.59%), Blastocystis hominis (9.19%) in 8 workers, and Giardia intestinalis (19.54%) in 17 workers.

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.

PubMed

Lemos, Vanessa; Graczyk, Thaddeus K; Alves, Margarida; Lobo, Maria Luísa; Sousa, Maria C; Antunes, Francisco; Matos, Olga

2005-12-01

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Evaluation of Two Library-Independent Microbial Source Tracking Methods To Identify Sources of Fecal Contamination in French Estuaries▿

PubMed Central

Gourmelon, Michèle; Caprais, Marie Paule; Ségura, Raphaël; Le Mennec, Cécile; Lozach, Solen; Piriou, Jean Yves; Rincé, Alain

2007-01-01

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193′), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers. PMID:17557850

Development and testing of real-time PCR assays for determining fecal loading and source identification (cattle, human, etc.) in surface water and groundwater

NASA Astrophysics Data System (ADS)

McKay, L. D.; Layton, A.; Gentry, R.

2004-12-01

A multi-disciplinary group of researchers at the University of Tennessee is developing and testing a series of microbial assay methods based on real-time PCR to detect fecal bacterial concentrations and host sources in water samples. Real-time PCR is an enumeration technique based on the unique and conserved nucleic acid sequences present in all organisms. The first research task was development of an assay (AllBac) to detect total amount of Bacteroides, which represents up to 30 percent of fecal mass. Subsequent assays were developed to detect Bacteroides from cattle (BoBac) and humans (HuBac) using 16sRNA genes based on DNA sequences in the national GenBank, as well as sequences from local fecal samples. The assays potentially have significant advantages over conventional bacterial source tracking methods because: 1. unlike traditional enumeration methods, they do not require bacterial cultivation; 2. there are no known non-fecal sources of Bacteroides; 3. the assays are quantitative with results for total concentration and for each species expressed in mg/l; and 4. they show little regional variation within host species, meaning that they do not require development of extensive local gene libraries. The AllBac and BoBac assays have been used in a study of fecal contamination in a small rural watershed (Stock Creek) near Knoxville, TN, and have proven useful in identification of areas where cattle represent a significant fecal input and in development of BMPs. It is expected that these types of assays (and future assays for birds, hogs, etc.) could have broad applications in monitoring fecal impacts from Animal Feeding Operations, as well as from wildlife and human sources.

Comparison of four sampling methods for the detection of Salmonella in broiler litter.

PubMed

Buhr, R J; Richardson, L J; Cason, J A; Cox, N A; Fairchild, B D

2007-01-01

Experiments were conducted to compare litter sampling methods for the detection of Salmonella. In experiment 1, chicks were challenged orally with a suspension of naladixic acid-resistant Salmonella and wing banded, and additional nonchallenged chicks were placed into each of 2 challenge pens. Nonchallenged chicks were placed into each nonchallenge pen located adjacent to the challenge pens. At 7, 8, 10, and 11 wk of age the litter was sampled using 4 methods: fecal droppings, litter grab, drag swab, and sock. For the challenge pens, Salmonella-positive samples were detected in 3 of 16 fecal samples, 6 of 16 litter grab samples, 7 of 16 drag swabs samples, and 7 of 16 sock samples. Samples from the nonchallenge pens were Salmonella positive in 2 of 16 litter grab samples, 9 of 16 drag swab samples, and 9 of 16 sock samples. In experiment 2, chicks were challenged with Salmonella, and the litter in the challenge and adjacent nonchallenge pens were sampled at 4, 6, and 8 wk of age with broilers remaining in all pens. For the challenge pens, Salmonella was detected in 10 of 36 fecal samples, 20 of 36 litter grab samples, 14 of 36 drag swab samples, and 26 of 36 sock samples. Samples from the adjacent nonchallenge pens were positive for Salmonella in 6 of 36 fecal droppings samples, 4 of 36 litter grab samples, 7 of 36 drag swab samples, and 19 of 36 sock samples. Sock samples had the highest rates of Salmonella detection. In experiment 3, the litter from a Salmonella-challenged flock was sampled at 7, 8, and 9 wk by socks and drag swabs. In addition, comparisons with drag swabs that were stepped on during sampling were made. Both socks (24 of 36, 67%) and drag swabs that were stepped on (25 of 36, 69%) showed significantly more Salmonella-positive samples than the traditional drag swab method (16 of 36, 44%). Drag swabs that were stepped on had comparable Salmonella detection level to that for socks. Litter sampling methods that incorporate stepping on the sample material while in contact with the litter appear to detect Salmonella in greater incidence than traditional sampling methods of dragging swabs over the litter surface.

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

PubMed

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

Quantitative CrAssphage PCR Assays for Human Fecal ...

EPA Pesticide Factsheets

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

ENHANCED CONCENTRATION AND ISOLATION OF CYCLOSPORA CAYETANENSIS OOCYSTS FROM HUMAN FECAL SAMPLES

EPA Science Inventory

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infections disease. A new method for the purification of Cycloposra cayetanensis oocysts from fecal matter has been developed, using a modified detachment solution and a Renocal-sucrose gradient sedimen...

Spiral Plating Method To Quantify the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces.

PubMed

Shridhar, Pragathi B; Noll, Lance W; Cull, Charley A; Shi, Xiaorong; Cernicchiaro, Natalia; Renter, David G; Bai, Jianfa; Nagaraja, T G

2017-04-17

Cattle are a major reservoir of the six major Shiga toxin-producing non-O157 Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) responsible for foodborne illnesses in humans. Besides prevalence in feces, the concentrations of STEC in cattle feces play a major role in their transmission dynamics. A subset of cattle, referred to as super shedders, shed E. coli O157 at high concentrations (≥4 log CFU/g of feces). It is not known whether a similar pattern of fecal shedding exists for non-O157. Our objectives were to initially validate the spiral plating method to quantify the six non-O157 E. coli serogroups with pure cultures and culture-spiked fecal samples and then determine the applicability of the method and compare it with multiplex quantitative PCR (mqPCR) assays for the quantification of the six non-O157 E. coli serogroups in cattle fecal samples collected from commercial feedlots. Quantification limits of the spiral plating method were 3 log, 3 to 4 log, and 3 to 5 log CFU/mL or CFU/g for individual cultures, pooled pure cultures, and cattle fecal samples spiked with pooled pure cultures, respectively. Of the 1,152 cattle fecal samples tested from eight commercial feedlots, 122 (10.6%) and 320 (27.8%) harbored concentrations ≥4 log CFU/g of one or more of the six serogroups of non-O157 by spiral plating and mqPCR methods, respectively. A majority of quantifiable samples, detected by either spiral plating (135 of 137, 98.5%) or mqPCR (239 of 320, 74.7%), were shedding only one serogroup. Only one of the quantifiable samples was positive for a serogroup carrying Shiga toxin (stx 1 ) and intimin (eae) genes; 38 samples were positive for serogroups carrying the intimin gene. In conclusion, the spiral plating method can be used to quantify non-O157 serogroups in cattle feces, and our study identified a subset of cattle that was super shedders of non-O157 E. coli . The method has the advantage of quantifying non-O157 STEC, unlike mqPCR that quantifies serogroups only.

Evaluation of a real-time quantitative PCR method with propidium monazide treatment for analyses of viable fecal indicator bacteria in wastewater samples

EPA Science Inventory

The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating cri...

Comparison of Enterococcus Species Diversity in Marine Water and Wastewater Using Enterolert and EPA Method 1600

PubMed Central

Ferguson, Donna M.; Griffith, John F.; McGee, Charles D.; Weisberg, Stephen B.; Hagedorn, Charles

2013-01-01

EPA Method 1600 and Enterolert are used interchangeably to measure Enterococcus for fecal contamination of public beaches, but the methods occasionally produce different results. Here we assess whether these differences are attributable to the selectivity for certain species within the Enterococcus group. Both methods were used to obtain 1279 isolates from 17 environmental samples, including influent and effluent of four wastewater treatment plants, ambient marine water from seven different beaches, and freshwater urban runoff from two stream systems. The isolates were identified to species level. Detection of non-Enterococcus species was slightly higher using Enterolert (8.4%) than for EPA Method 1600 (5.1%). E. faecalis and E. faecium, commonly associated with human fecal waste, were predominant in wastewater; however, Enterolert had greater selectivity for E. faecalis, which was also shown using a laboratory-created sample. The same species selectivity was not observed for most beach water and urban runoff samples. These samples had relatively higher proportions of plant associated species, E. casseliflavus (18.5%) and E. mundtii (5.7%), compared to wastewater, suggesting environmental inputs to beaches and runoff. The potential for species selectivity among water testing methods should be considered when assessing the sanitary quality of beaches so that public health warnings are based on indicators representative of fecal sources. PMID:23840233

The impact of freeze-drying infant fecal samples on measures of their bacterial community profiles and milk-derived oligosaccharide content.

PubMed

Lewis, Zachery T; Davis, Jasmine C C; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B; Mills, David A

2016-01-01

Infant fecal samples are commonly studied to investigate the impacts of breastfeeding on the development of the microbiota and subsequent health effects. Comparisons of infants living in different geographic regions and environmental contexts are needed to aid our understanding of evolutionarily-selected milk adaptations. However, the preservation of fecal samples from individuals in remote locales until they can be processed can be a challenge. Freeze-drying (lyophilization) offers a cost-effective way to preserve some biological samples for transport and analysis at a later date. Currently, it is unknown what, if any, biases are introduced into various analyses by the freeze-drying process. Here, we investigated how freeze-drying affected analysis of two relevant and intertwined aspects of infant fecal samples, marker gene amplicon sequencing of the bacterial community and the fecal oligosaccharide profile (undigested human milk oligosaccharides). No differences were discovered between the fecal oligosaccharide profiles of wet and freeze-dried samples. The marker gene sequencing data showed an increase in proportional representation of Bacteriodes and a decrease in detection of bifidobacteria and members of class Bacilli after freeze-drying. This sample treatment bias may possibly be related to the cell morphology of these different taxa (Gram status). However, these effects did not overwhelm the natural variation among individuals, as the community data still strongly grouped by subject and not by freeze-drying status. We also found that compensating for sample concentration during freeze-drying, while not necessary, was also not detrimental. Freeze-drying may therefore be an acceptable method of sample preservation and mass reduction for some studies of microbial ecology and milk glycan analysis.

Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

USGS Publications Warehouse

Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

2012-01-01

During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water

PubMed Central

Jiménez-Clavero, Miguel Angel; Fernández, Carlos; Ortiz, José Antonio; Pro, Javier; Carbonell, Gregoria; Tarazona, José Vicente; Roblas, Neftalí; Ley, Victoria

2003-01-01

Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination. PMID:14532098

Development of Cross-Assembly Phage PCR-Based Methods ...

EPA Pesticide Factsheets

Technologies that can characterize human fecal pollution in environmental waters offer many advantages over traditional general indicator approaches. However, many human-associated methods cross-react with non-human animal sources and lack suitable sensitivity for fecal source identification applications. The genome of a newly discovered bacteriophage (~97 kbp), the Cross-Assembly phage or “crAssphage”, assembled from a human gut metagenome DNA sequence library is predicted to be both highly abundant and predominately occur in human feces suggesting that this double stranded DNA virus may be an ideal human fecal pollution indicator. We report the development of two human-associated crAssphage endpoint PCR methods (crAss056 and crAss064). A shotgun strategy was employed where 384 candidate primers were designed to cover ~41 kbp of the crAssphage genome deemed favorable for method development based on a series of bioinformatics analyses. Candidate primers were subjected to three rounds of testing to evaluate assay optimization, specificity, limit of detection (LOD95), geographic variability, and performance in environmental water samples. The top two performing candidate primer sets exhibited 100% specificity (n = 70 individual samples from 8 different animal species), >90% sensitivity (n = 10 raw sewage samples from different geographic locations), LOD95 of 0.01 ng/µL of total DNA per reaction, and successfully detected human fecal pollution in impaired envi

A human fecal contamination index for ranking impaired ...

EPA Pesticide Factsheets

Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk management. The transition from a research subject to a management tool requires the integration of standardized water sampling, laboratory, and data analysis procedures. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and Bayesian data algorithm to establish a human fecal contamination index that can be used to rank impaired recreational water sites polluted with human waste. Stability and bias of index predictions were investigated under various parameters including siteswith different pollution levels, sampling period time range (1-15 weeks), and number of qPCR replicates per sample (2-14 replicates). Sensitivity analyses were conducted with simulated data sets (100 iterations) seeded with HF183/BacR287 qPCR laboratory measurements from water samples collected from three Southern California sites (588 qPCR measurements). Findings suggest that site ranking is feasible and that all parameters tested influence stability and bias in human fecal contamination indexscoring. Trends identified by sensitivity analyses will provide managers with the information needed to design and conduct field studies to rank impaired recreational water sites based

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

PubMed Central

Ravaliya, Kruti; Garcia, Santos; Heredia, Norma; Fabiszewski de Aceituno, Anna; Bartz, Faith E.; Leon, Juan S.; Jaykus, Lee-Ann

2014-01-01

In 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 on Bacteroidales 16S 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 universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10 genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10 GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10 GEC/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 between Bacteroidales and generic Escherichia coli across all samples. This study provides evidence that Bacteroidales markers 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. PMID:24212583

Effects of Holding Time, Storage, and the Preservation of ...

EPA Pesticide Factsheets

The purpose of this project was to answer questions related to storage of samples to be analyzed by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. The project was divided into two parts. The first part was to determine if filters that were used to collect fecal indicators could be stored frozen and analyzed at a later date and the second part was to determine if refrigerated water samples could be held for 24 to 48 hours prior to analysis by qPCR. Both of these studies answer questions that were important in the analysis of fresh and marine surface water samples for beach monitoring purposes. 1) Develop and evaluate qPCR assays and test methods for the detection and quantification of genetic markers from indicator bacteria that are associated with human fecal waste and from two new groups of general fecal indicator bacteria (E. coli and Clostridia) that historically have been widely used or are favored in specific regions 2) Determine the occurrence and densities of genetic markers detected by new qPCR assays developed under objective 1 and compare with occurrence and densities of genetic markers detected by previously developed qPCR assays for enterococci and total Bacterioidalesin waste waters and fecal material from different animal sources. 3) Determine stability of fecal indicator bacteria target DNA sequences in freezer archived filter retentates of ambient surface water samples 4) Determine the densitie

Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

PubMed

Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

2015-09-01

Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS[S

PubMed Central

Kakiyama, Genta; Muto, Akina; Takei, Hajime; Nittono, Hiroshi; Murai, Tsuyoshi; Kurosawa, Takao; Hofmann, Alan F.; Pandak, William M.; Bajaj, Jasmohan S.

2014-01-01

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. PMID:24627129

Comparing protein and energy status of winter-fed white-tailed deer

USGS Publications Warehouse

Page, B.D.; Underwood, H.B.

2006-01-01

Although nutritional status in response to controlled feeding trials has been extensively studied in captive white-tailed deer (Odocoileus virginianus), there remains a considerable gap in understanding the influence of variable supplemental feeding protocols on free-ranging deer. Consequently, across the northern portion of the white-tailed deer range, numerous property managers are investing substantial resources into winter supplemental-feeding programs without adequate tools to assess the nutritional status of their populations. We studied the influence of a supplemental winter feeding gradient on the protein and energy status of free-ranging white-tailed deer in the Adirondack Mountains of New York. We collected blood and fecal samples from 31 captured fawns across 3 sites that varied considerably in the frequency, quantity, and method of supplemental feed distribution. To facilitate population-wide comparisons, we collected fresh fecal samples off the snow at each of the 3 sites with supplemental feeding and 1 reference site where no feeding occurred. Results indicated that the method of feed distribution, in addition to quantity and frequency, can affect the nutritional status of deer. The least intensively fed population showed considerable overlap in diet quality with the unfed population in a principal components ordination, despite the substantial time and financial resources invested in the feeding program. Data from fecal samples generally denoted a gradient in diet quality and digestibility that corresponded with the availability of supplements. Our results further demonstrated that fecal nitrogen and fecal fiber, indices of dietary protein and digestibility, can be estimated using regressions of fecal pellet mass, enabling a rapid qualitative assessment of diet quality.

INTERNAL AMPLIFICATION CONTROL FOR USE IN QUANTITATIVE POLYMERASE CHAIN REACTION FECAL INDICATOR BACTERIA ASSAYS

EPA Science Inventory

Quantitative polymerase chain reaction (QPCR) can be used as a rapid method for detecting fecal indicator bacteria. Because false negative results can be caused by PCR inhibitors that co-extract with the DNA samples, an internal amplification control (IAC) should be run with eac...

Confined cattle feeding trail to validate fecal DNA metabarcoding to inform rangeland free-roaming diet applications

USDA-ARS?s Scientific Manuscript database

Diet composition of free roaming livestock and wildlife in extensive rangelands are difficult to quantify. Recent technological advances now allow us to reconstruct plant species-specific dietary protein composition using fecal samples. However, it has been suggested that validation of the method i...

Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

EPA Science Inventory

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental wat...

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC MICROBIAL GENETIC MARKERS IN COW FECAL SAMPLES

EPA Science Inventory

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host specific markers. Here, we describe the application of a genome fragment enrichment met...

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC GENETIC MARKERS IN CATTLE FECAL SAMPLES - ABSTRACT

EPA Science Inventory

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host specific markers. Here, we describe the application of a genome fragment enrichment met...

Epidemiology of Salmonella sp. in California cull dairy cattle: prevalence of fecal shedding and diagnostic accuracy of pooled enriched broth culture of fecal samples

PubMed Central

Abu Aboud, Omran A.; Adaska, John M.; Williams, Deniece R.; Rossitto, Paul V.; Champagne, John D.; Lehenbauer, Terry W.; Atwill, Robert; Li, Xunde

2016-01-01

Background The primary objective of this cross-sectional study was to estimate the crude, seasonal and cull-reason stratified prevalence of Salmonella fecal shedding in cull dairy cattle on seven California dairies. A secondary objective was to estimate and compare the relative sensitivity (Se) and specificity (Sp) for pools of 5 and 10 enriched broth cultures of fecal samples for Salmonella sp. detection. Methods Seven dairy farms located in the San Joaquin Valley of California were identified and enrolled in the study as a convenience sample. Cull cows were identified for fecal sampling once during each season between 2014 and 2015, specifically during spring, summer, fall, and winter, and 10 cows were randomly selected for fecal sampling at the day of their sale. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous four month’s herd management. Fecal samples were frozen until testing for Salmonella. After overnight enrichment in liquid broth, pools of enrichment broth (EBP) were created for 5 and 10 samples. All individual and pooled broths were cultured on selective media with putative Salmonella colonies confirmed by biochemical testing before being serogrouped and serotyped. Results A total of 249 cull cows were enrolled into the study and their fecal samples tested for Salmonella. The survey-weighted period prevalence of fecal shedding of all Salmonella sp. in the cull cow samples across all study herds and the entire study period was 3.42% (N = 249; SE 1.07). The within herd prevalence of Salmonella shed in feces did not differ over the four study seasons (P = 0.074). The Se of culture of EBP of five samples was 62.5% (SE = 17.12), which was not statistically different from the Se of culture of EBP of 10 (37.5%, SE = 17.12, P = 0.48). The Sp of culture of EBP of five samples was 95.24% (SE = 3.29) and for pools of 10 samples was 100.00% (SE = 0). There was no statistical difference between the culture relative specificities of EBP of 5 and 10 (P > 0.99). Discussion Our study showed a numerically higher prevalence of Salmonella shedding in the summer, although the results were not significant, most likely due to a lack of power from the small sample size. A higher prevalence in summer months may be related to heat stress. To detect Salmonella, investigators may expect a 62.5% sensitivity for culture of EBP of five, relative to individual fecal sample enrichment and culture. In contrast, culture of EBP of 10 samples resulted in a numerically lower Se. Culture of EBP of size 5 or 10 samples, given similar prevalence and limit of detection, can be expected to yield specificities of 95 and 100%, respectively. PMID:27635350

Soiled-bedding Sentinel Detection of Murine Norovirus 4

PubMed Central

Manuel, Christopher A; Hsu, Charlie C; Riley, Lela K; Livingston, Robert S

2008-01-01

According to serologic surveys, murine norovirus (MNV) is the most prevalent viral pathogen infecting mice used in biomedical research. However, the use of sentinel mice to detect MNV-infected mouse populations has not been evaluated thoroughly. To this end, an experimental method of soiled bedding transfer was created to mimic a quarterly sentinel monitoring program. Soiled bedding (15 or 30 cm3) from ICR mice experimentally infected with MNV4 was transferred weekly to cages of pair-housed 4-wk-old ICR mice. After 12 wk, both mice in 80% (4 of 5) of cages receiving either 15 or 30 cm3 of soiled bedding were seropositive for MNV and were shedding virus in feces. To evaluate the stability of MNV RNA in mouse feces, fecal pellets from MNV-infected sentinel mice were stored at room temperature for as long as 14 d. After storage, all fecal samples tested positive for MNV by RT-PCR. To determine whether fecal samples could be pooled for MNV detection, 1 MNV-positive fecal pellet was combined with either 9 or 19 MNV-negative fecal pellets. All pooled fecal samples were positive for MNV by RT-PCR at both dilutions. These data indicate that although MNV-infected mouse populations can be detected by exposing sentinel mice to MNV-contaminated bedding, detection failures can occur. In addition, there was high agreement in the MNV infection status of cohoused sentinel mice. These data also demonstrate that MNV is readily detectable in pooled fecal samples and in mouse feces stored at room temperature for 2 wk. PMID:18459710

A comparative analysis of preservation techniques for the optimal molecular detection of hookworm DNA in a human fecal specimen

PubMed Central

Pilotte, Nils; Baumer, Ben; Grant, Jessica; Asbjornsdottir, Kristjana; Schaer, Fabian; Hu, Yan; Aroian, Raffi; Walson, Judd; Williams, Steven A.

2018-01-01

Background Proper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time. Methodology Quantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against “no preservative” controls and the “gold standard” of rapidly freezing samples at -20°C. The preservation methods were compared at both 4°C and at simulated tropical ambient temperature (32°C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA. Conclusions At 4°C there were no significant differences in DNA amplification efficiency (as measured by Cq values) regardless of the preservation method utilized over the 60-day period. At 32°C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cq value increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4°C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field. PMID:29346412

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method.

PubMed

Kageyama, A; Benno, Y

2001-01-01

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.

Optimization of scat detection methods for a social ungulate, the wild pig, and experimental evaluation of factors affecting detection of scat

USGS Publications Warehouse

Keiter, David A.; Cunningham, Fred L.; Rhodes, Olin E.; Irwin, Brian J.; Beasley, James

2016-01-01

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocols with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig (Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. Knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.

Optimization of scat detection methods for a social ungulate, the wild pig, and experimental evaluation of factors affecting detection of scat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keiter, David A.; Cunningham, Fred L.; Rhodes, Jr., Olin E.

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocolsmore » with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig ( Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. In conclusion, knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.« less

Optimization of Scat Detection Methods for a Social Ungulate, the Wild Pig, and Experimental Evaluation of Factors Affecting Detection of Scat.

PubMed

Keiter, David A; Cunningham, Fred L; Rhodes, Olin E; Irwin, Brian J; Beasley, James C

2016-01-01

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocols with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig (Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. Knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.

Optimization of scat detection methods for a social ungulate, the wild pig, and experimental evaluation of factors affecting detection of scat

DOE PAGES

Keiter, David A.; Cunningham, Fred L.; Rhodes, Jr., Olin E.; ...

2016-05-25

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocolsmore » with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig ( Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. In conclusion, knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.« less

Identification of feces by detection of Bacteroides genes.

PubMed

Nakanishi, Hiroaki; Shojo, Hideki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Adachi, Noboru; Saito, Kazuyuki

2013-01-01

In forensic science, the identification of feces is very important in a variety of crime investigations. However, no sensitive and simple fecal identification method using molecular biological techniques has been reported. Here, we focused on the fecal bacteria, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron, and developed a novel fecal identification method by detection of the gene sequences specific to these bacteria in various body (feces, blood, saliva, semen, urine, vaginal fluids and skin surfaces) and forensic (anal adhesions) specimens. Bacterial gene detection was performed by real-time PCR using a minor groove binding probe to amplify the RNA polymerase β-subunit gene of B. uniformis and B. vulgatus, and the α-1-6 mannanase gene of B. thetaiotaomicron. At least one of these bacteria was detected in the feces of 20 donors; the proportions of B. uniformis, B. vulgatus and B. thetaiotaomicron were 95, 85 and 60%, respectively. Bacteroides vulgatus was also detected in one of six vaginal fluid samples, but B. thetaiotaomicron and B. uniformis were not detected in body samples other than feces. Further, we applied this method to forensic specimens from 18 donors. Eighteen anal adhesions also contained at least one of three bacteria; B. uniformis, B. vulgatus and B. thetaiotaomicron were detected in 89, 78 and 56%, respectively, of the specimens. Thus, these bacteria were present at a high frequency in the fecal and forensic specimens, while either B. uniformis or B. vulgatus was detected in all samples. Therefore, B. uniformis and B. vulgatus represent more appropriate target species than B. thetaiotaomicron for the identification of fecal material. If B. vulgatus and/or B. uniformis are detected, it is likely that the sample contains feces. Taken together, our results suggest that the use of molecular biological techniques will aid the detection of feces in forensic practice, although it is possible that the samples contained both feces and vaginal fluid. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Influence of refrigeration and formalin on the floatability of Giardia duodenalis cysts.

PubMed

Moitinho, M d; Bertoli, M; Guedes, T A; Ferreira, C S

1999-01-01

Giardia duodenalis cysts obtained from fresh fecal samples, fecal samples kept under refrigeration and fecal samples treated with formalin were studied as to their floatability on sucrose solutions with the following specific gravities: 1,040 kg/m3; 1,050 kg/m3; 1, 060 kg/m3; 1,070 kg/m3; 1,080 kg/m3; 1,090 kg/m3; 1,100 kgm3; 1,150 kg/m3; 1,200 kg/m3; and 1,250 kg/m3, contained within counting-chambers 0.17 mm high. Cysts that floated on and those settled down as sediments were counted, and had their percentages estimated. Sucrose solutions of 1,200 kg/m3 specific gravity (the average specific gravity of diluting liquids employed in floatation techniques) caused to float 77.7%, 78.4% and 6.6% of the G. duodenalis cysts obtained, respectively, from fresh fecal samples, fecal samples kept under refrigeration, and fecal samples treated with formalin. Cysts obtained both from fresh fecal samples and fecal samples kept under refrigeration presented similar results concerning floatability. It was observed, however, that the treatment of feces with formalin diminished the cysts floatability under the various specific gravities studied. This results should influence, the recommendations for transport and storage of fecal samples used for parasitological coproscopy.

Male-specific coliphages for source tracking fecal contamination in surface waters and prevalence of Shiga-toxigenic Escherichia coli in a major produce production region of the Central Coast of California.

PubMed

Ravva, Subbarao V; Sarreal, Chester Z; Cooley, Michael B

2015-07-01

To provide data for traditional trace-back studies from fork to farm, it is necessary to determine the environmental sources for Shiga-toxigenic Escherichia coli. We developed SYBR green based reverse-transcriptase PCR methods to determine the prevalence of F+ RNA coliphages (FRNA) as indicators of fecal contamination. Male-specific coliphages, determined using a single-agar overlay method, were prevalent in all surface waters sampled for 8 months. F+ DNA coliphages (FDNA) were predominant compared to FRNA in water samples from majority of sampling locations. Most (90%) of the FRNA were sourced to humans and originated from human-impacted sites. Members of genogroup III represented 77% of FRNA originated from human sources. Furthermore, 93% of FRNA sourced to animals were also detected in water samples from human-impacted sites. Eighty percent of all FRNA were isolated during the winter months indicating seasonality in prevalence. In contrast, FDNA were more prevalent during summer months. E. coli O157:H7 and Shiga-toxigenic E. coli were detected in water samples from locations predominantly influenced by agriculture. Owing to their scarcity, their numbers could not be correlated with the prevalence of FRNA or FDNA in water samples. Both coliform bacteria and generic E. coli from agricultural or human-impacted sites were similar in numbers and thus could not be used to determine the sources of fecal contamination. Data on the prevalence of male-specific coliphages may be invaluable for predicting the sources of fecal contamination and aid in developing methods to prevent enteric pathogen contamination from likely sources during produce production.

Effects of fecal sampling on preanalytical and analytical phases in quantitative fecal immunochemical tests for hemoglobin.

PubMed

Rapi, Stefano; Berardi, Margherita; Cellai, Filippo; Ciattini, Samuele; Chelazzi, Laura; Ognibene, Agostino; Rubeca, Tiziana

2017-07-24

Information on preanalytical variability is mandatory to bring laboratories up to ISO 15189 requirements. Fecal sampling is greatly affected by lack of harmonization in laboratory medicine. The aims of this study were to obtain information on the devices used for fecal sampling and to explore the effect of different amounts of feces on the results from the fecal immunochemical test for hemoglobin (FIT-Hb). Four commercial sample collection devices for quantitative FIT-Hb measurements were investigated. The volume of interest (VOI) of the probes was measured from diameter and geometry. Quantitative measurements of the mass of feces were carried out by gravimetry. The effects of an increased amount of feces on the analytical environment were investigated measuring the Hb values with a single analytical method. VOI was 8.22, 7.1 and 9.44 mm3 for probes that collected a target of 10 mg of feces, and 3.08 mm3 for one probe that targeted 2 mg of feces. The ratio between recovered and target amounts of devices ranged from 56% to 121%. Different changes in the measured Hb values were observed, in adding increasing amounts of feces in commercial buffers. The amounts of collected materials are related to the design of probes. Three out 4 manufacturers declare the same target amount using different sampling volumes and obtaining different amounts of collected materials. The introduction of a standard probes to reduce preanalytical variability could be an useful step for fecal test harmonization and to fulfill the ISO 15189 requirements.

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants

NASA Technical Reports Server (NTRS)

Munoz, E. F.; Silverman, M. P.

1979-01-01

A single-step most-probable-number method for determining the number of fecal coliform bacteria present in sewage treatment plant effluents is discussed. A single growth medium based on that of Reasoner et al. (1976) and consisting of 5.0 gr. proteose peptone, 3.0 gr. yeast extract, 10.0 gr. lactose, 7.5 gr. NaCl, 0.2 gr. sodium lauryl sulfate, and 0.1 gr. sodium desoxycholate per liter is used. The pH is adjusted to 6.5, and samples are incubated at 44.5 deg C. Bacterial growth is detected either by measuring the increase with time in the electrical impedance ratio between the innoculated sample vial and an uninnoculated reference vial or by visual examination for turbidity. Results obtained by the single-step method for chlorinated and unchlorinated effluent samples are in excellent agreement with those obtained by the standard method. It is suggested that in automated treatment plants impedance ratio data could be automatically matched by computer programs with the appropriate dilution factors and most probable number tables already in the computer memory, with the corresponding result displayed as fecal coliforms per 100 ml of effluent.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

EPA Science Inventory

Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

EPA Science Inventory

Culture- and PCR-based measurements of fecal pollution were determined and compared to hydrologic and land use indicators. Stream water samples (n = 235) were collected monthly over a two year period from ten streams draining headwatersheds with different land use intensities ra...

A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR

PubMed Central

Umhang, Gérald; Bastien, Matthieu; Renault, Camille; Faisse, Marine; Caillot, Christophe; Boucher, Jean-Marc; Hormaz, Vanessa; Poulle, Marie-Lazarine; Boué, Franck

2017-01-01

Soil can be a source of human infection by many zoonotic helminth species including Echinococcus multilocularis and Toxocara spp. The prevention of alveolar echinococcosis could be greatly improved through the identification of at-risk areas. Yet very few data are available about the detection of E. multilocularis in soil, while more studies have been reported for Toxocara spp. Identification of soil contamination by E. multilocularis eggs requires the use of specific methods. This study describes the development of a method for the detection of E. multilocularis in soil samples with the concentration of eggs using a flotation/sieving method and detection by duplex real-time polymerase chain reaction (PCR). Toxocara spp. egg detection was also undertaken due to the widespread presence of this parasite in soil, despite it being considered less pathogenic. Method sensitivity of 100% was reached for the detection of 10 E. multilocularis eggs spiked in 10 g of soil. Concerning Toxocara spp., method sensitivity was lower but assumed to be due to the reduced effectiveness of the DNA extraction protocol. The parasitological status for E. multilocularis and Toxocara spp. of 63 carnivore fecal samples collected in highly endemic rural areas of France and of soil samples collected under and near these fecal samples was compared. The contamination of soil samples collected under positive fecal samples for E. multilocularis (n = 3) or Toxocara spp. (n = 19) confirmed the transfer of eggs from the definitive host to the environment. PMID:28737135

Effects of receiving-water quality and wastewater treatment on injury, survival, and regrowth of fecal-indicator bacteria and implications for assessment of recreational water quality

USGS Publications Warehouse

Francy, D.S.; Hart, T.L.; Virosteck, C.M.

1996-01-01

Bacterial injury, survival, and regrowth were investigated by use of replicate flow-through incubation chambers placed in the Cuyahoga River or Lake Erie in the greater Cleveland metropolitan area during seven 4-day field studies. The chambers contained wastewater or combined-sewer-overflow (CSO) effluents treated three ways-unchlorinated, chlorinated, and dechlorinated. At timestep intervals, the chamber contents were analyzed for concentrations of injured and healthy fecal coliforms by use of standard selective and enhanced-recovery membrane-filtration methods. Mean percent injuries and survivals were calculated from the fecal-coliform concentration data for each field study. The results of analysis of variance (ANOVA) indicated that treatment affected mean percent injury and survival, whereas site did not. In the warm-weather Lake Erie field study, but not in the warm-weather Cuyahoga River studies, the results of ANOVA indicated that dechlorination enhanced the repair of injuries and regrowth of chlorine-injured fecal coliforms on culture media over chlorination alone. The results of ANOVA on the percent injury from CSO effluent field studies indicated that dechlorination reduced the ability of organisms to recover and regrow on culture media over chlorination alone. However, because of atypical patterns of concentration increases and decreases in some CSO effluent samples, more work needs to be done before the effect of dechlorination and chlorination on reducing fecal-coliform concentrations in CSO effluents can be confirmed. The results of ANOVA on percent survivals found statistically significant differences among the three treatment methods for all but one study. Dechlorination was found to be less effective than chlorination alone in reducing the survival of fecal coliforms in wastewater effluent, but not in CSO effluent. If the concentration of fecal coliforms determined by use of the enhanced-recovery method can be predicted accurately from the concentration found by use of the standard method, then increased monitoring and expense to detect chlorine-injured organisms would be unnecessary. The results of linear regression analysis, however, indicated that the relation between enhanced-recovery and standard-method concentrations was best represented when the data were grouped by treatment. The model generated from linear regression of the unchlorinated data set provided an accurate estimate of enhanced-recovery concentrations from standard-method concentrations, whereas the models generated from the chlorinated and dechlorinated data sets did not. In addition, evaluation of fecal-coliform concentrations found in field studies in terms of Ohio recreational water-quality standards showed that concentrations obtained by standard and enhanced-recovery methods were not comparable. Sample treatment and analysis methods were found to affect the percentage of samples meeting and exceeding Ohio's bathing-water, primary-contact, and secondary-contact standards. Therefore, determining the health risk of swimming in receiving waters was often difficult without information on enhanced-recovery method concentrations and was especially difficult in waters receiving high proportions of chlorinated or dechlorinated effluents.

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

PubMed Central

Junick, Jana

2012-01-01

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR. PMID:22307308

Impact of exercise on fecal and cecal metabolome over aging: a longitudinal study in rats.

PubMed

Deda, Olga; Gika, Helen; Panagoulis, Theodoros; Taitzoglou, Ioannis; Raikos, Nikolaos; Theodoridis, Georgios

2017-01-01

Physical exercise can reduce adverse conditions during aging, while both exercise and aging act as metabolism modifiers. The present study investigates rat fecal and cecal metabolome alterations derived from exercise during rats' lifespan. Groups of rats trained life-long or for a specific period of time were under study. The training protocol consisted of swimming, 15-18 min per day, 3-5 days per week, with load of 4-0% of rat's weight. Fecal samples and cecal extracts were analyzed by targeted and untargeted metabolic profiling methods (GC-MS and LC-MS/MS). Effects of exercise and aging on the rats' fecal and cecal metabolome were observed. Fecal and cecal metabolomics are a promising field to investigate exercise biochemistry and age-related alterations.

Factors affecting genotyping success in giant panda fecal samples.

PubMed

Zhu, Ying; Liu, Hong-Yi; Yang, Hai-Qiong; Li, Yu-Dong; Zhang, He-Min

2017-01-01

Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH -20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at -20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates ( P < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies.

Factors affecting genotyping success in giant panda fecal samples

PubMed Central

Zhu, Ying; Liu, Hong-Yi; Yang, Hai-Qiong; Li, Yu-Dong

2017-01-01

Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH −20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at −20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates (P < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies. PMID:28560107

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples

PubMed Central

2012-01-01

Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits. PMID:23216873

A Synoptic Study of Fecal-Indicator Bacteria in the Wind River, Bighorn River, and Goose Creek Basins, Wyoming, June-July 2000

USGS Publications Warehouse

Clark, Melanie L.; Gamper, Merry E.

2003-01-01

A synoptic study of fecal-indicator bacteria was conducted during June and July 2000 in the Wind River, Bighorn River, and Goose Creek Basins in Wyoming as part of the U.S. Geological Survey's National Water-Quality Assessment Program for the Yellowstone River Basin. Fecal-coliform concentrations ranged from 2 to 3,000 col/100 mL (colonies per 100 milliliters) for 100 samples, and Escherichia coli concentrations ranged from 1 to 2,800 col/100 mL for 97 samples. Fecal-coliform concentrations exceeded the U.S. Environmental Protection Agency's recommended limit for a single sample for recreational contact with water in 37.0 percent of the samples. Escherichia coli concentrations exceeded the U.S. Environmental Protection Agency's recommended limit for a single sample for moderate use, full-body recreational contact with water in 38.1 percent of the samples and the recommended limit for infrequent use, full-body recreational contact with water in 24.7 percent of the samples. Fecal-indicator-bacteria concentrations varied by basin. Samples from the Bighorn River Basin had the highest median concentrations for fecal coliform of 340 col/100 mL and for Escherichia coli of 300 col/100 mL. Samples from the Wind River Basin had the lowest median concentrations for fecal coliform of 50 col/100 mL and for Escherichia coli of 62 col/100 mL. Fecal-indicator-bacteria concentrations varied by land cover. Samples from sites with an urban land cover had the highest median concentrations for fecal coliform of 540 col/100 mL and for Escherichia coli of 420 col/100 mL. Maximum concentrations for fecal coliform of 3,000 col/100 mL and for Escherichia coli of 2,800 col/100 mL were in samples from sites with an agricultural land cover. The lowest median concentrations for fecal coliform of 130 col/100 mL and for Escherichia coli of 67 col/100 mL were for samples from sites with a forested land cover. A strong and positive relation existed between fecal coliform and Escherichia coli (Spearman's Rho value of 0.976). The majority of the fecal coliforms were Escherichia coli during the synoptic study. Fecal-indicator-bacteria concentrations were not correlated to streamflow, water temperature, dissolved oxygen, pH, specific conduc-tance, and alkalinity. Fecal-indicator-bacteria concentrations were moderately correlated with turbidity (Spearman's Rho values of 0.662 and 0.640 for fecal coliform and Escherichia coli, respectively) and sediment (Spearman's Rho values of 0.628 and 0.636 for fecal coliform and Escherichia coli, respectively). Escherichia coli isolates analyzed by discriminant analysis of ribotype patterns for samples from the Bighorn River at Basin, Wyoming, and Bitter Creek near Garland, Wyoming, in the Bighorn River Basin were determined to be from nonhuman and human sources. Using a confidence interval of 90 percent, more of the isolates from both sites were classified as being from nonhuman than human sources; however, both samples had additional isolates that were classified as unknown sources. --------------------------------------------------------------------------------

Towards establishing a human fecal contamination index in microbial source tracking

EPA Science Inventory

There have been significant advances in development of PCR-based methods to detect source associated DNA sequences (markers), but method evaluation has focused on performance with individual challenge samples. Little attention has been given to integration of multiple samples fro...

Colonization potential to reconstitute a microbe community in patients detected early after fecal microbe transplant for recurrent C. difficile.

PubMed

Kumar, Ranjit; Maynard, Craig L; Eipers, Peter; Goldsmith, Kelly T; Ptacek, Travis; Grubbs, J Aaron; Dixon, Paula; Howard, Donna; Crossman, David K; Crowley, Michael R; Benjamin, William H; Lefkowitz, Elliot J; Weaver, Casey T; Rodriguez, J Martin; Morrow, Casey D

2016-01-13

Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient's gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice. A total of nine samples from three human donors, recipient's pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01%). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8%, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55%, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2-4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7% of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10%. We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.

Trail Creek I: Assessing the Water Quality of Streams using Fecal Indicator Bacteria and Microbial Source Tracking

NASA Astrophysics Data System (ADS)

Saintil, T.; Radcliffe, D. E.; Rasmussen, T. C.; Kannan, A.

2017-12-01

Fecal coliforms are indicators for disease-causing pathogens. The United States Environmental Protection Agency (US. EPA) recommends the use of E. coli and Enterococci because they are highly correlated with pathogenic organisms in recreational waters. This standard method helps to determine the overall water quality and the potential health risks. Studies have shown that it is difficult to estimate the exact sources of fecal contamination because both human and certain animal species contain E. coli and Enterococci in their waste. Certain strains of E. coli and Enterococci are also able to survive outside of their hosts, which should not be the case for an appropriate fecal indicator. As a result, microbial source tracking (MST) studies use gene specific markers to identify the possible contributors to water pollution whether human or animal. Trail Creek is a second-order stream located in Athens-Clarke County, GA. The 33-km2 watershed is approximately 64% forests, 18% pastures and 16% residential communities. Trail Creek is on the TMDL list and an extended study on the relationships between the different factors causing elevated fecal bacteria is needed. Synoptic sampling events were conducted during baseflow conditions at six locations. Storm sampling events (> 8 mm) were captured using automated samplers at two locations. These samplers were equipped with pressure transducers which record stage at 30-minute intervals. The samples were analyzed for fecal coliform, E. Coli and Enterococci. Water quality parameters including temperature, specific conductance, dissolved oxygen, pH, and turbidity were also recorded. Relationships between the parameters and fecal indicator bacteria show inconsistent patterns and high variability. Using quantitative PCR and MST techniques, the human specific marker (HF183) and ruminant marker (Rum2Bac) were used to identify the fecal sources in both baseflow and storm samples. The presence and abundance of the different markers at each site will be presented. The findings will be useful for adopting best management practices and implementing green infrastructure for the reduction of fecal pollution.

Application of F⁺RNA Coliphages as Source Tracking Enteric Viruses on Parsley and Leek Using RT-PCR.

PubMed

Shahrampour, Dina; Yavarmanesh, Masoud; Najafi, Mohammad Bagher Habibi; Mohebbi, Mohebbat

2015-12-01

The objective of this study was to identify sources of fecal contamination in leek and parsley, by using four different F(+)RNA coliphage genogroups (IV, I indicate animal fecal contamination and II, III indicate human fecal contamination). Three different concentrations (10(2), 10(4), 10(6) pfu/ml) of MS2 coliphage were inoculated on the surface of parsley and leek samples for detection of phage recovery efficiency among two methods of elution concentration (PEG-precipitation and Ultracentrifugation) by performing double agar layer (DAL) assay in three replications. Highest recovery of MS2 was observed in PEG method and in 10(6) inoculation concentration. Accordingly, the PEG method was used for washing and isolation of potentially contaminated phages of 30 collected samples (15 samples from the market and 15 samples from the farm). The final solutions of PEG method were tested for the enumeration of plaques by DAL assay. Total RNA was then extracted from recovered phages, and RT-PCR was performed by using four primer sets I, II, III, and IV. Incidence of F(+)RNA coliphages was observed in 12/15 (80 %) and 10/15 (66/6 %) of samples were obtained from farm and market, respectively, using both DAL and RT-PCR test methods. Different genotypes (I, II, and IV) of F(+)RNA coliphages were found in farm samples, while only genotype I was detected in market samples by using the primer sets. Due to the higher frequency of genotype I and IV, the absence of genotype III, and also the low frequency of genotype II, it is concluded that the contamination of vegetable (parsley and leek) in Neyshabour, Iran is most likely originated from animal sources.

Efficient Genome-Wide Sequencing and Low-Coverage Pedigree Analysis from Noninvasively Collected Samples

PubMed Central

Snyder-Mackler, Noah; Majoros, William H.; Yuan, Michael L.; Shaver, Amanda O.; Gordon, Jacob B.; Kopp, Gisela H.; Schlebusch, Stephen A.; Wall, Jeffrey D.; Alberts, Susan C.; Mukherjee, Sayan; Zhou, Xiang; Tung, Jenny

2016-01-01

Research on the genetics of natural populations was revolutionized in the 1990s by methods for genotyping noninvasively collected samples. However, these methods have remained largely unchanged for the past 20 years and lag far behind the genomics era. To close this gap, here we report an optimized laboratory protocol for genome-wide capture of endogenous DNA from noninvasively collected samples, coupled with a novel computational approach to reconstruct pedigree links from the resulting low-coverage data. We validated both methods using fecal samples from 62 wild baboons, including 48 from an independently constructed extended pedigree. We enriched fecal-derived DNA samples up to 40-fold for endogenous baboon DNA and reconstructed near-perfect pedigree relationships even with extremely low-coverage sequencing. We anticipate that these methods will be broadly applicable to the many research systems for which only noninvasive samples are available. The lab protocol and software (“WHODAD”) are freely available at www.tung-lab.org/protocols-and-software.html and www.xzlab.org/software.html, respectively. PMID:27098910

Current and future trends in fecal source tracking and deployment in the Lake Taihu Region of China

NASA Astrophysics Data System (ADS)

Hagedorn, Charles; Liang, Xinqiang

The emerging discipline of microbial and/or chemical source tracking (collectively termed fecal source tracking (FST)) is being used to identify origins of fecal contamination in polluted waters in many countries around the world. FST has developed rapidly because standard methods of measuring contamination in water by enumerating fecal indicator bacteria (FIB) such as fecal coliforms and enterococci do not identify the sources of the contamination. FST is an active area of research and development in both the academic and private sectors and includes: Developing and testing new microbial and chemical FST methods. Determining the geographic application and animal host ranges of existing and emerging FST techniques. Conducting experimental comparisons of FST techniques. Combining direct monitoring of human pathogens associated with waterborne outbreaks and zoonotic pathogens responsible for infections among people, wildlife, or domesticated animals with the use of FST techniques. Applying FST to watershed analysis and coastal environments. Designing appropriate statistical and probability analysis of FST data and developing models for mass loadings of host-specific fecal contamination. This paper includes a critical review of FST with emphasis on the extent to which methods have been tested (especially in comparison with other methods and/or with blind samples), which methods are applicable to different situations, their shortcomings, and their usefulness in predicting public health risk or pathogen occurrence. In addition, the paper addresses the broader question of whether FST and fecal indicator monitoring is the best approach to regulate water quality and protect human health. Many FST methods have only been tested against sewage or fecal samples or isolates in laboratory studies (proof of concept testing) and/or applied in field studies where the “real” answer is not known, so their comparative performance and accuracy cannot be assessed. For FST to be quantitative, stability of ratios between host-specific markers in the environment must be established. In addition, research is needed on the correlation between host-specific markers and pathogens, and survival of markers after waste treatments. As a result of the exclusive emphasis on FIB by regulatory agencies, monitoring and FST development has concentrated on FIB rather than the actual pathogens. A more rational approach to regulating water quality might be to use available epidemiological data to identify pathogens of concern in a particular water body, and then use targeted pathogen monitoring coupled with very specific FST approaches to control the pathogens. Baseline monitoring of FIB would be just one tool among many in this example.

Prevalence and Level of Enterohemorrhagic Escherichia coli in Culled Dairy Cows at Harvest.

PubMed

Stromberg, Zachary R; Lewis, Gentry L; Aly, Sharif S; Lehenbauer, Terry W; Bosilevac, Joseph M; Cernicchiaro, Natalia; Moxley, Rodney A

2016-03-01

The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n = 300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm(2) in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (κ = 0.58, P < 0.001), EHEC O121 (κ = 0.50, P < 0.001), and EHEC O157 (κ = 0.40, P < 0.001). No significant agreement was observed between NS and Atlas results or between culture and Atlas results. Detection of an EHEC serogroup in fecal samples was significantly associated with detection of the same EHEC serogroup in hide samples for EHEC O26 (P = 0.001), EHEC O111 (P = 0.002), EHEC O121 (P < 0.001), and EHEC-6 (P = 0.029) based on NS detection and for EHEC O121 (P < 0.001) based on detection by culture. This study provides evidence that non-O157 EHEC are ubiquitous on hides of culled dairy cattle and that feces are an important source of non-O157 EHEC hide contamination.

BACTERIOLOGICAL ANALYSIS WITH SAMPLING AND SAMPLE PRESERVATION SPECIFICS

EPA Science Inventory

Current federal regulations (40CFR 503) specify that under certain conditions treated municipal biosolids must be analyzed for fecal coliform or salmonellae. The regulations state that representative samples of biosolids must be collected and analyzed using standard methods. Th...

Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers▿

PubMed Central

Mieszkin, Sophie; Furet, Jean-Pierre; Corthier, Gérard; Gourmelon, Michèle

2009-01-01

The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution. PMID:19329663

Chapter 11. Community analysis-based methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Y.; Wu, C.H.; Andersen, G.L.

2010-05-01

Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities appear distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. Inmore » increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.« less

Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds

PubMed Central

ONUMA, Manabu; KAKOGAWA, Masayoshi; YANAGISAWA, Masae; HAGA, Atsushi; OKANO, Tomomi; NEAGARI, Yasuko; OKANO, Tsukasa; GOKA, Koichi; ASAKAWA, Mitsuhiko

2017-01-01

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop–mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required. PMID:28484128

Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage.

PubMed

Carroll, Ian M; Ringel-Kulka, Tamar; Siddle, Jennica P; Klaenhammer, Todd R; Ringel, Yehuda

2012-01-01

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

PubMed Central

Davies, Cheryl M.; Kaucner, Christine; Deere, Daniel; Ashbolt, Nicholas J.

2003-01-01

Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C. parvum loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 102 oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces−1 must be present to be detected. PMID:12732556

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

PubMed Central

Ryu, Hodon; Lu, Jingrang; Vogel, Jason; Elk, Michael; Chávez-Ramírez, Felipe; Ashbolt, Nicholas

2012-01-01

While 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 to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus 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. PMID:22492437

Comparison of fecal pooling strategies for detection of Mycobacterium avium ssp. paratuberculosis in cattle.

PubMed

McKenna, S L B; Ritter, C; Dohoo, I; Keefe, G P; Barkema, H W

2018-05-23

In herds with typical moderate to low within-herd prevalence, testing for Mycobacterium avium ssp. paratuberculosis (MAP), the infectious agent of Johne's disease, will be more cost-effective if individual fecal samples are cultured in composite pools. However, sensitivity to classify a pool containing 1 or more positive individual samples as positive may depend on pool size and number of individual positive samples within a pool. Fecal samples collected from 994 dairy cows sampled at slaughter were cultured to detect MAP. Culturing was done both individually and as composite pooled samples using the TREK ESP Culture System II broth medium (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH). Composite samples consisted of pools containing feces from 3, 5, 8, 10, or 15 cows. The number of individual fecal culture-positive cows within each pool ranged from 0 to 4. Culture of individual fecal samples detected MAP in 36 (3.6%) of the 994 cows. Individual samples that were detected within the first 50 d by TREK ESP Culture System II were more likely to lead to a positive pool result. In total, 840 pooled fecal samples were examined for presence of MAP, and of those, 272 pools actually contained feces from fecal culture-positive cows. The crude sensitivity (proportion of pools that contained at least 1 fecal-positive cow that tested positive) for pools of 3, 5, 8, 10, and 15 was 47, 67, 44, 59, and 39%, respectively. Across pools, an increase of the number of fecal culture-positive samples from 1 to 2 enhanced overall crude sensitivity from 44 to 71%. However, sensitivity did not further increase for pools with 3 or 4 fecal culture-positive samples (63 and 60%, respectively). Additionally, a simulation analysis assessing probability of pooled fecal samples being positive in herds of 50 and 100 cows was conducted. The simulation assumed that 1, 2, or 5 cows per herd were MAP fecal culture-positive and that pools of 5 and 10 were used. This low-prevalence herd simulation indicated that weighted mean herd probabilities of detecting a positive herd ranged between 52 and 99.3%, with the lowest probability for pools of 10 with 1 positive cow in the herd and the highest probability for pools of 5 with 5 positive cows in the herd. However, overall, pools of 5 and 10 had similar diagnostic capabilities, enabling cost savings by utilizing pools of 10. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Relationship Between Microbiota of the Colonic Mucosa vs Feces and Symptoms, Colonic Transit, and Methane Production in Female Patients With Chronic Constipation

PubMed Central

Parthasarathy, Gopanandan; Chen, Jun; Chen, Xianfeng; Chia, Nicholas; O'Connor, Helen M.; Wolf, Patricia G.; Gaskins, H. Rex; Bharucha, Adil E.

2015-01-01

Background & Aims In fecal samples from patients with chronic constipation, the microbiota differs from that of healthy subjects. However, the profiles of fecal microbiota only partially replicate those of the mucosal microbiota. It is not clear whether these differences are caused by variations in diet or colonic transit, or are associated with methane production (measured by breath tests). We compared the colonic mucosal and fecal microbiota in patients with chronic constipation and in healthy subjects to investigate the relationships between microbiota and other parameters. Methods Sigmoid colonic mucosal and fecal microbiota samples were collected from 25 healthy women (controls) and 25 women with chronic constipation and evaluated by 16S ribosomal RNA gene sequencing (average of 49,186 reads/sample). We assessed associations between microbiota (overall composition and operational taxonomic units) and demographic variables, diet, constipation status, colonic transit, and methane production (measured in breath samples after oral lactulose intake). Results Fourteen patients with chronic constipation had slow colonic transit. The profile of the colonic mucosal microbiota differed between constipated patients and controls (P<.05). The overall composition of the colonic mucosal microbiota was associated with constipation, independent of colonic transit (P<.05) and discriminated between patients with constipation and controls with 94% accuracy. Genera from Bacteroidetes were more abundant in the colonic mucosal microbiota of patients with constipation. The profile of the fecal microbiota was associated with colonic transit before adjusting for constipation, age, body mass index, and diet; genera from Firmicutes (Faecalibacterium, Lactococcus, and Roseburia) correlated with faster colonic transit. Methane production was associated with the composition of the fecal microbiota, but not with constipation or colonic transit. Conclusions After adjusting for diet and colonic transit, the profile of the microbiota in the colonic mucosa could discriminate patients with constipation from healthy individuals. The profile of the fecal microbiota was associated with colonic transit and methane production (measured in breath), but not constipation. PMID:26460205

The influence of serial fecal sampling on the diagnosis of giardiasis in humans, dogs, and cats.

PubMed

Uchôa, Flávia Fernandes de Mendonça; Sudré, Adriana Pittella; Macieira, Daniel de Barros; Almosny, Nádia Regina Pereira

2017-08-24

Giardia infection is a common clinical problem in humans and pets. The diagnosis of giardiasis is challenging as hosts intermittently excrete protozoan cysts in their feces. In the present study, we comparatively evaluated two methods of serial fecal sampling in humans, dogs, and cats from Rio de Janeiro, Brazil. The Faust et al. technique was used to examine fecal specimens collected in triplicate from 133 patients (52 humans, 60 dogs, and 21 cats). Specimens from 74 patients were received from the group assigned to carry out sampling on consecutive days - 34 humans, 35 dogs, and 5 cats, and specimens from 59 patients were received from the group assigned to carry out sampling on non-consecutive, separate days - 18 human beings, 25 dogs, and 16 cats. G. duodenalis cysts were found in stools of 30 individuals. Multiple stool sampling resulted in an increase in the number of samples that were positive for Giardia in both groups. The authors therefore conclude that multiple stool sampling increases the sensitivity of the Faust et al . technique to detect G. duodenalis cysts in samples from humans, cats and dogs.

Patterns and sources of fecal coliform bacteria in three streams in Virginia, 1999-2000

USGS Publications Warehouse

Hyer, Kenneth; Moyer, Douglas

2003-01-01

Surface-water impairment by fecal coliform bacteria is a water-quality issue of national scope and importance. In Virginia, more than 175 stream segments are on the Commonwealth's 1998 303(d) list of impaired waters because of elevated concentrations of fecal coliform bacteria. These fecal coliform-impaired stream segments require the development of total maximum daily load (TMDL) and associated implementation plans, but accurate information on the sources contributing these bacteria usually is lacking. The development of defendable fecal coliform TMDLs and management plans can benefit from reliable information on the bacteria sources that are responsible for the impairment. Bacterial source tracking (BST) recently has emerged as a powerful tool for identifying the sources of fecal coliform bacteria that impair surface waters. In a demonstration of BST technology, three watersheds on Virginia's 1998 303(d) list with diverse land-use practices (and potentially diverse bacteria sources) were studied. Accotink Creek is dominated by urban land uses, Christians Creek by agricultural land uses, and Blacks Run is affected by both urban and agricultural land uses. During the 20-month field study (March 1999?October 2000), water samples were collected from each stream during a range of flow conditions and seasons. For each sample, specific conductance, dissolved oxygen concentration, pH, turbidity, flow, and water temperature were measured. Fecal coliform concentrations of each water sample were determined using the membrane filtration technique. Next, Escherichia coli (E. coli) were isolated from the fecal coliform bacteria and their sources were identified using ribotyping (a method of 'genetic fingerprinting'). Study results provide enhanced understanding of the concentrations and sources of fecal coliform bacteria in these three watersheds. Continuum sampling (sampling along the length of the streams) indicated that elevated concentrations of fecal coliform bacteria (maximum observed concentration of 290,000 colonies/100 milliliters (col/100mL) could occur along the entire length of each stream, and that the samples collected at the downstream monitoring station of each stream were generally representative of the entire upstream reach. Seasonal patterns were observed in the base-flow fecal coliform concentrations of all streams; concentrations were typically highest in the summer and lowest in the winter. Fecal coliform concentrations were lowest during periods of base flow (typically 200?2,000 col/100mL) and increased by 3?4 orders of magnitude during storm events (as high as 700,000 col/100mL). Multiple linear regression models were developed to predict fecal coliform concentrations as a function of streamflow and other water-quality parameters. The source tracking technique provided identification of bacteria contributions from diverse sources that included (but were not limited to) humans, cattle, poultry, horses, dogs, cats, geese, ducks, raccoons, and deer. Seasonal patterns were observed in the contributions of cattle and poultry sources. There were relations between the identified sources of fecal coliform bacteria and the land-use practices within each watershed. There were only minor differences in the distribution of bacteria sources between low-flow periods and high-flow periods. A coupled approach that utilized both a large available source library and a smaller, location-specific source library provided the most success in identifying the unknown E. coli isolates. BST data should provide valuable support and guidance for producing more defendable and scientifically rigorous watershed models. Incorporation of these bacteria-source data into watershed management strategies also should result in the selection of more efficient source-reduction scenarios for improving water quality.

Sample treatment optimization for fish stool metabolomics.

PubMed

Hano, Takeshi; Ito, Mana; Ito, Katsutoshi; Uchida, Motoharu

2018-06-07

Gut microbiota play an essential role in an organism's health. The fecal metabolite profiling content reflects these microbiota-mediated physiological changes in various organisms, including fish. Therefore, metabolomics analysis of fish feces should provide insight into the dynamics linking physiology and gut microbiota. However, metabolites are often unstable in aquatic environments, making fecal metabolites difficult to examine in fish. In this study, a novel method using gas chromatography-mass spectrometry (GC-MS) was developed and optimized for the preparation of metabolomics samples from the feces of the marine fish, red sea bream (Pagrus major). The preparation methodology was optimized, focusing on rinsing frequency and rinsing solvent. Feces (collected within 4 h of excretion) were rinsed three times with sterilized 2.5% NaCl solution or 3.0% artificial seawater (ASW). Among the 86 metabolites identified in the NaCl-rinsed samples, 57 showed superior recovery to that in ASW-rinsed samples, indicating that NaCl is a better rinsing solvent, particularly for amino acids, organic acids, and fatty acids. To evaluate rinsing frequency, fecal samples were rinsed with NaCl solution 0, 1, 3, or 5 times. The results indicate that three or more rinses enabled robust and stable detection of metabolites encapsulated within the solid fecal residue. Furthermore, these data suggest that rinsing is unnecessary when studying sugars, amino acids, and sterols, again highlighting the need for appropriate rinsing solvent and frequency. This study provides further insight into the use of fecal samples to evaluate and promote fish health during farming and supports the application of this and similar analyses to study the effects of environmental fluctuations and/or contamination. Copyright © 2018 Elsevier B.V. All rights reserved.

A New Sampling Strategy for the Detection of Fecal Bacteria Integrated with USEPA Method 1622/1623

EPA Science Inventory

USEPA Method 1622/1623 requires the concentration of Cryptosporidium and Giardia from 10 liters of water samples prior to detection. During this process the supernatant is discarded because it is assumed that most protozoa are retained in the filtration and centrifugation steps....

Detection of feline coronavirus in cheetah (Acinonyx jubatus) feces by reverse transcription-nested polymerase chain reaction in cheetahs with variable frequency of viral shedding.

PubMed

Gaffney, Patricia M; Kennedy, Melissa; Terio, Karen; Gardner, Ian; Lothamer, Chad; Coleman, Kathleen; Munson, Linda

2012-12-01

Cheetahs (Acinonyx jubatus) are a highly threatened species because of habitat loss, human conflict, and high prevalence of disease in captivity. An epidemic of feline infectious peritonitis and concern for spread of infectious disease resulted in decreased movement of cheetahs between U.S. zoological facilities for managed captive breeding. Identifying the true feline coronavirus (FCoV) infection status of cheetahs is challenging because of inconsistent correlation between seropositivity and fecal viral shedding. Because the pattern of fecal shedding of FCoV is unknown in cheetahs, this study aimed to assess the frequency of detectable fecal viral shedding in a 30-day period and to determine the most efficient fecal sampling strategy to identify cheetahs shedding FCoV. Fecal samples were collected from 16 cheetahs housed at seven zoological facilities for 30 to 46 consecutive days; the samples were evaluated for the presence of FCoV by reverse transcription-nested polymerase chain reaction (RT-nPCR). Forty-four percent (7/16) of cheetahs had detectable FCoV in feces, and the proportion of positive samples for individual animals ranged from 13 to 93%. Cheetahs shed virus persistently, intermittently, or rarely over 30-46 days. Fecal RT-nPCR results were used to calculate the probability of correctly identifying a cheetah known to shed virus given multiple hypothetical fecal collection schedules. The most efficient hypothetical fecal sample collection schedule was evaluation of five individual consecutive fecal samples, resulting in a 90% probability of identifying a known shedder. Demographic and management risk factors were not significantly associated (P < or = 0.05) with fecal viral shedding. Because some cheetahs shed virus intermittently to rarely, fecal sampling schedules meant to identify all known shedders would be impractical with current tests and eradication of virus from the population unreasonable. Managing the captive population as endemically infected with FCoV may be a more feasible approach.

Identifying fecal pollution sources using 3M(™) Petrifilm (™) count plates and antibiotic resistance analysis in the Horse Creek Watershed in Aiken County, SC (USA).

PubMed

Harmon, S Michele; West, Ryan T; Yates, James R

2014-12-01

Sources of fecal coliform pollution in a small South Carolina (USA) watershed were identified using inexpensive methods and commonly available equipment. Samples from the upper reaches of the watershed were analyzed with 3M(™) Petrifilm(™) count plates. We were able to narrow down the study's focus to one particular tributary, Sand River, that was the major contributor of the coliform pollution (both fecal and total) to a downstream reservoir that is heavily used for recreation purposes. Concentrations of total coliforms ranged from 2,400 to 120,333 cfu/100 mL, with sharp increases in coliform counts observed in samples taken after rain events. Positive correlations between turbidity and fecal coliform counts suggested a relationship between fecal pollution and stormwater runoff. Antibiotic resistance analysis (ARA) compared antibiotic resistance profiles of fecal coliform isolates from the stream to those of a watershed-specific fecal source library (equine, waterfowl, canines, and untreated sewage). Known fecal source isolates and unknown isolates from the stream were exposed to six antibiotics at three concentrations each. Discriminant analysis grouped known isolates with an overall average rate of correct classification (ARCC) of 84.3 %. A total of 401 isolates from the first stream location were classified as equine (45.9 %), sewage (39.4 %), waterfowl (6.2 %), and feline (8.5 %). A similar pattern was observed at the second sampling location, with 42.6 % equine, 45.2 % sewage, 2.8 % waterfowl, 0.6 % canine, and 8.8 % feline. While there were slight weather-dependent differences, the vast majority of the coliform pollution in this stream appeared to be from two sources, equine and sewage. This information will contribute to better land use decisions and further justify implementation of low-impact development practices within this urban watershed.

High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

PubMed Central

Waldron, Anna M.; Galea, Francesca; Whittington, Ann-Michele; Saunders, Vanessa F.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

2014-01-01

Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611–622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand. PMID:24352996

A Rapid and Simple Real-Time PCR Assay for Detecting Foodborne Pathogenic Bacteria in Human Feces.

PubMed

Hanabara, Yutaro; Ueda, Yutaka

2016-11-22

A rapid, simple method for detecting foodborne pathogenic bacteria in human feces is greatly needed. Here, we examined the efficacy of a method that employs a combination of a commercial PCR master mix, which is insensitive to PCR inhibitors, and a DNA extraction method which used sodium dodecyl benzene sulfonate (SDBS), and Tween 20 to counteract the inhibitory effects of SDBS on the PCR assay. This method could detect the target genes (stx1 and stx2 of enterohemorrhagic Escherichia coli, invA of Salmonella Enteritidis, tdh of Vibrio parahaemolyticus, gyrA of Campylobacter jejuni, ceuE of Campylobacter coli, SEA of Staphylococcus aureus, ces of Bacillus cereus, and cpe of Clostridium perfringens) in a fecal suspension containing 1.0 × 10 1 to 1.0 × 10 3 CFU/ml. Furthermore, the assay was neither inhibited nor influenced by individual differences among the fecal samples of 10 subjects or fecal concentration (40-160 mg/ml in the fecal suspension). When we attempted to detect the genes of pathogenic bacteria in 4 actual clinical cases, we found that this method was more sensitive than standard culture method. These results showed that this assay is a rapid, simple detection method for foodborne pathogenic bacteria in human feces.

Comparison of DNA extraction methods for human gut microbial community profiling.

PubMed

Lim, Mi Young; Song, Eun-Ji; Kim, Sang Ho; Lee, Jangwon; Nam, Young-Do

2018-03-01

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

The Right Tool for the Job: Detection of Soil-Transmitted Helminths in Areas Co-endemic for Other Helminths

PubMed Central

Periago, Maria V.; Diniz, Renata C.; Pinto, Simone A.; Yakovleva, Anna; Correa-Oliveira, Rodrigo; Diemert, David J.; Bethony, Jeffrey M.

2015-01-01

Background Due to the recent increased use of the McMaster (MM) fecal egg counting method for assessing benzimidazole drug efficacy for treating soil-transmitted helminth (STH) infections, the aim of the current study was to determine the operational value of including the MM method alongside the Kato-Katz (KK) fecal thick smear to increase the diagnostic sensitivity when STHs are co-endemic with trematode helminths (e.g., Schistosoma mansoni). Methods A cross-sectional study was conducted in school-aged children aged 4-18 years in the northeastern region of the State of Minas Gerais (Brazil), where Necator americanus, Ascaris lumbricoides, Trichuris trichiura, and S. mansoni are co-endemic. One fecal sample from each participant was collected and transported to the field laboratory for analysis. Coprological diagnosis was performed on each fecal sample by three different methods: Formalin-Ether Sedimentation (FES), KK and the MM technique. The diagnostic sensitivity and negative predictive value (NPV) of each technique was calculated using the combination of all three techniques as the composite standard. In order to determine the agreement between the three techniques Fleiss´ kappa was used. Both the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) were calculated using the two quantification techniques (i.e., the MM and KK). Results Fecal samples from 1260 children were analyzed. The KK had higher diagnostic sensitivity than the MM for the detection of both A. lumbricoides (KK 97.3%, MM 69.5%) and hookworm (KK 95.1%, MM 80.8%). The CR of a single dose of mebendazole varied significantly between the KK and MM for both A. lumbricoides (p = 0.016) and hookworm (p = 0.000), with lower rates obtained with the KK. On the other hand, the FECR was very similar between both techniques for both A. lumbricoides and hookworm. Conclusion The MM did not add any diagnostic value over the KK in areas where both STHs and trematodes were co-endemic. The lower sensitivity of the MM would have an important impact on the administration of selective school-based treatment in this area since if only the MM were used, 36 (13.9%) children diagnosed with A. lumbricoides would have gone untreated. PMID:26241329

The presence and near-shore transport of human fecal pollution in Lake Michigan beaches

USGS Publications Warehouse

Molloy, S.L.; Liu, L.B.; Phanikumar, M.S.; Jenkins, T.M.; Wong, M.V.; Rose, J.B.; Whitman, R.L.; Shively, D.A.; Nevers, M.B.

2005-01-01

The Great Lakes are a source of water for municipal, agricultural and industrial use, and support significant recreation, commercial and sport fishing industries. Every year millions of people visit the 500 plus recreational beaches in the Great Lakes. An increasing public health risk has been suggested with increased evidence of fecal contamination at the shoreline. To investigate the transport and fate of fecal pollution at Great Lakes beaches and the health risk associated with swimming at these beaches, the near-shore waters of Mt Baldy Beach, Lake Michigan and Trail Creek, a tributary discharging into the lake were examined for fecal pollution indicators. A model of surf zone hydrodynamics coupled with a transport model with first-order inactivation of pollutant was used to understand the relative importance of different processes operating in the surf zone (e.g. physical versus biological processes). The Enterococcus human fecal pollution marker, which targets a putative virulence factor, the enterococcal surface protein (esp) in Enterococcus faecium, was detected in 2/28 samples (7%) from the tributaries draining into Lake Michigan and in 6/30 samples (20%) from Lake Michigan beaches. Preliminary analysis suggests that the majority of fecal indicator bactateria variation and water quality changes at the beaches can be explained by inputs from the influential stream and hydrometeorological conditions. Using modeling methods to predict impaired water quality may help reduce potential health threats to recreational visitors.

Patterns of Antimicrobial Resistance Observed in Escherichia coli Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water

PubMed Central

Sayah, Raida S.; Kaneene, John B.; Johnson, Yvette; Miller, RoseAnn

2005-01-01

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria. PMID:15746342

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers.

PubMed

Derrien, M; Jardé, E; Gruau, G; Pourcher, A M; Gourmelon, M; Jadas-Hécart, A; Pierson Wickmann, A C

2012-09-01

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5β-cholestan-3β-ol (coprostanol), 5β-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5β-cholestan-3β-ol (24-ethylcoprostanol) and 24-ethyl-5β-cholestan-3α-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)" methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate. Copyright © 2012 Elsevier Ltd. All rights reserved.

Survival of fecal coliforms in dry-composting toilets.

PubMed

Redlinger, T; Graham, J; Corella-Barud, V; Avitia, R

2001-09-01

The dry-composting toilet, which uses neither water nor sewage infrastructure, is a practical solution in areas with inadequate sewage disposal and where water is limited. These systems are becoming increasingly popular and are promoted to sanitize human excreta and to recycle them into fertilizer for nonedible plants, yet there are few data on the safety of this technology. This study analyzed fecal coliform reduction in approximately 90 prefabricated, dry-composting toilets (Sistema Integral de Reciclamiento de Desechos Orgánicos [SIRDOs]) that were installed on the U.S.-Mexico border in Ciudad Juárez, Chihuahua, Mexico. The purpose of this study was to determine fecal coliform reduction over time and the most probable method of this reduction. Biosolid waste samples were collected and analyzed at approximately 3 and 6 months and were classified based on U.S. Environmental Protection Agency standards. Results showed that class A compost (high grade) was present in only 35.8% of SIRDOs after 6 months. The primary mechanism for fecal coliform reduction was found to be desiccation rather than biodegradation. There was a significant correlation (P = 0.008) between classification rating and percent moisture categories of the biosolid samples: drier samples had a greater proportion of class A samples. Solar exposure was critical for maximal class A biosolid end products (P = 0.001). This study only addressed fecal coliforms as an indicator organism, and further research is necessary to determine the safety of composting toilets with respect to other pathogenic microorganisms, some of which are more resistant to desiccation.

Quantitative Real-Time PCR Fecal Source Identification in the ...

EPA Pesticide Factsheets

Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to allow for safe use leading to economic loss, endangerment of local wildlife, and poor conditions for recreational use. In this study, we employ host-associated qPCR methods for human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution combined with high-resolution geographic information system (GIS) land use data and general indicator bacteria measurements to elucidatewater quality spatial and temporal trends. Water samples (n=584) were collected over a 1-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries (Tillamook Basin, OR). A total of 16.6% of samples (n=97) yielded E. coli levels considered impaired based on Oregon Department of Environmental Quality bacteria criteria (406 MPN/100mL). Hostassociated genetic indicators were detected at frequencies of 39.2% (HF183/BacR287), 16.3% (HumM2), 74.6% (Rum2Bac), 13.0% (CowM2), 26.7% (CowM3), 19.8% (DG3), 3.2% (DG37), and 53.4% (GFD) across all water samples (n=584). Seasonal trends in avian, cattle, and human fecal pollution sources were evident over the study area. On a sample site basis, quantitative fecal source identification and

Survival of Fecal Coliforms in Dry-Composting Toilets

PubMed Central

Redlinger, Thomas; Graham, Jay; Corella-Barud, Verónica; Avitia, Raquel

2001-01-01

The dry-composting toilet, which uses neither water nor sewage infrastructure, is a practical solution in areas with inadequate sewage disposal and where water is limited. These systems are becoming increasingly popular and are promoted to sanitize human excreta and to recycle them into fertilizer for nonedible plants, yet there are few data on the safety of this technology. This study analyzed fecal coliform reduction in approximately 90 prefabricated, dry-composting toilets (Sistema Integral de Reciclamiento de Desechos Orgánicos [SIRDOs]) that were installed on the U.S.-Mexico border in Ciudad Juárez, Chihuahua, Mexico. The purpose of this study was to determine fecal coliform reduction over time and the most probable method of this reduction. Biosolid waste samples were collected and analyzed at approximately 3 and 6 months and were classified based on U.S. Environmental Protection Agency standards. Results showed that class A compost (high grade) was present in only 35.8% of SIRDOs after 6 months. The primary mechanism for fecal coliform reduction was found to be desiccation rather than biodegradation. There was a significant correlation (P = 0.008) between classification rating and percent moisture categories of the biosolid samples: drier samples had a greater proportion of class A samples. Solar exposure was critical for maximal class A biosolid end products (P = 0.001). This study only addressed fecal coliforms as an indicator organism, and further research is necessary to determine the safety of composting toilets with respect to other pathogenic microorganisms, some of which are more resistant to desiccation. PMID:11526002

Depletion of Stercobilin in Fecal Matter from a Mouse Model of Autism Spectrum Disorders

PubMed Central

Sekera, Emily R.; Rudolph, Heather L.; Carro, Stephen D.; Morales, Michael J.; Bett, Glenna C. L.; Rasmusson, Randall L.; Wood, Troy D.

2017-01-01

Introduction Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders lacking a clinical biomarker for diagnosis. Emerging evidence shows that intestinal microflora from ASD subjects can be distinguished from controls, suggesting metabolite differences due to the action of intestinal microbes may provide a means for identifying potential biomarkers for ASD. Objectives The aim of this study was to determine if quantitative differences in levels of stercobilin and stercobilinogen, metabolites produced by biological action of intestinal microflora, exist in the fecal matter between an ASD mouse model population and controls. Methods Pairs of fecal samples were collected from two mouse groups, an ASD model group with Timothy syndrome 2 (TS2-NEO) and a gender-matched control group. After centrifugation, supernatant was spiked with an 18O-labeled stercobilin isotopomer and subjected to solid phase extraction for processing. Extracted samples were spotted on a stainless steel plate and subjected to matrix-assisted laser desorption and ionization mass spectrometry using dihydroxybenzoic acid as the matrix (n = 5). Peak areas for bilins and 18O-stercobilin isotopomers were determined in each fecal sample. Results A 40–45% depletion in stercobilin in TS2-NEO fecal samples compared with controls was observed with p < 0.05; a less dramatic depletion was observed for stercobilinogen. Conclusions The results show that stercobilin depletion in feces is observed for an ASD mouse model vs. controls. This may help to explain recent observations of a less diverse microbiome in humans with ASD and may prove helpful in developing a clinical ASD biomarker. PMID:29147105

Evaluation of Porcelain Cup Soil Water Samplers for Bacteriological Sampling1

PubMed Central

Dazzo, Frank B.; Rothwell, Donald F.

1974-01-01

The validity of obtaining soil water for fecal coliform analyses by porcelain cup soil water samplers was examined. Numbers from samples of manure slurry drawn through porcelain cups were reduced 100- to 10,000,000-fold compared to numbers obtained from the external manure slurry, and 65% of the cups yielded coliform-free samples. Fecal coliforms adsorbed to cups apparently were released, thus influencing the counts of subsequent samples. Fecal coliforms persisted in soil water samplers buried in soil and thus could significantly influence the coliform counts of water samples obtained a month later. These studies indicate that porcelain cup soil water samplers do not yield valid water samples for fecal coliform analyses. Images PMID:16349998

Occurrence and distribution of fecal indicator bacteria and gene markers of pathogenic bacteria in Great Lakes tributaries, March-October 2011

USGS Publications Warehouse

Brennan, Angela K.; Johnson, Heather E.; Totten, Alexander R.; Duris, Joseph W.

2015-01-01

Water samples were analyzed for the presence of FIB concentrations (FIB; fecal coliform bacteria, Escherichia coli [E. coli], and enterococci) by using membrane filtration and serial dilution methods. The resulting enrichments from standard culturing of the samples were then analyzed by using polymerase chain reaction (PCR) to determine the occurrence of pathogen gene markers for Shigella species, Campylobacter jejuni and coli, Salmonellaspecies, and pathogenic E. coli, including Shiga toxin-producing E. coli (STEC).

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

PubMed Central

Mellerup, Anders; Ståhl, Marie

2015-01-01

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

Comparison of Cultivation and PCR-Hybridization for Detection of Salmonella in Porcine Fecal and Water Samples†

PubMed Central

Feder, Ingrid; Nietfeld, Jerome C.; Galland, John; Yeary, Teresa; Sargeant, Jan M.; Oberst, Richard; Tamplin, Mark L.; Luchansky, John B.

2001-01-01

A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37°C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viable Salmonella bacteria. Presumptive Salmonella isolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invA gene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement between Salmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study. PMID:11427557

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PubMed Central

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps. PMID:8919784

A simple modification of the Baermann method for diagnosis of strongyloidiasis.

PubMed

Hernández-Chavarría, F; Avendaño, L

2001-08-01

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37 degrees C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.

Utility of Helicobacter spp. associated GFD markers for detecting avian fecal pollution in natural waters of two continents.

PubMed

Ahmed, W; Harwood, V J; Nguyen, K; Young, S; Hamilton, K; Toze, S

2016-01-01

Avian fecal droppings may negatively impact environmental water quality due to the presence of high concentrations of fecal indicator bacteria (FIB) and zoonotic pathogens. This study was aimed at evaluating the performance characteristics and utility of a Helicobacter spp. associated GFD marker by screening 265 fecal and wastewater samples from a range of avian and non-avian host groups from two continents (Brisbane, Australia and Florida, USA). The host-prevalence and -specificity of this marker among fecal and wastewater samples tested from Brisbane were 0.58 and 0.94 (maximum value of 1.00). These values for the Florida fecal samples were 0.30 (host-prevalence) and 1.00 (host-specificity). The concentrations of the GFD markers in avian and non-avian fecal nucleic acid samples were measured at a test concentration of 10 ng of nucleic acid at Brisbane and Florida laboratories using the quantitative PCR (qPCR) assay. The mean concentrations of the GFD marker in avian fecal nucleic acid samples (5.2 × 10(3) gene copies) were two orders of magnitude higher than non-avian fecal nucleic acid samples (8.6 × 10(1) gene copies). The utility of this marker was evaluated by testing water samples from the Brisbane River, Brisbane and a freshwater creek in Florida. Among the 18 water samples tested from the Brisbane River, 83% (n = 18) were positive for the GFD marker, and the concentrations ranged from 6.0 × 10(1)-3.2 × 10(2) gene copies per 100 mL water. In all, 92% (n = 25) water samples from the freshwater creek in Florida were also positive for the GFD marker with concentrations ranging from 2.8 × 10(1)-1.3 × 10(4) gene copies per 100 mL water. Based on the results, it can be concluded that the GFD marker is highly specific to avian host groups, and could be used as a reliable marker to detect the presence and amount of avian fecal pollution in environmental waters. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Factors Influencing Fecal Contamination in Pond of Bangladesh

NASA Astrophysics Data System (ADS)

Knappett, P. S.; Escamilla, V.; Layton, A.; McKay, L. D.; Emch, M.; Mailloux, B. J.; Williams, D. E.; Huq, M. R.; Alam, M.; Farhana, L.; Ferguson, A. S.; Sayler, G. S.; Ahmed, K.; Serre, M. L.; Akita, Y.; Yunus, M.; van Geen, A.

2010-12-01

Occurrence of diarrheal disease in villages in rural Bangladesh remains relatively common, even though many households have switched to tubewell water for drinking and cooking. One factor contributing to this may be exposure to fecal contamination in ponds, which are often used for bathing and fishing. The objective of this study is to determine the dominant sources of fecal pollution in typical ponds and to explore the relationship between local population, latrine density, latrine quality and concentrations of fecal bacteria and pathogens in pond water. Forty-three ponds were sampled and analyzed for E. coli using culture-based methods and for E. coli, Bacteroides and adenovirus using quantitative PCR. Population and sanitation infrastructure were surveyed and compared to levels of pond fecal contamination. Molecular fecal source tracking using Bacteroides, determined that humans were the dominant source of fecal contamination in 79% of the ponds. Ponds directly receiving latrine effluent had the highest concentrations of fecal indicator bacteria. Concentrations of fecal indicator bacteria correlated with population surveyed within a distance of 30-70 m (p<0.01) and total latrines surveyed within 50-70 m (p<0.05). Unsanitary latrines with visible effluent within the pond drainage basin were also significantly correlated to fecal indicator concentrations (p<0.05). The vast majority of the surveyed ponds contained unsafe levels of fecal contamination primarily due to unsanitary latrines, and to lesser extent to sanitary latrines and cattle. Since the majority of fecal pollution is from humans, use of pond water could help explain the persistence of diarrheal disease in rural Bangladesh.

Annual ovarian activity monitored by the noninvasive measurement of fecal concentrations of progesterone and 17β-estradiol metabolites in rusa deer (Rusa timorensis)

PubMed Central

SUDSUKH, Apichaya; TAYA, Kazuyoshi; WATANABE, Gen; WAJJWALKU, Worawidh; THONGPHAKDEE, Ampika; THONGTIP, Nikorn

2016-01-01

To clarify the reproductive cycle of female Rusa deer (Rusa timorensis), the fecal concentrations of progesterone and 17β-estradiol metabolites were measured. Fecal samples were collected on a weekly basis for one year (between October, 2012 and September, 2013) from five healthy adult hinds in Thailand. At the beginning of the study, three hinds were pregnant. Two hinds delivered one healthy offspring, and one hind delivered a stillborn calf. The mating period of Rusa hinds in Thailand is from November to April. In pregnant hinds, fecal progesterone metabolite concentration was high in late pregnancy and abruptly declined to the baseline around parturition, suggesting that the placenta secretes a large amount of progesterone. Fecal 17β-estradiol metabolite concentration remained elevated around the day of parturition. Both concentrations of fecal progesterone and 17β-estradiol metabolites in non-lactating hinds were significantly higher than those in lactating hinds, indicating that ovarian activity of lactating hinds is suppressed by the suckling stimulus of fawn during lactation. The present study demonstrated that monitoring of fecal steroid hormones is useful method for assessing ovarian function in this species. PMID:27570098

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

PubMed

Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

2014-01-01

Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.

Assessment of microbiological contamination of fresh, minimally processed, and ready-to-eat lettuces (Lactuca sativa), Rio de Janeiro State, Brazil.

PubMed

Brandão, Marcelo L L; Almeida, Davi O; Bispo, Fernanda C P; Bricio, Silvia M L; Marin, Victor A; Miagostovich, Marize P

2014-05-01

This study aimed to assess the microbiological contamination of lettuces commercialized in Rio de Janeiro, Brazil, in order to investigate detection of norovirus genogroup II (NoV GII), Salmonella spp., total and fecal coliforms, such as Escherichia coli. For NoV detection samples were processed using the adsorption-elution concentration method associated to real-time quantitative polymerase chain reaction (qPCR). A total of 90 samples of lettuce including 30 whole fresh lettuces, 30 minimally processed (MP) lettuces, and 30 raw ready-to-eat (RTE) lettuce salads were randomly collected from different supermarkets (fresh and MP lettuce samples), food services, and self-service restaurants (RTE lettuce salads), all located in Rio de Janeiro, Brazil, from October 2010 to December 2011. NoV GII was not detected and PP7 bacteriophage used as internal control process (ICP) was recovered in 40.0%, 86.7%, and 76.7% of those samples, respectively. Salmonella spp. was not detected although fecal contamination has been observed by fecal coliform concentrations higher than 10(2) most probable number/g. E. coli was detected in 70.0%, 6.7%, and 30.0% of fresh, MP, and RTE samples, respectively. This study highlights the need to improve hygiene procedures at all stages of vegetable production and to show PP7 bacteriophage as an ICP for recovering RNA viruses' methods from MP and RTE lettuce samples, encouraging the evaluation of new protocols that facilitate the establishment of methodologies for NoV detection in a greater number of food microbiology laboratories. The PP7 bacteriophage can be used as an internal control process in methods for recovering RNA viruses from minimally processed and ready-to-eat lettuce samples. © 2014 Institute of Food Technologists®

Comparison of the Multiple-sample means with composite sample results for fecal indicator bacteria by quantitative PCR and culture

EPA Science Inventory

ABSTRACT: Few studies have addressed the efficacy of composite sampling for measurement of indicator bacteria by QPCR. In this study, composite results were compared to single sample results for culture- and QPCR-based water quality monitoring. Composite results for both methods ...

Effect of pH on fecal recovery of energy derived from volatile fatty acids.

PubMed

Kien, C L; Liechty, E A

1987-01-01

We assessed the effect of pH on volatilization of short-chain fatty acids during lyophilization. Acetic, propionic, valeric, and butyric acids were added to a fecal homogenate in amounts sufficient to raise the energy density by 18-27%. Fecal homogenate samples were either acidified (pH 2.8-3.2), alkalinized (pH 7.9-8.7), or left unchanged (4.0-4.8) prior to lyophilization and subsequent bomb calorimetry. Alkalinizing the fecal samples prevented the 20% loss of energy derived from each of these volatile fatty acids observed in samples either acidified or without pH adjustment. These data suggest that in energy balance studies involving subjects with active colonic fermentation, fecal samples should be alkalinized prior to lyophilization and bomb calorimetry.

Community Structures of Fecal Bacteria in Cattle from Different Animal Feeding Operations▿†

PubMed Central

Shanks, Orin C.; Kelty, Catherine A.; Archibeque, Shawn; Jenkins, Michael; Newton, Ryan J.; McLellan, Sandra L.; Huse, Susan M.; Sogin, Mitchell L.

2011-01-01

The fecal microbiome of cattle plays a critical role not only in animal health and productivity but also in food safety, pathogen shedding, and the performance of fecal pollution detection methods. Unfortunately, most published molecular surveys fail to provide adequate detail about variability in the community structures of fecal bacteria within and across cattle populations. Using massively parallel pyrosequencing of a hypervariable region of the rRNA coding region, we profiled the fecal microbial communities of cattle from six different feeding operations where cattle were subjected to consistent management practices for a minimum of 90 days. We obtained a total of 633,877 high-quality sequences from the fecal samples of 30 adult beef cattle (5 individuals per operation). Sequence-based clustering and taxonomic analyses indicate less variability within a population than between populations. Overall, bacterial community composition correlated significantly with fecal starch concentrations, largely reflected in changes in the Bacteroidetes, Proteobacteria, and Firmicutes populations. In addition, network analysis demonstrated that annotated sequences clustered by management practice and fecal starch concentration, suggesting that the structures of bovine fecal bacterial communities can be dramatically different in different animal feeding operations, even at the phylum and family taxonomic levels, and that the feeding operation is a more important determinant of the cattle microbiome than is the geographic location of the feedlot. PMID:21378055

Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

PubMed Central

Koskey, Amber M.; Fisher, Jenny C.; Traudt, Mary F.; Newton, Ryan J.

2014-01-01

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment. PMID:24242244

Survey of Bovine Enterovirus in Biological and Environmental Samples by a Highly Sensitive Real-Time Reverse Transcription-PCR

PubMed Central

Jiménez-Clavero, Miguel Angel; Escribano-Romero, Estela; Mansilla, Carmen; Gómez, Nuria; Córdoba, Laura; Roblas, Neftal; Ponz, Fernando; Ley, Victoria; Sáiz, Juan-Carlos

2005-01-01

Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination. PMID:16000759

Coccidia of whooping cranes

USGS Publications Warehouse

Forrester, Donald J.; Carpenter, J.W.; Blankinship, D.R.

1978-01-01

Coccidial oocysts were observed in 6 of 19 fecal samples from free-ranging whooping cranes (Grus americana) and 4 of 16 samples from captive whooping cranes. Eimeria gruis occurred in four free-ranging whooping cranes and E. reichenowi in two free-ranging and two captive whooping cranes. Fecal samples from two captive cranes contained oocysts of Isospora lacazei which was considered a spurious parasite. Oocysts of both species of Eimeria were prevalent in fecal samples collected from three free-ranging Canadian sandhill cranes (G. canadensis rowani) from whooping crane wintering grounds in Texas. These coccidia were prevalent also in fecal samples from 14 sandhill cranes (of 4 subspecies) maintained in captivity at the Patuxent Wildlife Research Center in Maryland.

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples

PubMed Central

Sharifdini, Meysam; Mirhendi, Hossein; Ashrafi, Keyhan; Hosseini, Mostafa; Mohebali, Mehdi; Khodadadi, Hossein; Kia, Eshrat Beigom

2015-01-01

This study was performed to evaluate nested polymerase chain reaction (PCR) and real-time PCR methods for detection of Strongyloides stercoralis in fecal samples compared with parasitological methods. A total of 466 stool samples were examined by conventional parasitological methods (formalin ether concentration [FEC] and agar plate culture [APC]). DNA was extracted using an in-house method, and mitochondrial cytochrome c oxidase subunit 1 and 18S ribosomal genes were amplified by nested PCR and real-time PCR, respectively. Among 466 samples, 12.7% and 18.2% were found infected with S. stercoralis by FEC and APC, respectively. DNA of S. stercoralis was detected in 18.9% and 25.1% of samples by real-time PCR and nested PCR, respectively. Considering parasitological methods as the diagnostic gold standard, the sensitivity and specificity of nested PCR were 100% and 91.6%, respectively, and that of real-time PCR were 84.7% and 95.8%, respectively. However, considering sequence analyzes of the selected nested PCR products, the specificity of nested PCR is increased. In general, molecular methods were superior to parasitological methods. They were more sensitive and more reliable in detection of S. stercoralis in comparison with parasitological methods. Between the two molecular methods, the sensitivity of nested PCR was higher than real-time PCR. PMID:26350449

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

PubMed Central

Rothrock, Michael J.; Hiett, Kelli L.; Gamble, John; Caudill, Andrew C.; Cicconi-Hogan, Kellie M.; Caporaso, J. Gregory

2014-01-01

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection.

PubMed

Weingarden, Alexa R; Chen, Chi; Bobr, Aleh; Yao, Dan; Lu, Yuwei; Nelson, Valerie M; Sadowsky, Michael J; Khoruts, Alexander

2014-02-15

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

PubMed

Kistler, Whitney M; Parlos, Julie A; Peper, Steven T; Dunham, Nicholas R; Kendall, Ronald J

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53-0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus)

PubMed Central

Kistler, Whitney M.; Parlos, Julie A.; Peper, Steven T.; Dunham, Nicholas R.; Kendall, Ronald J.

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53–0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population. PMID:27893772

Levels of plasma and fecal glucocorticoid metabolites following an ACTH challenge in male and female coyotes (Canis latrans).

PubMed

Stevenson, Erika T; Gese, Eric M; Neuman-Lee, Lorin A; French, Susannah S

2018-03-01

Knowledge of endocrine stress responses can be advantageous for understanding how animals respond to their environment. One tool in wildlife endocrinology is to measure the adrenocortical activity as a parameter of disturbance of animals. Fecal glucocorticoid metabolites (GCMs) provide a noninvasive assessment of adrenocortical activity. Using an adrenocorticotropic hormone (ACTH) challenge administered to 28 captive coyotes (Canis latrans), we measured the levels of plasma cortisol, and fecal cortisol and corticosterone metabolites (i.e., GCMs). Our goal was to determine the dose-response in the plasma and fecal samples following the injection and determine if there were effects of sex, age, and time of day. Specifically, animals were anesthetized for ~ 90 min with treatment animals intravenously injected with exogenous ACTH and control animals receiving saline. We collected blood samples prior to injection and at 4 different time points post-injection. We also collected fecal samples 2 days pre- and 2 days post-injection to measure fecal GCMs and determine if an endocrine stress response could be detected in fecal samples. We found a definite response in cortisol levels in the plasma for coyotes to the ACTH challenge. There was a response in fecal corticosterone 1 day post-injection, but the control males showed a similar response indicating a handling effect. Fecal cortisol levels did not indicate a response to the ACTH challenge, and were significantly lower than corticosterone concentrations. We also found significant sex, but not age or diurnal, differences in fecal GCMs. Radioimmunoassays for fecal corticosterone levels appeared to be a reliable indicator of physiological stress in coyotes.

Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.

PubMed

Peng, Xin; Yu, Ke-Qiang; Deng, Guan-Hua; Jiang, Yun-Xia; Wang, Yu; Zhang, Guo-Xia; Zhou, Hong-Wei

2013-12-01

Low cost and high throughput capacity are major advantages of using next generation sequencing (NGS) techniques to determine metagenomic 16S rRNA tag sequences. These methods have significantly changed our view of microorganisms in the fields of human health and environmental science. However, DNA extraction using commercial kits has shortcomings of high cost and time constraint. In the present study, we evaluated the determination of fecal microbiomes using a direct boiling method compared with 5 different commercial extraction methods, e.g., Qiagen and MO BIO kits. Principal coordinate analysis (PCoA) using UniFrac distances and clustering showed that direct boiling of a wide range of feces concentrations gave a similar pattern of bacterial communities as those obtained from most of the commercial kits, with the exception of the MO BIO method. Fecal concentration by boiling method affected the estimation of α-diversity indices, otherwise results were generally comparable between boiling and commercial methods. The operational taxonomic units (OTUs) determined through direct boiling showed highly consistent frequencies with those determined through most of the commercial methods. Even those for the MO BIO kit were also obtained by the direct boiling method with high confidence. The present study suggested that direct boiling could be used to determine the fecal microbiome and using this method would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity. © 2013 Elsevier B.V. All rights reserved.

Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

PubMed

Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

2015-01-01

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling

PubMed Central

Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

2015-01-01

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples. PMID:26035837

Using host-associated genetic markers to investigate sources of fecal contamination in two Vermont streams

USGS Publications Warehouse

Medalie, Laura; Matthews, Leslie J.; Stelzer, Erin A.

2011-01-01

The use of host-associated Bacteroidales-based 16S ribosomal ribonucleic acid genetic markers was investigated as a tool for providing information to managers on sources of bacterial impairment in Vermont streams. The study was conducted during 2009 in two watersheds on the U.S. Environmental Protection Agency's 303(d) List of Impaired Waters, the Huntington and the Mettawee Rivers. Streamwater samples collected during high-flow and base-flow conditions were analyzed for concentrations of Escherichia coli (E. coli) and Bacteroidales genetic markers (General AllBac, Human qHF183 and BacHum, Ruminant BoBac, and Canid BacCan) to identify humans, ruminants, and canids as likely or unlikely major sources of fecal contamination. Fecal reference samples from each of the potential source groups, as well as from common species of wildlife, were collected during the same season and from the same watersheds as water samples. The results were combined with data from other states to assess marker cross reaction and to relate marker results to E. coli, the regulated water-quality parameter, with a higher degree of statistical significance. Results from samples from the Huntington River collected under different flow conditions on three dates indicated that humans were unlikely to be a major source of fecal contamination, except for a single positive result at one station that indicated the potential for human sources. Ruminants (deer, moose, cow, or sheep) were potential sources of fecal contamination at all six stations on the Huntington River during one high-flow event and at all but two stations during the other high-flow event. Canids were potential sources of fecal contamination at some stations during two high-flow events, with genetic-marker concentrations in samples from two of the six stations showing consistent positive results for canids for both storm dates. A base-flow sample showed no evidence of major fecal contamination in the Huntington River from humans, ruminants, or canids. Results from samples from the Mettawee River watershed collected during high-flow conditions (12 storm samples on 2 dates at 6 stations) indicated that there was no evidence of fecal contamination from humans in seven samples and possible evidence in five samples. Results for humans were positive for only one station during both storm events. For two of the five samples with evidence for human fecal contamination, results for two different human genetic markers agreed, but results from three samples were inconsistent. In samples from five of the six Mettawee stations, ruminants were a potential source of fecal contamination on at least one of the three sampled dates, including three positive results for the base-flow sample. Yet samples from all of the stations that showed positive results for ruminants did so for only one or two of the three sampled dates. Samples from only one of the six stations gave consistent results, which were negative for ruminants for all three dates. In the Mettawee River base-flow sample, humans were an unlikely source of major fecal contamination. Factors that may influence results and conclusions include the timing of sample collection relative to the storm event; variability of E. coli and Bacteroidales concentrations in fecal reference samples and in water; sampling and analytical errors; the potential cross reactivity of host-associated genetic markers; and different persistence and survival rates of E. coli bacteria and Bacteroidales genetic markers on land, in water, and by season. These factors interfere with the ability to directly relate Bacteroidales concentrations to E. coli concentrations in river samples. It must be recognized that while use of Bacteroidales genetic markers as a source tracking tool coupled with the interpretive approach described in this report cannot be used quantitatively to pinpoint sources, it can be used to exclude potential sources as major contributors to fecal contamination.

Correlation of quantitative PCR for a poultry-specific brevibacterium marker gene with bacterial and chemical indicators of water pollution in a watershed impacted by land application of poultry litter.

PubMed

Weidhaas, Jennifer L; Macbeth, Tamzen W; Olsen, Roger L; Harwood, Valerie J

2011-03-01

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

Conclusions on measurement uncertainty in microbiology.

PubMed

Forster, Lynne I

2009-01-01

Since its first issue in 1999, testing laboratories wishing to comply with all the requirements of ISO/IEC 17025 have been collecting data for estimating uncertainty of measurement for quantitative determinations. In the microbiological field of testing, some debate has arisen as to whether uncertainty needs to be estimated for each method performed in the laboratory for each type of sample matrix tested. Queries also arise concerning the estimation of uncertainty when plate/membrane filter colony counts are below recommended method counting range limits. A selection of water samples (with low to high contamination) was tested in replicate with the associated uncertainty of measurement being estimated from the analytical results obtained. The analyses performed on the water samples included total coliforms, fecal coliforms, fecal streptococci by membrane filtration, and heterotrophic plate counts by the pour plate technique. For those samples where plate/membrane filter colony counts were > or =20, uncertainty estimates at a 95% confidence level were very similar for the methods, being estimated as 0.13, 0.14, 0.14, and 0.12, respectively. For those samples where plate/membrane filter colony counts were <20, estimated uncertainty values for each sample showed close agreement with published confidence limits established using a Poisson distribution approach.

Detection of antibodies against classical swine fever virus in fecal samples from wild boar.

PubMed

Seo, Sang won; Sunwoo, Sun young; Hyun, Bang hoon; Lyoo, Young S

2012-12-28

Classical swine fever (CSF) is a contagious viral disease that affects pigs. Wild boars can play an important epidemiological role in CSF outbreaks. In the past decades, studies conducted in many countries have reported that the CSF virus (CSFV) may persist in wild boar populations. The existence of CSFV in the free-ranging wild boar populations was indirectly confirmed by determining the prevalence of antibodies against CSFV in the serum of hunted wild boars. However, analyzing sero-prevalence in hunted wild boars to study the risk of CSF outbreaks is difficult due to insufficient number of samples, limitation of hunting area and biased age distribution of hunted wild boars. To improve this survey method, we collected feces of wild boars from their habitat and tested them using CSFV antibody enzyme-linked immunosorbent assay (ELISA) and CSF virus neutralization (VN) test. In this study, ELISA was found to be highly sensitive for detecting antibodies against CSFV in fecal samples. Most of doubtful or positive results obtained in CSFV ELISA were confirmed by VN tests. Despite the high coincidence rate of antibody-positive samples between CSFV ELISA and VN test, the possibility of false positive reaction should be considered. In the regional distribution, a fact that antibody-positive fecal and serum samples were found in geographically close area was shown. Hence, presence of antibodies in fecal samples may provide vital information regarding the risk of CSF outbreaks in wild boar groups in geographical proximity. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacteriological and Immunological Profiling of Meconium and Fecal Samples from Preterm Infants: A Two-Year Follow-Up Study

PubMed Central

Gómez, Marta; Moles, Laura; Espinosa-Martos, Irene; Bustos, Gerardo; de Vos, Willem M.; Rodríguez, Juan M.; Fuentes, Susana

2017-01-01

An abnormal colonization pattern of the preterm gut may affect immune maturation and exert a long-term influence on the intestinal bacterial composition and host health. However, follow-up studies assessing the evolution of the fecal microbiota of infants that were born preterm are very scarce. In this work, the bacterial compositions of fecal samples, obtained from sixteen 2-year-old infants were evaluated using a phylogenetic microarray; subsequently, the results were compared with those obtained in a previous study from samples of meconium and feces collected from the same infants while they stayed in the neonatal intensive care unit (NICU). In parallel, the concentration of a wide range of cytokines, chemokines, growth factors and immunoglobulins were determined in meconium and fecal samples. Globally, a higher bacterial diversity and a lower interindividual variability were observed in 2-year-olds’ feces, when compared to the samples obtained during their first days of life. Hospital-associated fecal bacteria, that were dominant during the NICU stay, seemed to be replaced, two years later, by genera, which are usually predominant in the healthy adult microbiome. The immune profile of the meconium and fecal samples differed, depending on the sampling time, showing different immune maturation statuses of the gut. PMID:29186903

Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces.

PubMed

Shridhar, P B; Noll, L W; Shi, X; An, B; Cernicchiaro, N; Renter, D G; Nagaraja, T G; Bai, J

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P > 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

Distribution of human fecal marker GB-124 bacteriophages in urban sewage and reclaimed water of São Paulo city, Brazil.

PubMed

Prado, Tatiana; Bruni, Antônio de Castro; Barbosa, Mikaela Renata Funada; Bonanno, Vilma Marques Santos; Garcia, Suzi Cristina; Sato, Maria Inês Zanoli

2018-04-01

Bacteriophages infecting Bacteroides fragilis GB-124 have been described as potential markers of human fecal contamination in water sources. The aim of this study was to evaluate the occurrence of GB-124 phages in raw sewage, secondary effluents and reclaimed water of the São Paulo city using a low-cost microbial source tracking method. Samples were collected monthly from April 2015 to March 2016 in four municipal wastewater treatment plants that operate with activated sludge processes followed by different tertiary treatments (sand-anthracite filtration, membrane bioreactor/reverse osmosis) and final chlorination. GB-124 phages were detected in 100% of the raw sewage samples, with viral loads varying from 7.5 × 10 3 to 1.32 × 10 6 PFU/L. Virus removal efficiency in activated sludge processes ranged from 1.89 to 2.31 log 10 . Frequencies of phage detection were lower in reclaimed water samples (0-22.2%). The results indicated that GB-124 phage could be a complementary low-cost viral marker for the detection of human fecal pollution in waters impacted with urban sewage in this region. However, the datasets of tertiary effluents resulted in several samples with concentrations below the detection limit (DL ≤1 PFU/mL) suggesting the need to obtain analytical methods with lower DL for greater accuracy of negative results.

Helminth Fauna in Captive European Gray Wolves (Canis lupus lupus) in Germany.

PubMed

Bindke, Johanna Daniela; Springer, Andrea; Böer, Michael; Strube, Christina

2017-01-01

Captive as well as free-ranging wolves, which are currently recolonizing Germany, may harbor a variety of gastrointestinal parasites. This study investigated endoparasites in captive European gray wolves ( Canis lupus lupus ) using coproscopical methods. Fecal samples were collected monthly between October 2012 and November 2013 from 18 wolf enclosures in 14 German zoological gardens, representing 72 individual wolves. In total, 1,041 fecal samples including 26 bulk samples were analyzed by the sedimentation and flotation method. The most frequently detected egg morphotypes included five nematodes [Ancylostomatidae ( Ancylostoma or Uncinaria spp.), Toxocara canis, Toxascaris leonina, Trichuris vulpis , and Capillaria / Eucoleus spp.], one cestode (Taeniidae) and one trematode ( Alaria alata ). 44.76% of all samples were positive for at least one of these egg morphotypes. Overall, Ancylostomatidae showed the highest frequency (30.84% of all samples), followed by Capillaria/Eucoleus spp. (19.88%), Toxocara canis (5.19%), taeniids (3.75%), Trichuris vulpis and Alaria alata (3.65% each), and Toxascaris leonina (1.25%). As fecal samples were collected from the environment and could not be assigned to individual wolves, sample results were combined per zoo and month. General linear mixed models were employed to analyze the effect of season and management factors on the occurrence of Ancylostomatidae, Capillaria/Eucoleus spp., Toxocara canis and taeniids. No statistically significant effect of season was found, whereas anthelmintic treatment negatively affected Ancylostomatidae egg excretion. Detected parasites and their prevalences are comparable to previous studies on wolf parasitism conducted elsewhere in Europe. As many of the most prevalent helminths are of zoonotic importance, routine anthelmintic treatment of captive wolves should be recommended.

Ground-water quality and effects of poultry confined animal feeding operations on shallow ground water, upper Shoal Creek basin, Southwest Missouri, 2000

USGS Publications Warehouse

Mugel, Douglas N.

2002-01-01

Forty-seven wells and 8 springs were sampled in May, October, and November 2000 in the upper Shoal Creek Basin, southwest Missouri, to determine if nutrient concentrations and fecal bacteria densities are increasing in the shallow aquifer as a result of poultry confined animal feeding operations (CAFOs). Most of the land use in the basin is agricultural, with cattle and hay production dominating; the number of poultry CAFOs has increased in recent years. Poultry waste (litter) is used as a source of nutrients on pasture land as much as several miles away from poultry barns.Most wells in the sample network were classified as ?P? wells, which were open only or mostly to the Springfield Plateau aquifer and where poultry litter was applied to a substantial acreage within 0.5 mile of the well both in spring 2000 and in several previous years; and ?Ag? wells, which were open only or mostly to the Springfield Plateau aquifer and which had limited or no association with poultry CAFOs. Water-quality data from wells and springs were grouped for statistical purposes as P1, Ag1, and Sp1 (May 2000 samples) and P2, Ag2, and Sp2 (October or November 2000 samples). The results of this study do not indicate that poultry CAFOs are affecting the shallow ground water in the upper Shoal Creek Basin with respect to nutrient concentrations and fecal bacteria densities. Statistical tests do not indicate that P wells sampled in spring 2000 have statistically larger concentrations of nitrite plus nitrate or fecal indicator bacteria densities than Ag wells sampled during the same time, at a 95-percent confidence level. Instead, the Ag wells had statistically larger concentrations of nitrite plus nitrate and fecal coliform bacteria densities than the P wells.The results of this study do not indicate seasonal variations from spring 2000 to fall 2000 in the concentrations of nutrients or fecal indicator bacteria densities from well samples. Statistical tests do not indicate statistically significant differences at a 95-percent confidence level for nitrite plus nitrate concentrations or fecal indicator bacteria densities between either P wells sampled in spring and fall 2000, or Ag wells sampled in spring and fall 2000. However, analysis of samples from springs shows that fecal streptococcus bacteria densities were statistically smaller in fall 2000 than in spring 2000 at a 95-percent confidence level.Nitrite plus nitrate concentrations in spring 2000 samples ranged from less than the detection level [0.02 mg/L (milligram per liter) as nitrogen] to 18 mg/L as nitrogen. Seven samples from three wells had nitrite plus nitrate concentrations at or larger than the maximum contaminant level (MCL) of 10 mg/L as nitrogen. The median nitrite plus nitrate concentrations were 0.28 mg/L as nitrogen for P1 samples, 4.6 mg/L as nitrogen for Ag1 samples, and 3.9 mg/L as nitrogen for Sp1 samples.Fecal coliform bacteria were detected in 1 of 25 P1 samples and 5 of 15 Ag1 samples. Escherichia coli (E. coli) bacteria were detected in 3 of 24 P1 samples and 1 of 13 Ag1 samples. Fecal streptococcus bacteria were detected in 8 of 25 P1 samples and 6 of 15 Ag1 samples. Bacteria densities in samples from wells ranged from less than 1 to 81 col/100 mL (colonies per 100 milliliters) of fecal coliform, less than 1 to 140 col/100 mL of E. coli, and less than 1 to 130 col/100 mL of fecal streptococcus. Fecal indicator bacteria densities in samples from springs were substantially larger than in samples from wells. In Sp1 samples, bacteria densities ranged from 12 to 3,300 col/100 mL of fecal coliform, 40 to 2,700 col/100 mL of E. coli, and 42 to 3,100 col/100 mL of fecal streptococcus.

Fecal-indicator bacteria in the Allegheny, Monongahela, and Ohio Rivers, near Pittsburgh, Pennsylvania, July-September 2001

USGS Publications Warehouse

Fulton, John W.; Buckwalter, Theodore F.

2004-01-01

This report presents the results of a study by the Allegheny County Health Department (ACHD) and the U.S. Geological Survey (USGS) to determine the concentrations of fecal-indicator bacteria in the Allegheny, Monongahela, and Ohio Rivers (Three Rivers) in Allegheny County, Pittsburgh, Pa. Water-quality samples and river-discharge measurements were collected from July to September 2001 during dry- (72-hour dry antecedent period), mixed-, and wet-weather (48-hour dry antecedent period and at least 0.3 inch of rain in a 6-hour period) conditions at five sampling sites on the Three Rivers in Allegheny County. Water samples were collected weekly to establish baseline conditions and during successive days after three wet-weather events. Water samples were analyzed for fecal-indicator organisms including fecal-coliform (FC) bacteria, Escherichia coli (E. coli), and enterococci bacteria. Water samples were collected by the USGS and analyzed by the ACHD Laboratory. At each site, left-bank and right-bank surface-water samples were collected in addition to a composite sample (discharge-weighted sample representative of the channel cross section as a whole) at each site. Fecal-indicator bacteria reported in bank and composite samples were used to evaluate the distribution and mixing of bacteria-source streams in receiving waters such as the Three Rivers. Single-event concentrations of enterococci, E. coli, and FC during dry-weather events were greater than State and Federal water-quality standards (WQS) in 11, 28, and 28 percent of the samples, respectively; during mixed-weather events, concentrations of fecal-indicator bacteria were greater than WQS in 28, 37, and 43 percent of the samples, respectively; and during wet-weather events, concentrations of fecal-indicator bacteria were greater than WQS in 56, 71, and 81 percent of samples, respectively. Single-event, wet-weather concentrations exceeded those during dry-weather events for all sites except the Allegheny River at Oakmont. For this site, dilution during wet-weather events or the lack of source streams upgradient of the site may have caused this anomaly. Additionally, single-event concentrations of E. coli and FC frequently exceeded the WQS reported during wet-weather events. It is difficult to establish a short-term trend in fecal-indicator bacteria concentrations as a function of time after a wet-weather event due to factors including the spatial variability of sources contributing fecal material, dry-weather discharges, resuspension of bottom sediments, and flow augmentation from reservoirs. Relative to E. coli and enterococci, FC concentrations appeared to decrease with time, which may be attributed to the greater die-off rate for FC bacteria. Fecal-indicator bacteria concentrations at a site are dependent on the spatial distribution of point sources upstream of the station, the time-of-travel, rate of decay, and the degree of mixing and resuspension. Therefore, it is difficult to evaluate whether the left, right, and composite concentrations reported at a particular site are significantly different. To evaluate the significance of the fecal-indicator bacteria concentrations and turbidity reported in grab and composite samples during dry-, mixed-, and wet-weather events, data sets were evaluated using Wilcoxon rank sum tests. Tests were conducted using the fecal-indicator bacteria colonies and turbidity reported for each station for a given weather event. For example, fecal coliform counts reported in the left-bank sample were compared against the right-bank and composite samples, respectively, for the Ohio River at Sewickley site during dry-, mixed-, and wet-weather events. The statistical analyses suggest that, depending on the sampling site, the fecal-bacteria concentrations measured at selected locations vary spatially within a channel (left bank compared to right, right bank compared to composite). The most significant differences occurred between feca

Distribution and variability of fecal-indicator bacteria in Scioto and Olentangy rivers in the Columbus, Ohio, area

USGS Publications Warehouse

Myers, Donna N.

1992-01-01

This report presents the results of a study by the U.S. Geological Survey, in cooperation with the City of Columbus, Ohio, to determine the distribution and variability of fecal-indicator bacteria in Scioto and Olentangy Rivers. Fecal-indicator bacteria are among the contaminants of concern to recreational users of these rivers in the Columbus area. Samples were collected to be analyzed for fecal-coliform and Escherichia coli (E. coli) bacteria and selected water-quality constituents and physical properties at 10 sites-- 4 on the Olentangy River and 6 on the Scioto River during the recreational seasons in 1987, 1988, and 1989. Measurements of streamflow also were made at these sites at various frequencies during base flow and runoff. The concentrations of fecal-coliform and E. coli bacteria in the Scioto and Olentangy Rivers spanned a range of five orders of magnitude, from less than 20 to greater than 2,000,000 col/100 mL (colonies per 100 milliliters). In addition, the concentrations of fecal coliform and E. coli bacteria are well correlated (r=0.97) in the study area. At times, relatively high concentrations, for fecal-indicator bacteria (concentrations greater than 51,000 col/100 mL for fecal-coliform and E. coli ) were found in Olentangy River at Woody Hayes Drive and at Goodale Street, and in Scioto River at Greenlawn Avenue and at Columbus. Intermediate concentrations of fecal-indicator bacteria (from 5,100 to 50,000 col/100 mL for fecal coliform and (from 510 to 50,000 col/100 mL for E. coli ) were found in Scioto River at Town Street and below O'Shaughnessy Dam near Dublin, Ohio, and in Olentangy River at Henderson Road. The lowest (median) concentrations of fecal-indicator bacteria (from 20 to 5,000 col/100 mL for fecal coliform and from 20 to 500 col/100 mL for E. coli ) were found at Olentangy River near Worthington, Ohio, Scioto River at Dublin Road Water Treatment Plant and below Griggs Reservoir. Fecal-coliform concentrations exceeded the geometric mean and single-sample Ohio Water Quality Standards for recreation less frequently than E. coli concentrations. The E. coli numerical water-quality standards are more difficult to meet than the fecal coliform standards because they are as much as an order of magnitude lower in some instances. The geometric mean bathing-water and primary-contact standards for fecal-coliform and E. coli bacteria were exceeded in more samples for Olentangy River at Goodale Street than for any other site. The single-sample bathing-water standard for fecal-coliform bacteria was exceeded in 83 percent of all samples and for E. coli in 91 percent of samples for Olentangy River at Goodale Street. Compared to Olentangy River at Goodale Street, geometric means and single-samples exceeded the bathing-water standards somewhat less frequently for Scioto River at Town Street and far less frequently for Scioto River at Dublin Road Water Treatment Plant. In contrast to results for fecal-indicator bacteria, the differences between sites for pH and for concentrations for total alkalinity, total chloride, total nonfilterable residue, total nitrate plus nitrite as nitrogen, total phosphorus, and total organic carbon were small. The large contribution of streamflow and discharge of fecal-indicator bacteria from Olentangy River to Scioto River has a major effect on the Scioto River downstream from the confluence of Olentangy River during periods of rainfall and runoff. Fecal-indicator discharges were calculated for times before, during, and at 24-hour intervals for 48 to 72 hours after two runoff-producing storms. Fecal-coliform and E. coli concentrations were lower in samples collected before runoff and during receding streamflows at 24- to 48-hours after the storms than in samples collected during runoff. The fecal-indicator discharges entering Scioto River from Olentangy River ranged from 22.6 to nearly 100 percent of the total for two storms studied. Controlling nonpoint, unregulated,

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

PubMed

Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

2017-09-19

Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

PubMed

V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

2016-12-01

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.

Field-based evaluation of a male-specific (F+) RNA coliphage ...

EPA Pesticide Factsheets

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Thus, sensitive, reliable and easy to use methods for the detection of microorganisms are needed to evaluate water quality. In this study, we performed a field evaluation of an anion-exchange resin based platform to concentrate F-RNA coliphages (fecal/enteric virus indicators) from diverse fecally impacted environmental waters. In this platform, F-RNA coliphages are adsorbed to anion-exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase PCR detection. Matrix-dependent inhibition was evaluated using known quantities of spiked F-RNA coliphage genogroups GI, GII, GII and GIV. Detection was successful in 97%, 72%, 85% and 98% of the samples for spiked F-RNA coliphage GI, GII, GIII and GIV, respectively, and was differentially affected by inhibitory properties specific to each water sample. No association between inhibition and the water samples’ physicochemical properties was apparent. Parallel evaluations of the spiked samples with internal amplification control (IAC) reactions (a widely used control to assess inhibition) demonstrated that IAC reaction inhibition was not agreement with that observed for spiked samples, suggesting that testing of spiked samples allows for better assessments of matrix-dependent inhibition. Additionally, the anion-

Antibiotic-Resistant Genes and Pathogens Shed by Wild Deer Correlate with Land Application of Residuals.

PubMed

Rogers, Shane W; Shaffer, Carrie E; Langen, Tom A; Jahne, Michael; Welsh, Rick

2018-03-09

The purpose of this study was to investigate genetic biomarkers of zoonotic enteric pathogens and antibiotic-resistant genes (ARGs) in the feces of white-tailed deer (Odocoileus virginianus) as related to proximity of deer to land that receives livestock manure or human waste biosolid fertilizers. Deer feces were collected in the St. Lawrence River Valley and Adirondack State Park of New York. Campylobacter spp. 16S rDNA was detected in 12 of 232 fecal samples (8 of 33 sites). Salmonellae were cultivated from 2 of 182 fecal samples (2 of 29 sites). Genetic virulence markers for Shiga-like toxin I (stx 1 ) and enterohemolysin (hylA) were each detected in one isolate of Escherichia coli; E. coli O157 was not detected in any of 295 fecal samples. ARGs detected in deer feces included ermB (erythromycin-resistant gene; 9 of 295 fecal samples, 5 of 38 sites), vanA (vancomycin-resistant gene; 93 of 284 samples, 33 of 38 sites), tetQ (tetracycline-resistant gene; 93 of 295 samples, 25 of 38 sites), and sul(I) (sulfonamide-resistant gene; 113 of 292 samples, 28 of 38 sites). Genetic markers of pathogens and ARGs in deer feces were spatially associated with collection near concentrated animal feeding operations (CAFOs; Campylobacter spp., tetQ, and ermB) and land-applied biosolids (tetQ). These results indicate that contact with human waste biosolids or animal manure may be an important method of pathogen and ARG transmission and that deer in proximity to land-applied manure and human waste biosolids pose increased risk to nearby produce and water quality.

Optimization of fecal cytology in the dog: comparison of three sampling methods.

PubMed

Frezoulis, Petros S; Angelidou, Elisavet; Diakou, Anastasia; Rallis, Timoleon S; Mylonakis, Mathios E

2017-09-01

Dry-mount fecal cytology (FC) is a component of the diagnostic evaluation of gastrointestinal diseases. There is limited information on the possible effect of the sampling method on the cytologic findings of healthy dogs or dogs admitted with diarrhea. We aimed to: (1) establish sampling method-specific expected values of selected cytologic parameters (isolated or clustered epithelial cells, neutrophils, lymphocytes, macrophages, spore-forming rods) in clinically healthy dogs; (2) investigate if the detection of cytologic abnormalities differs among methods in dogs admitted with diarrhea; and (3) investigate if there is any association between FC abnormalities and the anatomic origin (small- or large-bowel diarrhea) or the chronicity of diarrhea. Sampling with digital examination (DE), rectal scraping (RS), and rectal lavage (RL) was prospectively assessed in 37 healthy and 34 diarrheic dogs. The median numbers of isolated ( p = 0.000) or clustered ( p = 0.002) epithelial cells, and of lymphocytes ( p = 0.000), differed among the 3 methods in healthy dogs. In the diarrheic dogs, the RL method was the least sensitive in detecting neutrophils, and isolated or clustered epithelial cells. Cytologic abnormalities were not associated with the origin or the chronicity of diarrhea. Sampling methods differed in their sensitivity to detect abnormalities in FC; DE or RS may be of higher sensitivity compared to RL. Anatomic origin or chronicity of diarrhea do not seem to affect the detection of cytologic abnormalities.

Pepper Mild Mottle Virus as an Indicator of Fecal Pollution ▿

PubMed Central

Rosario, Karyna; Symonds, Erin M.; Sinigalliano, Christopher; Stewart, Jill; Breitbart, Mya

2009-01-01

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments. PMID:19767474

Influence of Coliform Source on Evaluation of Membrane Filters

PubMed Central

Brodsky, M. H.; Schiemann, D. A.

1975-01-01

Four brands of membrane filters were examined for total and fecal coliform recovery performance by two experimental approaches. Using diluted EC broth cultures of water samples, Johns-Manville filters were superior to Sartorius filters for fecal coliform but equivalent for total coliform recovery. Using river water samples, Johns-Manville filters were superior to Sartorius filters for total coliform but equivalent for fecal coliform recovery. No differences were observed between Johns-Manville and Millipore or Millipore and Sartorius filters for total or fecal coliform recoveries using either approach, nor was any difference observed between Millipore and Gelman filters for fecal coliform recovery from river water samples. These results indicate that the source of the coliform bacteria has an important influence on the conclusions of membrane filter evaluation studies. PMID:1106318

Fecal corticosterone reflects serum corticosterone in Florida sandhill cranes.

PubMed

Ludders, J W; Langenberg, J A; Czekala, N M; Erb, H N

2001-07-01

Florida sandhill cranes (Grus canadensis pratensis) were conditioned to confinement 6 hr/day for 7 days. On day 8, each bird's jugular vein was catheterized, blood samples were drawn, and each crane was confined for 6 hr. Using a randomized, restricted cross-over design, cranes were injected intravenously with either 0.9% NaCl solution or ACTH (cosyntropin; Cortrosyn; 0.25 mg). During the 6 hr of confinement, fecal samples (feces and urine) were collected from each of five cranes immediately after defecation. Individual fecal samples were collected approximately at hourly intervals and assayed for corticosterone. We showed previously that serum corticosterone did not vary significantly following saline injection, but peaked significantly 60 min after ACTH injection. Maximal fecal corticosterone concentrations (ng/g) were greater (P < 0.10; median 1087 ng/g) following ACTH stimulation compared to maximal fecal corticosterone concentrations at the end of acclimation (day 7; median 176) and following saline treatment (median 541). In cranes under controlled conditions, fecal corticosterone concentration reflects serum corticosterone levels, fecal corticosterone, Grus canadensis pratensis, sandhill cranes, serum corticosterone levels.

NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER

EPA Science Inventory

Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly...

Using Elderly Educators to Increase Colorectal Cancer Screening.

ERIC Educational Resources Information Center

Weinrich, Sally P.; And Others

1993-01-01

Used elderly educator method for increasing rate of return of fecal occult blood sampling in colorectal screening among 171 socioeconomically disadvantaged older persons. Two methods using elderly educators had overall response rate of more than 60%. Found statistically significant difference between two methods that used elderly educators and two…

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

PubMed Central

Staley, C.; Sadowsky, M. J.; Gyawali, P.; Sidhu, J. P. S.; Palmer, A.; Beale, D. J.; Toze, S.

2015-01-01

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. PMID:26231650

The diagnostic value of a new fecal marker, matrix metalloprotease-9, in different types of inflammatory bowel diseases.

PubMed

Farkas, Klaudia; Saródi, Zoltán; Bálint, Anita; Földesi, Imre; Tiszlavicz, László; Szűcs, Mónika; Nyári, Tibor; Tajti, János; Nagy, Ferenc; Szepes, Zoltán; Bor, Renáta; Annaházi, Anita; Róka, Richárd; Molnár, Tamás

2015-03-01

Only limited data are available regarding the diagnostic accuracy of fecal matrix metalloprotease-9 [MMP-9] for inflammatory bowel disease [IBD]. The aims of our study were to assess the diagnostic accuracy of fecal MMP-9 in patients with active Crohn's disease [CD], ulcerative colitis [UC], and pouchitis, and to compare the diagnostic accuracy of fecal MMP-9 and fecal calprotectin [CP] in IBD. Stool and blood samples were collected in 50 CD, 54 UC, and 34 ileal pouch-anal anastomosis patients before control endoscopies were performed. Biopsies were taken for histologic purposes. The activities of CD, UC, and pouchitis were defined with the use of clinical, endoscopic, and histologic activity scores. Fecal CP and MMP-9 levels were quantified by enzyme-linked immunosorbent assay. Active CD, UC, and pouchitis were detected in 38%, 54%, and 29% of the patients, respectively. A significant correlation was revealed between fecal CP and the clinical activities of CD and UC, and between fecal CP and the endoscopic activity of UC and pouchitis. Fecal MMP-9 did not correlate with any of the activity indices of CD; however, strong associations were shown between fecal MMP-9 and clinical, endoscopic, and histologic activities of both UC and pouchitis. This is the first study assessing the diagnostic accuracy of MMP-9 in different types of IBD. Our results showed that fecal MMP-9 has high sensitivity in the detection of endoscopically active UC and pouchitis. These non-invasive methods help assess intestinal inflammation. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm.

PubMed

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.

Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation, Deep Sequencing, and a Novel Iterative Sequence Classification Algorithm

PubMed Central

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J.; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. PMID:24695106

Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach.

PubMed

Durso, Lisa M; Harhay, Gregory P; Bono, James L; Smith, Timothy P L

2011-02-01

The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using "universal" primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats. Published by Elsevier B.V.

Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies.

PubMed

Song, Se Jin; Amir, Amnon; Metcalf, Jessica L; Amato, Katherine R; Xu, Zhenjiang Zech; Humphrey, Greg; Knight, Rob

2016-01-01

Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

Epidemiology of Salmonella sp. in California cull dairy cattle: prevalence of fecal shedding and diagnostic accuracy of pooled enriched broth culture of fecal samples.

PubMed

Abu Aboud, Omran A; Adaska, John M; Williams, Deniece R; Rossitto, Paul V; Champagne, John D; Lehenbauer, Terry W; Atwill, Robert; Li, Xunde; Aly, Sharif S

2016-01-01

The primary objective of this cross-sectional study was to estimate the crude, seasonal and cull-reason stratified prevalence of Salmonella fecal shedding in cull dairy cattle on seven California dairies. A secondary objective was to estimate and compare the relative sensitivity (Se) and specificity (Sp) for pools of 5 and 10 enriched broth cultures of fecal samples for Salmonella sp. detection. Seven dairy farms located in the San Joaquin Valley of California were identified and enrolled in the study as a convenience sample. Cull cows were identified for fecal sampling once during each season between 2014 and 2015, specifically during spring, summer, fall, and winter, and 10 cows were randomly selected for fecal sampling at the day of their sale. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous four month's herd management. Fecal samples were frozen until testing for Salmonella. After overnight enrichment in liquid broth, pools of enrichment broth (EBP) were created for 5 and 10 samples. All individual and pooled broths were cultured on selective media with putative Salmonella colonies confirmed by biochemical testing before being serogrouped and serotyped. A total of 249 cull cows were enrolled into the study and their fecal samples tested for Salmonella. The survey-weighted period prevalence of fecal shedding of all Salmonella sp. in the cull cow samples across all study herds and the entire study period was 3.42% (N = 249; SE 1.07). The within herd prevalence of Salmonella shed in feces did not differ over the four study seasons (P = 0.074). The Se of culture of EBP of five samples was 62.5% (SE = 17.12), which was not statistically different from the Se of culture of EBP of 10 (37.5%, SE = 17.12, P = 0.48). The Sp of culture of EBP of five samples was 95.24% (SE = 3.29) and for pools of 10 samples was 100.00% (SE = 0). There was no statistical difference between the culture relative specificities of EBP of 5 and 10 (P > 0.99). Our study showed a numerically higher prevalence of Salmonella shedding in the summer, although the results were not significant, most likely due to a lack of power from the small sample size. A higher prevalence in summer months may be related to heat stress. To detect Salmonella, investigators may expect a 62.5% sensitivity for culture of EBP of five, relative to individual fecal sample enrichment and culture. In contrast, culture of EBP of 10 samples resulted in a numerically lower Se. Culture of EBP of size 5 or 10 samples, given similar prevalence and limit of detection, can be expected to yield specificities of 95 and 100%, respectively.

Quality evaluation of processed clay soil samples

PubMed Central

Steiner-Asiedu, Matilda; Harrison, Obed Akwaa; Vuvor, Frederick; Tano-Debrah, Kwaku

2016-01-01

Introduction This study assessed the microbial quality of clay samples sold on two of the major Ghanaian markets. Methods The study was a cross-sectional assessing the evaluation of processed clay and effects it has on the nutrition of the consumers in the political capital town of Ghana. The items for the examination was processed clay soil samples. Results Staphylococcus spp and fecal coliforms including Klebsiella, Escherichia, and Shigella and Enterobacterspp were isolated from the clay samples. Samples from the Kaneshie market in Accra recorded the highest total viable counts 6.5 Log cfu/g and Staphylococcal count 5.8 Log cfu/g. For fecal coliforms, Madina market samples had the highest count 6.5 Log cfu/g and also recorded the highest levels of yeast and mould. For Koforidua, total viable count was highest in the samples from the Zongo market 6.3 Log cfu/g. Central market samples had the highest count of fecal coliforms 4.6 Log cfu/g and yeasts and moulds 6.5 Log cfu/g. “Small” market recorded the highest staphylococcal count 6.2 Log cfu/g. The water activity of the clay samples were low, and ranged between 0.65±0.01 and 0.66±0.00 for samples collected from Koforidua and Accra respectively. Conclusion The clay samples were found to contain Klebsiella spp. Escherichia, Enterobacter, Shigella spp. staphylococcus spp., yeast and mould. These have health implications when consumed. PMID:27642456

Occurrence of fecal coliform bacteria in selected streams in Wyoming, 1990-99

USGS Publications Warehouse

Clark, Melanie L.; Norris, Jodi R.

2000-01-01

The U.S. Geological Survey (USGS), in cooperation with the Wyoming Department of Environmental Quality (WDEQ), is collecting water samples for analysis of fecal coliform bacteria at 18 stream sites as part of a statewide network. Contamination by bacteria of fecal origin in streams where contact recreation is a designated water use is a concern because of potential public-health risk from the presence of enteric pathogens. Fecal coliform concentrations are temporally and spatially variable in Wyoming streams-concentrations ranged from less than 1 to 45,000 colonies per 100 milliliters of water during 1990-99. Fecal coliform concentrations were less than the water-quality criterion of 400 colonies per 100 milliliters in 83 percent of the samples, indicating fecal coliform contamination is not a widespread problem in these Wyoming streams. However, 14 of the 18 monitoring sites had at least one sample in which the fecal coliform concentration exceeded 400 colonies per 100 milliliters at some time during the 10-year period. Fecal coliform concentrations generally are higher during April through September than during October through March. The higher concentrations coincide with the time period when the public-health risk is higher because summer months are when contact recreation use is more likely occurring. Fecal coliform concentrations were positively correlated with discharge and stream temperature and generally were negatively correlated with pH, specific conductance, and dissolved oxygen.

Mycobacterium avium subsp. paratuberculosis from free-ranging deer and rabbits surrounding Minnesota dairy herds

PubMed Central

2005-01-01

Abstract The objectives of this study were to estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) among deer and rabbits surrounding infected and noninfected Minnesota dairy farms using fecal culture, and to describe the frequency that farm management practices were used that could potentially lead to transmission of infection between these species. Fecal samples from cows and the cow environment were collected from 108 Minnesota dairy herds, and fecal pellets from free-ranging white-tailed deer and eastern cottontail rabbits were collected from locations surrounding 114 farms; all samples were tested using bacterial culture. In addition, a questionnaire was administered to 114 herd owners. Sixty-two percent of the dairy herds had at least 1 positive fecal pool or environmental sample. A total of 218 rabbit samples were collected from 90% of the herds, and 309 deer samples were collected from 47% of the herds. On 2 (4%) of the farms sampled, 1 deer fecal sample was MAP positive. Both farms had samples from the cow fecal pool and cow environment that were positive by culture. On 2 (2%) other farms, 1 rabbit fecal sample was positive by culture to MAP, with one of these farms having positive cow fecal pools and cow environmental samples. Pasture was used on 79% of the study farms as a grazing area for cattle, mainly for dry cows (75%) and bred or prebred heifers (87%). Of the 114 farms, 88 (77%) provided access to drylot for their cattle, mainly for milking cows (77/88; 88%) and bred heifers (87%). Of all study farms, 90 (79%) used some solid manure broadcasting on their crop fields. Of all 114 farms, the estimated probability of daily physical contact between cattle manure and deer or rabbits was 20% and 25%, respectively. Possible contact between cattle manure and deer or rabbits was estimated to occur primarily from March through December. The frequency of pasture or drylot use and manure spreading on crop fields may be important risk factors for transmission of MAP among dairy cattle, deer, and rabbits. Although the MAP prevalence among rabbits and deer is low, their role as MAP reservoirs should be considered. PMID:15745220

EVALUATION OF BIOSOLID SAMPLE PROCESSING TECHNIQUES TO MAXIMIZE RECOVERY OF BACTERIA

EPA Science Inventory

Current federal regulations (40 CFR 503) require enumeration of fecal coliform or Salmoella prior to land application of Class A biosolids. This regulation specifies use of enumeration methods included in "Standard Methods for the Examination of Water and Wastewater 18th Edition,...

Plasma Septin9 versus fecal immunochemical testing for colorectal cancer screening: a prospective multicenter study.

PubMed

Johnson, David A; Barclay, Robert L; Mergener, Klaus; Weiss, Gunter; König, Thomas; Beck, Jürgen; Potter, Nicholas T

2014-01-01

Screening improves outcomes related to colorectal cancer (CRC); however, suboptimal participation for available screening tests limits the full benefits of screening. Non-invasive screening using a blood based assay may potentially help reach the unscreened population. To compare the performance of a new Septin9 DNA methylation based blood test with a fecal immunochemical test (FIT) for CRC screening. In this trial, fecal and blood samples were obtained from enrolled patients. To compare test sensitivity for CRC, patients with screening identified colorectal cancer (n = 102) were enrolled and provided samples prior to surgery. To compare test specificity patients were enrolled prospectively (n = 199) and provided samples prior to bowel preparation for screening colonoscopy. Plasma and fecal samples were analyzed using the Epi proColon and OC Fit-Check tests respectively. For all samples, sensitivity for CRC detection was 73.3% (95% CI 63.9-80.9%) and 68.0% (95% CI 58.2-76.5%) for Septin9 and FIT, respectively. Specificity of the Epi proColon test was 81.5% (95% CI 75.5-86.3%) compared with 97.4% (95% CI 94.1-98.9%) for FIT. For paired samples, the sensitivity of the Epi proColon test (72.2% -95% CI 62.5-80.1%) was shown to be statistically non-inferior to FIT (68.0%-95% CI 58.2-76.5%). When test results for Epi proColon and FIT were combined, CRC detection was 88.7% at a specificity of 78.8%. At a sensitivity of 72%, the Epi proColon test is non- inferior to FIT for CRC detection, although at a lower specificity. With negative predictive values of 99.8%, both methods are identical in confirming the absence of CRC. ClinicalTrials.gov NCT01580540.

Combination of Metagenomics and Culture-Based Methods to Study the Interaction Between Ochratoxin A and Gut Microbiota

PubMed Central

Guo, Mingzhang; Huang, Kunlun; Chen, Siyuan; Qi, Xiaozhe; He, Xiaoyun; Cheng, Wen-Hsing; Luo, Yunbo; Xia, Kai; Xu, Wentao

2014-01-01

Gut microbiota represent an important bridge between environmental substances and host metabolism. Here we reported a comprehensive study of gut microbiota interaction with ochratoxin A (OTA), a major food-contaminating mycotoxin, using the combination of metagenomics and culture-based methods. Rats were given OTA (0, 70, or 210 μg/kg body weight) by gavage and fecal samples were collected at day 0 and day 28. Bacterial genomic DNA was extracted from the fecal samples and both 16S rRNA and shotgun sequencing (two main methods of metagenomics) were performed. The results indicated OTA treatment decreased the within-subject diversity of the gut microbiota, and the relative abundance of Lactobacillus increased considerably. Changes in functional genes of gut microbiota including signal transduction, carbohydrate transport, transposase, amino acid transport system, and mismatch repair were observed. To further understand the biological sense of increased Lactobacillus, Lactobacillus selective medium was used to isolate Lactobacillus species from fecal samples, and a strain with 99.8% 16S rRNA similarity with Lactobacillus plantarum strain PFK2 was obtained. Thin-layer chromatography showed that this strain could absorb but not degrade OTA, which was in agreement with the result in metagenomics that no genes related to OTA degradation increased. In conclusion, combination of metagenomics and culture-based methods can be a new strategy to study intestinal toxicity of toxins and find applicable bacterial strains for detoxification. When it comes to OTA, this kind of mycotoxin can cause compositional and functional changes of gut microbiota, and Lactobacillus are key genus to detoxify OTA in vivo. PMID:24973096

Establishing Statistical Equivalence of Data from Different Sampling Approaches for Assessment of Bacterial Phenotypic Antimicrobial Resistance

PubMed Central

2018-01-01

ABSTRACT To assess phenotypic bacterial antimicrobial resistance (AMR) in different strata (e.g., host populations, environmental areas, manure, or sewage effluents) for epidemiological purposes, isolates of target bacteria can be obtained from a stratum using various sample types. Also, different sample processing methods can be applied. The MIC of each target antimicrobial drug for each isolate is measured. Statistical equivalence testing of the MIC data for the isolates allows evaluation of whether different sample types or sample processing methods yield equivalent estimates of the bacterial antimicrobial susceptibility in the stratum. We demonstrate this approach on the antimicrobial susceptibility estimates for (i) nontyphoidal Salmonella spp. from ground or trimmed meat versus cecal content samples of cattle in processing plants in 2013-2014 and (ii) nontyphoidal Salmonella spp. from urine, fecal, and blood human samples in 2015 (U.S. National Antimicrobial Resistance Monitoring System data). We found that the sample types for cattle yielded nonequivalent susceptibility estimates for several antimicrobial drug classes and thus may gauge distinct subpopulations of salmonellae. The quinolone and fluoroquinolone susceptibility estimates for nontyphoidal salmonellae from human blood are nonequivalent to those from urine or feces, conjecturally due to the fluoroquinolone (ciprofloxacin) use to treat infections caused by nontyphoidal salmonellae. We also demonstrate statistical equivalence testing for comparing sample processing methods for fecal samples (culturing one versus multiple aliquots per sample) to assess AMR in fecal Escherichia coli. These methods yield equivalent results, except for tetracyclines. Importantly, statistical equivalence testing provides the MIC difference at which the data from two sample types or sample processing methods differ statistically. Data users (e.g., microbiologists and epidemiologists) may then interpret practical relevance of the difference. IMPORTANCE Bacterial antimicrobial resistance (AMR) needs to be assessed in different populations or strata for the purposes of surveillance and determination of the efficacy of interventions to halt AMR dissemination. To assess phenotypic antimicrobial susceptibility, isolates of target bacteria can be obtained from a stratum using different sample types or employing different sample processing methods in the laboratory. The MIC of each target antimicrobial drug for each of the isolates is measured, yielding the MIC distribution across the isolates from each sample type or sample processing method. We describe statistical equivalence testing for the MIC data for evaluating whether two sample types or sample processing methods yield equivalent estimates of the bacterial phenotypic antimicrobial susceptibility in the stratum. This includes estimating the MIC difference at which the data from the two approaches differ statistically. Data users (e.g., microbiologists, epidemiologists, and public health professionals) can then interpret whether that present difference is practically relevant. PMID:29475868

Establishing Statistical Equivalence of Data from Different Sampling Approaches for Assessment of Bacterial Phenotypic Antimicrobial Resistance.

PubMed

Shakeri, Heman; Volkova, Victoriya; Wen, Xuesong; Deters, Andrea; Cull, Charley; Drouillard, James; Müller, Christian; Moradijamei, Behnaz; Jaberi-Douraki, Majid

2018-05-01

To assess phenotypic bacterial antimicrobial resistance (AMR) in different strata (e.g., host populations, environmental areas, manure, or sewage effluents) for epidemiological purposes, isolates of target bacteria can be obtained from a stratum using various sample types. Also, different sample processing methods can be applied. The MIC of each target antimicrobial drug for each isolate is measured. Statistical equivalence testing of the MIC data for the isolates allows evaluation of whether different sample types or sample processing methods yield equivalent estimates of the bacterial antimicrobial susceptibility in the stratum. We demonstrate this approach on the antimicrobial susceptibility estimates for (i) nontyphoidal Salmonella spp. from ground or trimmed meat versus cecal content samples of cattle in processing plants in 2013-2014 and (ii) nontyphoidal Salmonella spp. from urine, fecal, and blood human samples in 2015 (U.S. National Antimicrobial Resistance Monitoring System data). We found that the sample types for cattle yielded nonequivalent susceptibility estimates for several antimicrobial drug classes and thus may gauge distinct subpopulations of salmonellae. The quinolone and fluoroquinolone susceptibility estimates for nontyphoidal salmonellae from human blood are nonequivalent to those from urine or feces, conjecturally due to the fluoroquinolone (ciprofloxacin) use to treat infections caused by nontyphoidal salmonellae. We also demonstrate statistical equivalence testing for comparing sample processing methods for fecal samples (culturing one versus multiple aliquots per sample) to assess AMR in fecal Escherichia coli These methods yield equivalent results, except for tetracyclines. Importantly, statistical equivalence testing provides the MIC difference at which the data from two sample types or sample processing methods differ statistically. Data users (e.g., microbiologists and epidemiologists) may then interpret practical relevance of the difference. IMPORTANCE Bacterial antimicrobial resistance (AMR) needs to be assessed in different populations or strata for the purposes of surveillance and determination of the efficacy of interventions to halt AMR dissemination. To assess phenotypic antimicrobial susceptibility, isolates of target bacteria can be obtained from a stratum using different sample types or employing different sample processing methods in the laboratory. The MIC of each target antimicrobial drug for each of the isolates is measured, yielding the MIC distribution across the isolates from each sample type or sample processing method. We describe statistical equivalence testing for the MIC data for evaluating whether two sample types or sample processing methods yield equivalent estimates of the bacterial phenotypic antimicrobial susceptibility in the stratum. This includes estimating the MIC difference at which the data from the two approaches differ statistically. Data users (e.g., microbiologists, epidemiologists, and public health professionals) can then interpret whether that present difference is practically relevant. Copyright © 2018 Shakeri et al.

Improving the Understanding of Schistosomiasis among Adolescents in Endemic Areas in Brazil: a Comparison of Educational Methods

PubMed Central

Gazzinelli, Maria Flávia; Lobato, Lucas; Andrade, Gisele; Matoso, Leonardo Ferreira; Diemert, David J.; Gazzinelli, Andréa

2016-01-01

Objective To evaluate the effectiveness of two teaching strategies, both guided by the concept of dialogicity, on adolescents’ knowledge about schistosomiasis and adherence to diagnostic fecal testing. Methods Two teaching strategies related to schistosomiasis were developed, an educational video and group conversation, which were tested in two groups of students aged 10–15 years old. Before and after the intervention, a questionnaire was applied to assess participants' knowledge about schistosomiasis and, after the intervention, two fecal samples were requested from each participant. Comparisons were performed by paired t- and McNemar tests. Results Both strategies resulted in statistically significant improvements in knowledge between the pre- and post-tests. Students who watched the video had a higher return rate of fecal samples and percentage of correct questionnaire answers, mainly on questions about schistosomiasis infection. Conclusion teaching strategies based on dialogue favored the construction of concepts about schistosomiasis that can influence the adoption of positives attitudes related to health. Practical Implications Using teaching strategies based on the concept of dialogicity can favor the increase of knowledge of school age children about schistosomiasis and can influence behavioral change related to health. PMID:27180618

Evaluation of microbiological accumulation capability of the commercial sponge Spongia officinalis var. adriatica (Schmidt) (Porifera, Demospongiae).

PubMed

Stabili, Loredana; Licciano, Margherita; Longo, Caterina; Corriero, Giuseppe; Mercurio, Maria

2008-05-01

This study was carried out to evaluate the microbiological accumulation capability of the demosponge Spongia officinalis var. adriatica. Six microbiological parameters were researched in two sampling periods in the water and in reared sponge samples coming from sites with different degrees of microbial contamination: an off-shore fish farm displaced off the Apulian coast (Southern Adriatic Sea) and a no-impacted area displaced into the Marine Protected Area of Porto Cesareo (Apulian coast-Ionian Sea). We detected the density of culturable heterotrophic bacteria by spread plate on marine agar, total culturable bacteria at 37 degrees C on Plate Count Agar and vibrios on thiosulphate-citrate-bile-sucrose-salt (TCBS) agar. Total and fecal coliforms as well as fecal streptococci concentrations were detected by the MPN method. Bacterial densities were always higher in the sponge homogenates compared with the corresponding seawater in the sampling points and in both sampling periods. As regard vibrios, total culturable bacteria at 37 degrees C and fecal streptococci concentrations, the highest values were observed in the sponge samples coming from the off-shore fish farm during the summer period. The ability of Spongia officinalis var. adriatica to accumulate the microbial pollution indicators suggests that this species can be employed as a bioindicator for monitoring water quality.

Naturally colonized beef cattle populations fed combinations of yeast culture and an ionophore in finishing diets containing dried distiller's grains with solubles had similar fecal shedding of Escherichia coli O157:H7.

PubMed

Swyers, K L; Carlson, B A; Nightingale, K K; Belk, K E; Archibeque, S L

2011-06-01

Beef steers (n = 252) were used to evaluate the effects of dietary supplement on fecal shedding of Escherichia coli O157:H7. Seven pens of 9 steers (63 steers per treatment) were fed diets supplemented with or without yeast culture (YC) or monensin (MON) and their combination (YC × MON). YC and MON were offered at 2.8 g/kg and 33 mg/kg of dry matter intake, respectively. Environmental sponge samples (from each pen floor, feed bunk, and water trough) were collected on day 0. Rectal fecal grab samples were collected on days 0, 28, 56, 84, 110, and 125. Samples were collected and pooled by pen and analyzed for presumptive E. coli O157:H7 colonies, which were confirmed by a multiplex PCR assay and characterized by pulsed-field gel electrophoresis (PFGE) typing. On day 0, E. coli O157:H7 was detected in 7.0% of feed bunk samples and 14.3% of pen floor samples but in none of the water trough samples. The 71.4% prevalence of E. coli O157:H7 in fecal samples on day 0 decreased significantly (P < 0.05) over time. E. coli O157:H7 fecal shedding was not associated with dietary treatment (P > 0.05); however, in cattle fed YC and YC × MON fecal shedding was 0% by day 28. Eight Xba I PFGE subtypes were identified, and a predominant subtype and three closely related subtypes (differing by three or fewer bands) accounted for 78.7% of environmental and fecal isolates characterized. Results from this study indicate that feeding YC to cattle may numerically decrease but not eliminate fecal shedding of E. coli O157:H7 at the onset of treatment and that certain E. coli O157 subtypes found in the feedlot environment may persist in feedlot cattle.

Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies

PubMed Central

Amir, Amnon; Metcalf, Jessica L.; Amato, Katherine R.; Xu, Zhenjiang Zech; Humphrey, Greg

2016-01-01

ABSTRACT Immediate freezing at −20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods—95% ethanol, FTA cards, and the OMNIgene Gut kit—can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive. PMID:27822526

Diet composition and terrestrial prey selection of the Laysan teal on Laysan Island

USGS Publications Warehouse

Reynolds, M.H.; Slotterback, J.W.; Walters, J.R.

2006-01-01

The Laysan teal (Anas laysanensis) is an endangered dabbling duck endemic to the Hawaiian Archipelago but currently restricted to a single breeding population on Laysan Island. We studied its diet using fecal analysis and behavioral observations. Laysan teal fecal samples (N=118) contained prey items in 15 primary prey categories with a mean of 2.9 (range 0-7) taxa per sample. Sixty-two of these fecal samples were quantified with 2,270 prey items identified (mean items per sample 37; range 0-205). Based on fecal analysis and behavioral observations, we learned that the Laysan teal is not strictly a macroinsectivore as previously reported, but consumed seeds, succulent leaves, and algae, in addition to adult and larval diptera, ants, lepidoptera, coleoptera, and Artemia. We compared abundance of invertebrates from two terrestrial foraging substrates, soil and standing vegetation, to the abundance of invertebrate prey items counted in fecal samples collected from these habitats for the same period. In the soil substrate, Laysan teal selected two of the most abundant invertebrates, lepidoptera larvae and coleoptera. In the standing vegetation, Laysan teal selected the most abundant taxa: coleoptera. Amphipods were consumed in proportion to their abundance, and small gastropods (Tornatellides sp.), isopods, and arachnids were avoided or were identified in fecal matter in disproportion to their abundance in the foraging habitat. We compared fecal composition of samples collected in aquatic and terrestrial habitats and detected significant differences in samples' species compositions. The conservation implications of the adult Laysan teal's diet are positive, since results indicate that the Laysan teal are opportunistic insectivores, and exhibit dietary flexibility that includes seeds and other food. Dietary flexibility improves the possibility of successfully reestablishing populations on other predator-free islands.

Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients.

PubMed

Hamad, Ibrahim; Abou Abdallah, Rita; Ravaux, Isabelle; Mokhtari, Saadia; Tissot-Dupont, Hervé; Michelle, Caroline; Stein, Andreas; Lagier, Jean-Christophe; Raoult, Didier; Bittar, Fadi

2018-01-01

Research on the relationship between changes in the gut microbiota and human disease, including AIDS, is a growing field. However, studies on the eukaryotic component of the intestinal microbiota have just begun and have not yet been conducted in HIV-infected patients. Moreover, eukaryotic community profiling is influenced by the use of different methodologies at each step of culture-independent techniques. Herein, initially, four DNA extraction protocols were compared to test the efficiency of each method in recovering eukaryotic DNA from fecal samples. Our results revealed that recovering eukaryotic components from fecal samples differs significantly among DNA extraction methods. Subsequently, the composition of the intestinal eukaryotic microbiota was evaluated in HIV-infected patients and healthy volunteers through clone sequencing, high-throughput sequencing of nuclear ribosomal internal transcribed spacers 1 (ITS1) and 2 (ITS2) amplicons and real-time PCRs. Our results revealed that not only richness (Chao-1 index) and alpha diversity (Shannon diversity) differ between HIV-infected patients and healthy volunteers, depending on the molecular strategy used, but also the global eukaryotic community composition, with little overlapping taxa found between techniques. Moreover, our results based on cloning libraries and ITS1/ITS2 metabarcoding sequencing showed significant differences in fungal composition between HIV-infected patients and healthy volunteers, but without distinct clusters separating the two groups. Malassezia restricta was significantly more prevalent in fecal samples of HIV-infected patients, according to cloning libraries, whereas operational taxonomic units (OTUs) belonging to Candida albicans and Candida tropicalis were significantly more abundant in fecal samples of HIV-infected patients compared to healthy subjects in both ITS subregions. Finally, real-time PCR showed the presence of Microsporidia, Giardia lamblia, Blastocystis and Hymenolepis diminuta in different proportions in fecal samples from HIV patients as compared to healthy individuals. Our work revealed that the use of different sequencing approaches can impact the perceived eukaryotic diversity results of the human gut. We also provide a more comprehensive view of the eukaryotic community in the gut of HIV-infected patients through the complementarity of the different molecular techniques used. Combining these various methodologies may provide a gold standard for a more complete characterization of the eukaryotic microbiome in future studies.

Effects of a fire alarm strobe light on fecal corticosterone metabolite concentrations in mice.

PubMed

Godfrey, Denice; Silverman, Jerald

2009-02-01

The type and location of fire alarms are important considerations in animal facility design. The Guide for the Care and Use of Laboratory Animals recommends minimizing animal exposure to such alarms. Nevertheless, it is often necessary to maintain fire alarms within animal housing or procedural areas. The authors exposed male mice to the flashing strobe light component of a standard fire alarm and evaluated mouse fecal corticosterone concentration, which is known to be an indicator of stress. Mice were exposed to the strobe light for 5 min during either the light or the dark phase of the light:dark cycle. The authors collected fecal samples every 6 h for 24 h before exposing mice to the alarm and every 6 h for 24 h after exposure. Fecal samples taken before exposure (baseline samples) showed a normal circadian pattern of corticosterone metabolite excretion. In fecal samples taken after mice were exposed to the fire alarm, metabolite concentrations did not significantly differ from baseline concentrations over time.

Comparing wastewater chemicals, indicator bacteria concentrations, and bacterial pathogen genes as fecal pollution indicators

USGS Publications Warehouse

Haack, S.K.; Duris, J.W.; Fogarty, L.R.; Kolpin, D.W.; Focazio, M.J.; Furlong, E.T.; Meyer, M.T.

2009-01-01

The objective of this study was to compare fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli [EC], and enterococci [ENT]) concentrations with a wide array of typical organic wastewater chemicals and selected bacterial genes as indicators of fecal pollution in water samples collected at or near 18 surface water drinking water intakes. Genes tested included esp (indicating human-pathogenic ENT) and nine genes associated with various animal sources of shiga-toxin-producing EC (STEC). Fecal pollution was indicated by genes and/or chemicals for 14 of the 18 tested samples, with little relation to FIB standards. Of 13 samples with <50 EC 100 mL-1, human pharmaceuticals or chemical indicators of wastewater treatment plant effluent occurred in six, veterinary antibiotics were detected in three, and stx1 or stx2 genes (indicating varying animal sources of STEC) were detected in eight. Only the EC eaeA gene was positively correlated with FIB concentrations. Human-source fecal pollution was indicated by the esp gene and the human pharmaceutical carbamazepine in one of the nine samples that met all FIB recreational water quality standards. Escherichia coli rfbO157 and stx2c genes, which are typically associated with cattle sources and are of potential human health significance, were detected in one sample in the absence of tested chemicals. Chemical and gene-based indicators of fecal contamination may be present even when FIB standards are met, and some may, unlike FIB, indicate potential sources. Application of multiple water quality indicators with variable environmental persistence and fate may yield greater confidence in fecal pollution assessment and may inform remediation decisions. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

Nutrient digestibility and fecal characteristics are different among captive exotic felids fed a beef-based raw diet.

PubMed

Vester, Brittany M; Burke, Sarah L; Dikeman, Cheryl L; Simmons, Lee G; Swanson, Kelly S

2008-03-01

Nutrient digestibility has not been well characterized in exotic felids. The objective of this experiment was to evaluate differences in nutrient digestibility and fecal characteristics in five large exotic captive felid species, including bobcats, jaguars, cheetahs, Indochinese tigers, and Siberian tigers. All animals were individually housed and adapted to a beef-based raw diet (Nebraska Brand((R)) Special Beef Feline, North Platte, NE) for 16 d. Total fecal collections were conducted from days 17 to 20. Fecal samples were weighed and scored on collection. Diet and fecal samples were evaluated for dry matter, organic matter, protein, fat, and energy to determine total tract digestibility. Fresh fecal samples were collected to determine fecal pH, ammonia, phenol, indole, short-chain fatty acid, and branched-chain fatty acid concentrations. Fecal scores were greater (P<0.01) in Indochinese tigers when compared with all other species, and cheetahs had greater (P<0.01) fecal scores than jaguars and bobcats. Fat digestibility was greater (P<0.01) in Siberian tigers, Indochinese tigers, and bobcats (96%) compared with cheetahs and jaguars (94%). Digestible energy was greater (P<0.05) in bobcats and Indochinese tigers at 93.5 and 92.9%, respectively, compared with cheetahs and jaguars, 91.6%. Fecal pH was greater (P<0.01) in bobcats compared with all other species evaluated. Indole concentrations were greater (P<0.05) in cheetahs and jaguars compared with bobcats and Indochinese tigers. Fecal ammonia concentrations were increased (P<0.05) in cheetahs compared with all other species. The beef-based raw diet was highly digestible; however, differences in fat and digestible energy suggest that species should be considered when determining caloric needs of exotic felids. Zoo Biol 27:126-136, 2008. (c) 2008 Wiley-Liss, Inc.

Bayesian estimation of the sensitivity and specificity of individual fecal culture and Paralisa to detect Mycobacterium avium subspecies paratuberculosis infection in young farmed deer.

PubMed

Stringer, Lesley A; Jones, Geoff; Jewell, Chris P; Noble, Alasdair D; Heuer, Cord; Wilson, Peter R; Johnson, Wesley O

2013-11-01

A Bayesian latent class model was used to estimate the sensitivity and specificity of an immunoglobulin G1 serum enzyme-linked immunosorbent assay (Paralisa) and individual fecal culture to detect young deer infected with Mycobacterium avium subsp. paratuberculosis. Paired fecal and serum samples were collected, between July 2009 and April 2010, from 20 individual yearling (12-24-month-old) deer in each of 20 South Island and 18 North Island herds in New Zealand and subjected to culture and Paralisa, respectively. Two fecal samples and 16 serum samples from 356 North Island deer, and 55 fecal and 37 serum samples from 401 South Island deer, were positive. The estimate of individual fecal culture sensitivity was 77% (95% credible interval [CI] = 61-92%) with specificity of 99% (95% CI = 98-99.7%). The Paralisa sensitivity estimate was 19% (95% CI = 10-30%), with specificity of 94% (95% CI = 93-96%). All estimates were robust to variation of priors and assumptions tested in a sensitivity analysis. The data informs the use of the tests in determining infection status at the individual and herd level.

Longitudinal relationship between fecal culture, fecal quantitative PCR, and milk ELISA in Mycobacterium avium ssp. paratuberculosis-infected cows from low-prevalence dairy herds.

PubMed

Beaver, A; Sweeney, R W; Hovingh, E; Wolfgang, D R; Gröhn, Y T; Schukken, Y H

2017-09-01

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of ruminant Johne's disease, presents a particular challenge with regard to infection mitigation on dairy farms. Diagnostic testing strategies to identify and quantify MAP and associated antibodies are imperfect, and certain facets of the relationship between diagnostic tests remain to be explored. Additional repeated-measures data from known infected animals are needed to complement the body of cross-sectional research on Johne's disease-testing methods. Statistical models that accurately account for multiple diagnostic results while adjusting for the effects of individual animals and herds over time can provide a more detailed understanding of the interplay between diagnostic outcomes. Further, test results may be considered as continuous wherever possible so as to avoid the information loss associated with dichotomization. To achieve a broader understanding of the relationship between diagnostic tests, we collected a large number of repeated fecal and milk samples from 14 infected cows, in addition to bulk milk samples, from 2 low-prevalence dairy herds in the northeast United States. Predominately through the use of mixed linear modeling, we identified strong associations between milk ELISA optical density, fecal quantitative PCR, and fecal culture in individual animals while concurrently adjusting for variables that could alter these relationships. Notably, we uncovered subtleties in the predictive abilities of fecal shedding level on milk ELISA results, with animals categorized as disease progressors reaching higher ELISA optical density levels. Moreover, we observed that spikes in fecal shedding could predict subsequent high ELISA values up to 2 mo later. We also investigated the presence of MAP in individual milk samples via PCR and noted an association between poor udder hygiene and MAP positivity in milk, suggesting some level of environmental contamination. The paucity of positive milk samples and the complete absence of detectable MAP in the bulk tank throughout the study period indicate that contamination of milk with MAP may not be of chief concern in low-prevalence herds. An enhanced understanding of the interrelationships between diagnostic tests can only benefit the development of testing strategies and objectives, which in turn may lessen MAP infection prevalence in dairy herds. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

USE OF A MOLECULAR PROBE ASSAY FOR MONITORING SALMONELLA SPP. IN BIOSOLIDS SAMPLES

EPA Science Inventory

Current federal regulations (40 CFR 503) require enumeration of fecal coliform or salmonellae prior to land application of biosolids. This regulation specifies use of enumeration methods included in "Standard methods for the Examination of Water and Wastewater 18th Edition," (SM)...

Characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, C.H.; Sercu, B.; Van De Werhorst, L.C.

2010-03-01

Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomicmore » units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and a-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.« less

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

PubMed Central

Wu, Cindy H.; Sercu, Bram; Van De Werfhorst, Laurie C.; Wong, Jakk; DeSantis, Todd Z.; Brodie, Eoin L.; Hazen, Terry C.; Holden, Patricia A.; Andersen, Gary L.

2010-01-01

Background Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. Methodology/Principal Findings Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and α-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC∶A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. Conclusions/Significance This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health. PMID:20585654

Applicability of universal Bacteroidales genetic marker for microbial monitoring of drinking water sources in comparison to conventional indicators.

PubMed

Shahryari, A; Nikaeen, M; Khiadani Hajian, M; Nabavi, F; Hatamzadeh, M; Hassanzadeh, A

2014-11-01

Water quality monitoring is essential for the provision of safe drinking water. In this study, we compared a selection of fecal indicators with universal Bacteroidales genetic marker to identify fecal pollution of a variety of drinking water sources. A total of 60 samples were collected from water sources. The microbiological parameters included total coliforms, fecal coliforms, Escherichia coli and fecal streptococci as the fecal indicator bacteria (FIB), Clostridium perfringens and H2S bacteria as alternative indicators, universal Bacteroidales genetic marker as a promising alternative fecal indicator, and Salmonella spp., Shigella spp., and E. coli O157 as pathogenic bacteria. From 60 samples analyzed, Bacteroidales was the most frequently detected indicator followed by total coliforms. However, the Bacteroidales assay failed to detect the marker in nine samples positive for FIB and other alternative indicators. The results of our study showed that the absence of Bacteroidales is not necessarily an evidence of fecal and pathogenic bacteria absence and may be unable to ensure the safety of the water. Further research, however, is required for a better understanding of the use of a Bacteroidales genetic marker as an indicator in water quality monitoring programs.

Molecular Investigations of Bacteroides as Microbial Source Tracking Tools in Southeast Louisiana Watersheds

NASA Astrophysics Data System (ADS)

Schulz, C. J.; Childers, G. W.; Engel, A. S.

2006-12-01

Microbial Source Tracking (MST) is a developing field that is gaining increased attention. MST refers to a host of techniques that discriminates among the origins of fecal material found in natural waters from different sources (e.g. human, livestock, and wildlife) by using microbial indicator species with specificity to only certain host organisms. The development of species-specific molecular markers would allow for better evaluation of public health risks and tracking of nutrient sources impacting a watershed. Although several MST methods have been reported with varying levels of success, few offer general applicability for natural waters due to spatial and temporal constraints associated with these methods. One group of molecular MST markers that show promise for broad environmental applications are molecular 16S rDNA probes for Bacteroides. This method is based on 16S rDNA detection directly from environmental samples without the need for a preliminary cultivation step. In this study we have expanded previous sampling efforts to compile a database of over 1000 partial 16S rRNA Bacteroides genes retrieved from the fecal material of 15 different host species (human, cat, dog, pig, kangaroo). To characterize survival of Bacteroides outside of the host, survival time of the Bacteroides marker was compared to that of E.coli under varying natural environmental conditions (temperature and salinity). Bacteroides displayed a survival curve with shouldering and tailing similar to that of E.coli, but log reduction times differed with treatment. In summary, MST marker stability was identified within host species and the overall Bacteroides community structure correlated to host diet, suggesting that detection of a Bacteroides community could confidently identify fecal contamination point sources. Natural water samples from southeast Louisiana were collected for MST including the Tangipahoa River watershed where the source of fecal contamination has been hotly debated. The Bacteroides tool repeatedly demonstrated the presence of cattle related Bacteroides markers and the absence of human markers.This study is now being expanded to include the entire Lake Pontchartrain Basin.

Methods to improve routine bioassay monitoring for freshly separated, poorly transported plutonium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bihl, D.E.; Lynch, T.P.; Carbaugh, E.H.

1988-09-01

Several human cases involving inhalation of plutonium oxide at Hanford have shown clearance half-times from the lung that are much longer than the 500-day half-time recommended for class Y plutonium in Publication 30 of the International Commission on Radiological Protection(ICRP). The more tenaciously retained material is referred to as super class Y plutonium. The ability to detect super class Y plutonium by current routine bioassay measurements is shown to be poor. Pacific Northwest Laboratory staff involved in the Hanford Internal Dosimetry Program investigated four methods to se if improvements in routine monitoring of workers for fresh super class Y plutoniummore » are feasible. The methods were lung counting, urine sampling, fecal sampling, and use of diethylenetriaminepentaacetate (DTPA) to enhance urinary excretion. Use of DTPA was determined to be not feasible. Routine fecal sampling was found to be feasible but not recommended. Recommendations were made to improve the detection level for routine annual urinalysis and routine annual lung counting. 12 refs., 9 figs., 7 tabs.« less

Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut

PubMed Central

2015-01-01

The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories. PMID:25350865

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR ▿

PubMed Central

Imamovic, Lejla; Ballesté, Elisenda; Jofre, Juan; Muniesa, Maite

2010-01-01

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx2 genes and stx2 variants were detected in the viral fraction of some of the samples after sequencing of stx2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment. PMID:20622134

Fecal-indicator bacteria in the Allegheny, Monongahela, and Ohio Rivers and selected tributaries, Allegheny County, Pennsylvania, 2001-2005

USGS Publications Warehouse

Buckwalter, Theodore F.; Zimmerman, Tammy M.; Fulton, John W.

2006-01-01

Concentrations of fecal-indicator bacteria were determined in 1,027 water-quality samples collected from July 2001 through August 2005 during dry- (72-hour dry antecedent period) and wet-weather (48-hour dry antecedent period and at least 0.3 inch of rain in a 24-hour period) conditions in the Allegheny, Monongahela, and Ohio Rivers (locally referred to as the Three Rivers) and selected tributaries in Allegheny County. Samples were collected at five sampling sites on the Three Rivers and at eight sites on four tributaries to the Three Rivers having combined sewer overflows. Water samples were analyzed for three fecal-indicator organisms fecal coliform, Escherichia coli (E. coli), and enterococci bacteria. Left-bank and right-bank surface-water samples were collected in addition to a cross-section composite sample at each site. Concentrations of fecal coliform, E. coli, and enterococci were detected in 98.6, 98.5, and 87.7 percent of all samples, respectively. The maximum fecal-indicator bacteria concentrations were collected from Sawmill Run, a tributary to the Ohio River; Sawmill Run at Duquesne Heights had concentrations of fecal coliform, E. coli, and enterococci of 410,000, 510,000, and 180,000 col/100 mL, respectively, following a large storm. The samples collected in the Three Rivers and selected tributaries frequently exceeded established recreational standards and criteria for bacteria. Concentrations of fecal coliform exceeded the Pennsylvania water-quality standard (200 col/100 mL) in approximately 63 percent of the samples. Sample concentrations of E. coli and enterococci exceeded the U.S. Environmental Protection Agency (USEPA) water-quality criteria (235 and 61 col/100 mL, respectively) in about 53 and 47 percent, respectively, of the samples. Fecal-indicator bacteria were most strongly correlated with streamflow, specific conductance, and turbidity. These correlations most frequently were observed in samples collected from tributary sites. Fecal-indicator bacteria concentrations and turbidity were correlated to the location of sample collection in the cross section. Most differences were between bank and composite samples; differences between right-bank and left-bank samples were rarely observed. The Allegheny River sites had more significant correlations than the Monongahela or Ohio River sites. Comparisons were made between fecal-indicator bacteria in composite samples collected during dry-weather, wet-weather day-one, wet-weather day-two (tributary sites only), and wet-weather day-three (Three Rivers sites only) events in the Three Rivers and selected tributary sites. The lowest median bacteria concentrations generally were observed in the dry-weather composite samples. All median bacteria concentrations in dry-weather composite samples in the five Three Rivers sites were below water-quality standards and criteria; bacteria concentrations in the upstream tributary sites rarely met all standards or criteria. Only Turtle Creek, Thompson Run, and Chartiers Creek had at least one median bacteria concentration below water-quality standards or criteria. Median bacteria concentrations in the composite samples generally were higher the day after a wet-weather event compared to dry-weather composite samples and other wet-weather composite samples collected. In the five Three Rivers sites, median bacteria concentrations 3 days after a wet-weather event in composite samples tended to fall below the water-quality standards and criteria; in the eight tributary sites, median bacteria concentrations in the dry-weather and wet-weather composite samples generally were above the water-quality standards or criteria. Composite samples collected at the upstream sites on the Three Rivers and selected tributaries generally had lower median bacteria concentrations than composite samples collected at the downstream sites during dry- and wet-weather events. Higher concentrations downstream may be because o

Linking on-farm dairy management practices to storm-flow fecal coliform loading for California coastal watersheds.

PubMed

Lewis, D J; Atwill, E R; Lennox, M S; Hou, L; Karle, B; Tate, K W

2005-08-01

How and where to improve water quality within an agricultural watershed requires data at a spatial scale that corresponds with individual management decision units on an agricultural operation. This is particularly true in the context of water quality regulations, such as Total Maximum Daily Loads (TMDLs), that identify agriculture as one source of non-point source pollution through larger tributary watershed scale and above and below water quality investigations. We have conducted a systems approach study of 10 coastal dairies and ranches to document fecal coliform concentration and loading to surface waters at the management decision unit scale. Water quality samples were collected on a storm event basis from loading units that included: manure management systems; gutters; storm drains; pastures; and corrals and lots. In addition, in-stream samples were collected above and below the dairy facilities and from a control watershed, managed for light grazing and without a dairy facility or human residence and corresponding septic system. Samples were analyzed for fecal coliform concentration by membrane filtration. Instantaneous discharge was measured for each collected sample. Storm runoff was also calculated using the curve number method (SCS, 1985). Results for a representative dairy as well as the entire 10 dairy data set are presented. Fecal coliform concentrations demonstrate high variability both within and between loading units. Fecal coliform concentrations for pastures range from 206 to 2,288,888 cfu/100 ml and for lots from 1,933 to 166,105,000 cfu/100 ml. Mean concentrations for pastures and lots are 121,298 (SE = 62,222) and 3,155,584 (SE = 1,902,713) cfu/100 ml, respectively. Fecal coliform load from units of concentrated animals and manure are significantly more than units such as pastures while storm flow amounts were significantly less. Compared with results from earlier tributary scale studies in the watershed, this systems approach has generated water quality data that is beneficial for management decisions because of its scale and representation of current management activities. These results are facilitating on-farm changes through the cooperative efforts of dairy managers, regulatory agency staff, and sources of technical and financial assistance.

A Validation of Extraction Methods for Noninvasive Sampling of Glucocorticoids in Free-Living Ground Squirrels

PubMed Central

Mateo, Jill M.; Cavigelli, Sonia A.

2008-01-01

Fecal hormone assays provide a powerful tool for noninvasive monitoring of endocrine status in wild animals. In this study we validated a protocol for extracting and measuring glucocorticoids in free-living and captive Belding’s ground squirrels (Spermophilus beldingi). We first compared two commonly used extraction protocols to determine which performed better with commercially available antibodies. We next verified the preferred extraction method by correlating circulating and fecal glucocorticoid measures from a group of individuals over time. For this comparison, we used both a cortisol and a corticosterone antibody to determine which had greater affinity to the fecal metabolites. Cortisol was the primary circulating glucocorticoid, but both hormones were present in well above detectable concentrations in the blood, which does not occur in other sciurids. In addition, the cortisol antibody showed greater binding with the fecal extracts than did the corticosterone antibody. Finally, we used adrenocorticotropic hormone and dexamethasone challenges to demonstrate that changes in adrenal functioning are reflected in changing fecal corticoid levels. These results suggest that our extraction protocol provides a fast, reliable assay of stress hormones in free-living ground squirrels without the confounding influence of short-term rises in glucocorticoid concentrations caused by handling and restraint stress and that it can facilitate ecological and evolutionary studies of stress in wild species. PMID:16228945

[Presence of fecal coliforms, Escherichia coli and DNAse- and coagulase-positive Staphylococcus aureus, in "colonial" cheese sold in the city of Blumenau, Estado de Santa Catarina, Brazil].

PubMed

Reibnitz, M G; Tavares, L B; García, J A

1998-01-01

Twenty cheese samples were collected at Blumenau (SC) and were submitted to analysis in order to verify the presence of fecal coliforms, Escherichia coli and Staphylococcus aureus. Among the 20 samples of cheese, analysis revealed that 70% and 20% respectively, were not within present legal specifications (Norma 001/87-DNVSA) for fecal coliforms and Escherichia coli. For Staphylococcus, 95% of the samples were not within present legal specifications.

Specificity of a Bacteroides thetaiotaomicron marker for human feces

USGS Publications Warehouse

Carson, C.A.; Christiansen, J.M.; Yampara-Iquise, H.; Benson, V.W.; Baffaut, C.; Davis, J.V.; Broz, R.R.; Kurtz, W.B.; Rogers, W.M.; Fales, W.H.

2005-01-01

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese. Copyright ?? 2005, American Society for Microbiology. All Rights Reserved.

Sewage Reflects the Microbiomes of Human Populations

PubMed Central

Newton, Ryan J.; McLellan, Sandra L.; Dila, Deborah K.; Vineis, Joseph H.; Morrison, Hilary G.; Eren, A. Murat

2015-01-01

ABSTRACT Molecular characterizations of the gut microbiome from individual human stool samples have identified community patterns that correlate with age, disease, diet, and other human characteristics, but resources for marker gene studies that consider microbiome trends among human populations scale with the number of individuals sampled from each population. As an alternative strategy for sampling populations, we examined whether sewage accurately reflects the microbial community of a mixture of stool samples. We used oligotyping of high-throughput 16S rRNA gene sequence data to compare the bacterial distribution in a stool data set to a sewage influent data set from 71 U.S. cities. On average, only 15% of sewage sample sequence reads were attributed to human fecal origin, but sewage recaptured most (97%) human fecal oligotypes. The most common oligotypes in stool matched the most common and abundant in sewage. After informatically separating sequences of human fecal origin, sewage samples exhibited ~3× greater diversity than stool samples. Comparisons among municipal sewage communities revealed the ubiquitous and abundant occurrence of 27 human fecal oligotypes, representing an apparent core set of organisms in U.S. populations. The fecal community variability among U.S. populations was significantly lower than among individuals. It clustered into three primary community structures distinguished by oligotypes from either: Bacteroidaceae, Prevotellaceae, or Lachnospiraceae/Ruminococcaceae. These distribution patterns reflected human population variation and predicted whether samples represented lean or obese populations with 81 to 89% accuracy. Our findings demonstrate that sewage represents the fecal microbial community of human populations and captures population-level traits of the human microbiome. PMID:25714718

Validation of DESS as a DNA Preservation Method for the Detection of Strongyloides spp. in Canine Feces.

PubMed

Beknazarova, Meruyert; Millsteed, Shelby; Robertson, Gemma; Whiley, Harriet; Ross, Kirstin

2017-06-09

Strongyloides stercoralis is a gastrointestinal parasitic nematode with a life cycle that includes free-living and parasitic forms. For both clinical (diagnostic) and environmental evaluation, it is important that we can detect Strongyloides spp. in both human and non-human fecal samples. Real-time PCR is the most feasible method for detecting the parasite in both clinical and environmental samples that have been preserved. However, one of the biggest challenges with PCR detection is DNA degradation during the postage time from rural and remote areas to the laboratory. This study included a laboratory assessment and field validation of DESS (dimethyl sulfoxide, disodium EDTA, and saturated NaCl) preservation of Strongyloides spp. DNA in fecal samples. The laboratory study investigated the capacity of 1:1 and 1:3 sample to DESS ratios to preserve Strongyloides ratti in spike canine feces. It was found that both ratios of DESS significantly prevented DNA degradation compared to the untreated sample. This method was then validated by applying it to the field-collected canine feces and detecting Strongyloides DNA using PCR. A total of 37 canine feces samples were collected and preserved in the 1:3 ratio (sample: DESS) and of these, 17 were positive for Strongyloides spp. The study shows that both 1:1 and 1:3 sample to DESS ratios were able to preserve the Strongyloides spp. DNA in canine feces samples stored at room temperature for up to 56 days. This DESS preservation method presents the most applicable and feasible method for the Strongyloides DNA preservation in field-collected feces.

RNA-Based Methods Increase the Detection of Fecal Bacteria and Fecal Identifiers in Environmental Waters

EPA Science Inventory

We evaluated the use of qPCR RNA-based methods in the detection of fecal bacteria in environmental waters. We showed that RNA methods can increase the detection of fecal bacteria in multiple water matrices. The data suggest that this is a viable alternative for the detection of a...

Evaluating internal and external markers versus fecal sampling procedure interactions when estimating intake in dairy cows consuming a corn silage-based diet.

PubMed

Velásquez, A V; da Silva, G G; Sousa, D O; Oliveira, C A; Martins, C M M R; Dos Santos, P P M; Balieiro, J C C; Rennó, F P; Fukushima, R S

2018-04-18

Feed intake assessment is a valuable tool for herd management decisions. The use of markers, either internal or external, is currently the most used technique for estimating feed intake in production animals. The experiment used 10 multiparous Holstein cows fed a corn silage-based diet, with 55:45 forage-to-concentrate ratio, the average fecal recovery (FR) of TiO 2 was higher than FR of Cr 2 O 3 , and both FR were more than unity. With internal markers, acetyl bromide lignin and cutin FR were lower than unity, and average FR for indigestible neutral detergent fiber (iNDF) and indigestible acid detergent fiber (iADF) was 1.5. The FR was unaffected by the fecal sampling procedure and appears to be an intrinsic property of each molecule and how it interacts with digesta. Of the 2 external markers, only Cr 2 O 3 produced accurate fecal output (FO) estimates and the same happened to dry matter digestibility (DMD) when iNDF and iADF were used. Estimates for DMD and FO were affected by sampling procedure; 72-h bulk [sub-sample from total feces collection (TFC)] sampling consistently produced accurate results. The grab (sub-samples taken at specific times during the day) sampling procedures were accurate when using either of the indigestible fibers (iNDF or iADF) to estimate DMD. However, grab sampling procedures can only be recommended when concomitant TFC is performed on at least one animal per treatment to determine FR. Under these conditions, Cr 2 O 3 is a suitable marker for estimating FO, and iNDF and iADF are adequate for estimating DMD. Moreover, the Cr 2 O 3 +iADF marker pair produces accurate dry matter intake estimates and deserves further attention in ruminant nutrition studies. The method of dosing the external markers is extremely important and greatly affects and determines results. Whichever the method, it must allow the animals to display normal feeding behavior and not affect performance. The grab sampling procedures can replace TFC (once FR is established), which may open new possibilities for pasture-based or collectively housed animals. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Potential of fecal waste for the production of biomethane, bioethanol and biodiesel.

PubMed

Gomaa, Mohamed A; Abed, Raeid M M

2017-07-10

Fecal waste is an environmental burden that requires proper disposal, which ultimately becomes also an economic burden. Because fecal waste is nutrient-rich and contains a diverse methanogenic community, it has been utilized to produce biomethane via anaerobic digestion. Carbohydrates and lipids in fecal waste could reach up to 50% of the dry weight, which also suggests a potential as a feedstock for bioethanol and biodiesel production. We measured biomethane production from fecal waste of cows, chickens, goats and humans and compared the microbial community composition before and after anaerobic digestion. We also compared the fecal waste for cellulase production, saccharification and fermentation to produce bioethanol and for lipid content and fatty acid profiles to produce biodiesel. All fecal waste produced biomethane, with the highest yield of 433.4±77.1ml CH 4 /g VS from cow fecal waste. Production of bioethanol was achieved from all samples, with chicken fecal waste yielding as high as 1.6±0.25g/l. Sludge samples exhibited the highest extractable portion of lipids (20.9±0.08wt%) and conversion to fatty acid methyl esters (11.94wt%). Utilization of fecal waste for the production of biofuels is environmentally and economically beneficial. Copyright © 2017 Elsevier B.V. All rights reserved.

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

PubMed

Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

2015-10-01

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Performance of forty-one microbial source tracking methods: A twenty-seven lab evaluation study

EPA Science Inventory

The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 43 ...

Factors affecting isolation and identification of Mycobacterium avium subsp. paratuberculosis from fecal and tissue samples in a liquid culture system.

PubMed

Whittington, Richard J

2009-03-01

Culture of Mycobacterium avium subsp. paratuberculosis is the definitive diagnostic test for Johne's disease, a chronic granulomatous enteropathy of animals. Compared to solid media, the identification of all strains of the organism in liquid media can be more difficult because the appearance of colonies and mycobactin dependence are not observable, and the growth of other organisms needs to be distinguished, commonly by PCR. Factors affecting the isolation rate of S strains and the contamination rate in modified Middlebrook 7H9 broth (Bactec 12B) and 7H10 agar were studied using 11,598 fecal samples and 2,577 tissue samples from sheep from 1,421 farms over 10 years. Minimization of contamination in Bactec cultures required the avoidance of the carryover of fecal particles from the first sedimentation step in the double-incubation centrifugation method, and contamination was reduced significantly by incubating the sample in a solution containing vancomycin, amphotericin B, and nalidixic acid for 3 days compared to 2 days. The growth of irrelevant microorganisms confounded the identification of M. avium subsp. paratuberculosis in liquid culture by inhibiting IS900 PCR and in solid medium culture by inhibiting the growth of M. avium subsp. paratuberculosis or obscuring colonies. The contamination of samples was clustered in certain laboratory submissions and was reduced by including ampicillin in Bactec medium without affecting the odds of isolation of M. avium subsp. paratuberculosis. The long-term contamination rate for fecal cultures was about 7%, and that for tissue cultures was <0.2%. Liquid medium was more sensitive than solid medium culture for M. avium subsp. paratuberculosis. The applicability of these findings for C strains is discussed.

Identification of fecal contamination sources in water using host-associated markers.

PubMed

Krentz, Corinne A; Prystajecky, Natalie; Isaac-Renton, Judith

2013-03-01

In British Columbia, Canada, drinking water is tested for total coliforms and Escherichia coli, but there is currently no routine follow-up testing to investigate fecal contamination sources in samples that test positive for indicator bacteria. Reliable microbial source tracking (MST) tools to rapidly test water samples for multiple fecal contamination markers simultaneously are currently lacking. The objectives of this study were (i) to develop a qualitative MST tool to identify fecal contamination from different host groups, and (ii) to evaluate the MST tool using water samples with evidence of fecal contamination. Singleplex and multiplex polymerase chain reaction (PCR) were used to test (i) water from polluted sites and (ii) raw and drinking water samples for presence of bacterial genetic markers associated with feces from humans, cattle, seagulls, pigs, chickens, and geese. The multiplex MST assay correctly identified suspected contamination sources in contaminated waterways, demonstrating that this test may have utility for heavily contaminated sites. Most raw and drinking water samples analyzed using singleplex PCR contained at least one host-associated marker. Singleplex PCR was capable of detecting host-associated markers in small sample volumes and is therefore a promising tool to further analyze water samples submitted for routine testing and provide information useful for water quality management.

Modulation of gut microbiota and increase in fecal water content in mice induced by administration of Lactobacillus kefiranofaciens DN1.

PubMed

Jeong, Dana; Kim, Dong-Hyeon; Kang, Il-Byeong; Kim, Hyunsook; Song, Kwang-Young; Kim, Hong-Seok; Seo, Kun-Ho

2017-02-22

Lactobacillus kefiranofaciens is the key probiotic bacterium in kefir. In this study, we investigated the effects of oral consumption of L. kefiranofaciens on the fecal quality and intestinal microbiota of mice. Four-week-old Balb/c mice were divided into two groups (n = 8 each) and administered 0.2 mL of saline (control group) or saline containing 2 × 10 8 cfu L. kefiranofaciens DN1 (LKF_DN1 group) for two weeks. At the end of the experiment, their fecal samples were collected and the fecal quality and microbiota were assessed. The LKF_DN1 group exhibited higher total fecal weight and fecal weight per stool sample than the control group (p < 0.05). Interestingly, the fecal water content was significantly higher in the fecal samples of the LKF_DN1 group than in those of the control group (p < 0.05). The numbers of total bacteria, Firmicutes, Bacteroidetes, Lactobacillus, and Prevotella were significantly higher in the LKF_DN1 group than in the control group (p < 0.05). In contrast, the number of opportunistic pathogens, including Proteobacteria and Enterobacteriaceae, and the percentage of genus Clostridium among the total bacteria were significantly reduced in the LKF_DN1 group (p < 0.05). Our data suggest that regular L. kefiranofaciens DN1 administration could alleviate constipation and improve gut microbiota.

Effect of Temperature and Time on Fecal Hemoglobin Stability in 5 Fecal Immunochemical Test Methods and One Guaiac Method.

PubMed

Catomeris, Peter; Baxter, Nancy N; Boss, Sheila C; Paszat, Lawrence F; Rabeneck, Linda; Randell, Edward; Serenity, Mardie L; Sutradhar, Rinku; Tinmouth, Jill

2018-01-01

- Although promising for colorectal cancer screening, hemoglobin (Hb) stability remains a concern with fecal immunochemical tests. This study implemented a novel, standardized method to compare Hb stability across various fecal immunochemical tests. The method can be used to inform decisions when selecting a kit for use in colorectal cancer screening. In so doing, this work addressed a critical need for standardization in this field. - To compare the stability of Hb across 5 different immunochemical kits and one guaiac kit. - The stability of Hb was analyzed in collection devices inoculated with Hb-spiked feces and (1) stored at various temperatures (frozen, refrigerated, ambient, and elevated) for more than 60 days; (2) after undergoing 3 controlled, freeze-thaw cycles; and (3) after being transported by courier or postal services in uncontrolled temperature conditions from 3 locations in Ontario, Canada, to a central testing center. - The stability of Hb varied with time and temperature and by kit. Lower Hb recoveries occurred with increasing temperature and increasing time from sample collection to testing. Refrigeration provided the best stability, although results varied across kits (eg, from 4.2 days to >60 days before a prespecified threshold [<70% probability of the test results remaining positive] was reached). Freeze-thaw stability varied across kits and cycles (Hb recoveries: NS Plus [Alfresa Pharma, Chuo-ku, Osaka, Japan], 91.7% to 95.4%; OC Diana [Eiken Chemical, Taito-ku, Tokyo, Japan], 57.6% to 74.9%). Agreement regarding Hb levels before and after transportation varied across kits (from 57% to 100%). - Important differences in Hb stability were found across the included fecal immunochemical tests. These findings should inform practice-based and population-based colorectal cancer screening.

The impact of ecological variability on the reproductive endocrinology of wild female African elephants.

PubMed

Wittemyer, George; Ganswindt, André; Hodges, Keith

2007-03-01

Non-invasive endocrine methods enable investigation of the relationship between ecological variation and ovarian activity and how this impacts on demographic processes. The underlying physiological factors driving high variation in inter-calving intervals among multi-parous African elephants offer an interesting system for such an investigation. This study investigates the relationship between Normalized Differential Vegetation Index (NDVI), an ecosystem surrogate measure of primary productivity, and fecal progestin concentrations among wild female elephants. Matched fecal samples and behavioral data on reproductive activity were collected from 37 focal individuals during the two-year study. Linear mixed models were used to explore the relationship between fecal 5alpha-pregnane-3-ol-20-one concentrations and the independent variables of NDVI, calf sex, female age, gestation day, and time since last parturition. Among both non-pregnant and pregnant females, fecal 5alpha-pregnane-3-ol-20-one concentrations were significantly correlated with time-specific NDVI indicating a strong relationship between ecological conditions and endocrine activity regulating reproduction. In addition, the age of a female and time since her last parturition impacted hormone concentrations. These results indicate that the identification of an individual's reproductive status from a single hormone sample is possible, but difficult to achieve in practice since numerous independent factors, particularly season, impact fecal hormone concentrations. Regardless of season, however, fecal 5alpha-pregnane-3-ol-20-one concentrations below 1 microg/g were exclusively collected from non-pregnant females, which could be used as a threshold value to identify non-pregnant individuals. Collectively the information generated contributes to a better understanding of environmental regulation of reproductive endocrinology in wild elephant populations, information salient to the management and manipulation of population dynamics in this species.

Comparison and continuous estimates of fecal coliform and Escherichia coli bacteria in selected Kansas streams, May 1999 through April 2002

USGS Publications Warehouse

Rasmussen, Patrick P.; Ziegler, Andrew C.

2003-01-01

The sanitary quality of water and its use as a public-water supply and for recreational activities, such as swimming, wading, boating, and fishing, can be evaluated on the basis of fecal coliform and Escherichia coli (E. coli) bacteria densities. This report describes the overall sanitary quality of surface water in selected Kansas streams, the relation between fecal coliform and E. coli, the relation between turbidity and bacteria densities, and how continuous bacteria estimates can be used to evaluate the water-quality conditions in selected Kansas streams. Samples for fecal coliform and E. coli were collected at 28 surface-water sites in Kansas. Of the 318 samples collected, 18 percent exceeded the current Kansas Department of Health and Environment (KDHE) secondary contact recreational, single-sample criterion for fecal coliform (2,000 colonies per 100 milliliters of water). Of the 219 samples collected during the recreation months (April 1 through October 31), 21 percent exceeded the current (2003) KDHE single-sample fecal coliform criterion for secondary contact rec-reation (2,000 colonies per 100 milliliters of water) and 36 percent exceeded the U.S. Environmental Protection Agency (USEPA) recommended single-sample primary contact recreational criterion for E. coli (576 colonies per 100 milliliters of water). Comparisons of fecal coliform and E. coli criteria indicated that more than one-half of the streams sampled could exceed USEPA recommended E. coli criteria more frequently than the current KDHE fecal coliform criteria. In addition, the ratios of E. coli to fecal coliform (EC/FC) were smallest for sites with slightly saline water (specific conductance greater than 1,000 microsiemens per centimeter at 25 degrees Celsius), indicating that E. coli may not be a good indicator of sanitary quality for those streams. Enterococci bacteria may provide a more accurate assessment of the potential for swimming-related illnesses in these streams. Ratios of EC/FC and linear regression models were developed for estimating E. coli densities on the basis of measured fecal coliform densities for six individual and six groups of surface-water sites. Regression models developed for the six individual surface-water sites and six groups of sites explain at least 89 percent of the variability in E. coli densities. The EC/FC ratios and regression models are site specific and make it possible to convert historic fecal coliform bacteria data to estimated E. coli densities for the selected sites. The EC/FC ratios can be used to estimate E. coli for any range of historical fecal coliform densities, and in some cases with less error than the regression models. The basin- and statewide regression models explained at least 93 percent of the variance and best represent the sites where a majority of the data used to develop the models were collected (Kansas and Little Arkansas Basins). Comparison of the current (2003) KDHE geometric-mean primary contact criterion for fecal coliform bacteria of 200 col/100 mL to the 2002 USEPA recommended geometric-mean criterion of 126 col/100 mL for E. coli results in an EC/FC ratio of 0.63. The geometric-mean EC/FC ratio for all sites except Rattlesnake Creek (site 21) is 0.77, indicating that considerably more than 63 percent of the fecal coliform is E. coli. This potentially could lead to more exceedances of the recommended E. coli criterion, where the water now meets the current (2003) 200-col/100 mL fecal coliform criterion. In this report, turbidity was found to be a reliable estimator of bacteria densities. Regression models are provided for estimating fecal coliform and E. coli bacteria densities using continuous turbidity measurements. Prediction intervals also are provided to show the uncertainty associated with using the regression models. Eighty percent of all measured sample densities and individual turbidity-based estimates from the regression models were in agreement as exceedi

Characterizing the fecal microbiota of infants with botulism.

PubMed

Shirey, T Brian; Dykes, Janet K; Lúquez, Carolina; Maslanka, Susan E; Raphael, Brian H

2015-11-23

Infant botulism is the most prevalent form of botulism in the USA, representing 68.5 % of cases reported from 2001-2012. Infant botulism results when botulinum toxin-producing clostridia (BTPC) colonize the infant gut with concomitant in vivo production of the highly potent botulinum neurotoxin (BoNT). The gut microbiota of infants with botulism is largely uncharacterized; therefore, it remains unclear whether the microbiota profile of these patients are distinct in composition, abundance, or diversity. To address this uncertainty, we employed 16S rRNA gene profiling to characterize the fecal microbiota in 14 stool samples among laboratory-confirmed and non-confirmed infant botulism cases. Seven bacterial phyla were identified among all 14 infant stool samples examined. Compared to samples from non-confirmed cases, the fecal microbiota of infant botulism patients displayed significantly higher Proteobacteria abundance. Of the 20 bacterial families identified, Enterobacteriaceae was significantly more abundant in samples from infants with botulism. Firmicutes abundance and the abundance ratio of Firmicutes/Proteobacteria was significantly lower in samples from infants with botulism. Lactobacillus spp. abundance was notably reduced in 12 of the 14 samples. Clostridium botulinum and Clostridium baratii were identified in low relative abundances in confirmed and non-confirmed samples based on their 16S rRNA gene profiles, although their toxigenicity remained undetermined. No significant differences were observed in the number of operational taxonomic units (OTUs) observed or in fecal microbiota diversity between laboratory-confirmed and non-confirmed samples. Correlations between individual phylum abundances and infant age were variable, and no significant differences were shown in number of OTUs observed or in fecal microbiota diversity between samples delineated by overall mean age. Significant differences in Proteobacteria, Firmicutes, and Enterobacteriaceae abundances were identified in the fecal microbiota of infants with botulism when compared to samples from non-confirmed cases. Fecal microbiota diversity was not significantly altered in infants with botulism, and a limited presence of BTPC was shown. It could not be determined whether the fecal microbiota profiles shown here were comparable prior to patient illness, or whether they were the direct result of infant botulism. The results of this study do, however, provide a detailed and descriptive observation into the infant gut microbiota after intestinal colonization by BTPC.

Herd-level prevalence of Map infection in dairy herds of southern Chile determined by culture of environmental fecal samples and bulk-tank milk qPCR.

PubMed

Kruze, J; Monti, G; Schulze, F; Mella, A; Leiva, S

2013-09-01

Paratuberculosis, an infectious disease of domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), is an economically important disease in dairy herds worldwide. In Chile the disease has been reported in domestic and wildlife animals. However, accurate and updated estimations of the herd-prevalence in cattle at national or regional level are not available. The objectives of this study were to determine the herd-level prevalence of dairy herds with Map infected animals of Southern Chile, based on two diagnostic tests: culture of environmental fecal samples and bulk-tank milk qPCR. Two composite environmental fecal samples and one bulk-tank milk sample were collected during September 2010 and September 2011 from 150 dairy farms in Southern Chile. Isolation of Map from environmental fecal samples was done by culture of decontaminated samples on a commercial Herrold's Egg Yolk Medium (HEYM) with and without mycobactin J. Suspicious colonies were confirmed to be Map by conventional IS900 PCR. Map detection in bulk-tank milk samples was done by real time IS900 PCR assay. PCR-confirmed Map was isolated from 58 (19.3%) of 300 environmental fecal samples. Holding pens and manure storage lagoons were the two more frequent sites found positive for Map, representing 35% and 33% of total positive samples, respectively. However, parlor exits and cow alleyways were the two sites with the highest proportion of positive samples (40% and 32%, respectively). Herd prevalence based on environmental fecal culture was 27% (true prevalence 44%) compared to 49% (true prevalence 87%) based on bulk-tank milk real time IS900 PC. In both cases herd prevalence was higher in large herds (>200 cows). These results confirm that Map infection is wide spread in dairy herds in Southern Chile with a rough herd-level prevalence of 28-100% depending on the herd size, and that IS900 PCR on bulk-tank milk samples is more sensitive than environmental fecal culture to detect Map-infected dairy herds. Copyright © 2013 Elsevier B.V. All rights reserved.

The Adrenocortical Response of Greater Sage Grouse (Centrocercus urophasianus) to Capture, ACTH Injection, and Confinement, as Measured in Fecal Samples

PubMed Central

Jankowski, M. D.; Wittwer, D. J.; Heisey, D. M.; Franson, J. C.; Hofmeister, E. K.

2009-01-01

Investigators of wildlife populations often utilize demographic indicators to understand the relationship between habitat characteristics and population viability. Assessments of corticosterone may enable earlier detection of populations at risk of decline because physiological adjustments to habitat disturbance occur before reproductive diminutions. Noninvasive methods to accomplish these assesments are important in species of concern, such as the greater sage grouse (GRSG). Therefore, we validated a radioimmunoassay that measures immunoreactive corticosterone metabolites (ICM) in fecal samples and used it to characterize the adrenocortical response of 15 GRSG exposed to capture, intravenous injection of 50 IU/kg adrenocorticotrophic hormone (ACTH) or saline, and 22 h of confinement. Those animals injected with ACTH exhibited a more sustained (P = 0.0139) and less variable (P = 0.0012) response than those injected with saline, indicating different levels of adrenocortical activity. We also found that potential field-collection protocols of fecal samples did not alter ICM concentrations: samples held at 4°C for up to 16 h contained similar levels of ICM as those frozen (−20°C) immediately. This study demonstrates a multiphasic adrenocortical response that varied with the level of stimulation and indicates that the assay used to measure this phenomenon is applicable for studies of wild GRSG. PMID:19199814

The adrenocortical response of greater sage grouse (Centrocercus urophasianus) to capture, ACTH injection, and confinement, as measured in fecal samples

USGS Publications Warehouse

Jankowski, M.D.; Wittwer, D.J.; Heisey, D.M.; Franson, J. Christian; Hofmeister, Erik K.

2009-01-01

Investigators of wildlife populations often utilize demographic indicators to understand the relationship between habitat characteristics and population viability. Assessments of corticosterone may enable earlier detection of populations at risk of decline because physiological adjustments to habitat disturbance occur before reproductive diminutions. Noninvasive methods to accomplish these assesments are important in species of concern, such as the greater sage grouse (GRSG). Therefore, we validated a radioimmunoassay that measures immunoreactive corticosterone metabolites (ICM) in fecal samples and used it to characterize the adrenocortical response of 15 GRSG exposed to capture, intravenous injection of 50 IU/kg adrenocorticotrophic hormone (ACTH) or saline, and 22 h of confinement. Those animals injected with ACTH exhibited a more sustained (P = 0.0139) and less variable (P = 0.0012) response than those injected with saline, indicating different levels of adrenocortical activity. We also found that potential field-collection protocols of fecal samples did not alter ICM concentrations: samples held at 4??C for up to 16 h contained similar levels of ICM as those frozen (-20??C) immediately. This study demonstrates a multiphasic adrenocortical response that varied with the level of stimulation and indicates that the assay used to measure this phenomenon is applicable for studies of wild GRSG. ?? 2009 by The University of Chicago. All rights reserved.

Identification of fecal input sites in spring water by selection and genotyping of multiresistant Escherichia coli.

PubMed

Wicki, Melanie; Karabulut, Fatma; Auckenthaler, Adrian; Felleisen, Richard; Tanner, Marcel; Baumgartner, Andreas

2011-12-01

The localization of fecal input sites is important for water quality management. For this purpose, we have developed a new approach based on a three-step procedure, including a preparatory phase, the screening of multiresistant bacteria using selective agar plates, and a typing phase where selected Escherichia coli isolates are characterized by antibiotic resistance profiles and molecular fingerprinting techniques (pulsed-field gel electrophoresis [PFGE]). These two well-known source tracking methods were combined in order to reduce cost and effort. This approach was successfully applied under field conditions in a study area located in the north-western part of Switzerland. E. coli isolates from spring water and surface water samples collected in this area were screened with selective agar plates. In this way, 21 different groups, each consisting of strains with the same pattern of antibiotic resistance, were found. Of these, four groups were further analyzed using PFGE. Strains with identical PFGE profiles were detected repeatedly, demonstrating the suitability of this method for the localization of fecal input sites over an extended period of time. Identical PFGE patterns of strains detected in water from two different springs were also found in the stream flowing through the study area. These results demonstrated the applicability of the new approach for the examination of incidents of fecal contamination in drinking water. The advantages of the described approach over genotyping methods currently being used to identify sources of fecal contaminants are a reduction in time, costs, and the effort required. Identical isolates could be identified without the construction of large libraries.

Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Weili; Richard J. Giannone; Morowitz, Michael J.

The early-life microbiota establishment in the human infant gut is highly variable and plays a crucial role in host nutrients and immunity maturation. While high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the construction of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on Double Filtering (DF) to enhance microbial protein characterization in complex fecal samples from healthy premature infants. We improved the overall depth of infant gut proteome measurement, withmore » an increase in the number of identified low abundance proteins, and observed greater than twofold improvement in metrics for microbial protein identifications and quantifications with a relatively high rank correlation to control. We further showed the substantial enhancement of this approach for extensively interpreting microbial functional categories between infants by affording more detailed and confident identified categories. This approach provided an avenue for in-depth measurement in the microbial component of infant fecal samples and thus comprehensive characterization of infant gut microbiome functionality.« less

Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut

DOE PAGES

Xiong, Weili; Richard J. Giannone; Morowitz, Michael J.; ...

2014-10-28

The early-life microbiota establishment in the human infant gut is highly variable and plays a crucial role in host nutrients and immunity maturation. While high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the construction of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on Double Filtering (DF) to enhance microbial protein characterization in complex fecal samples from healthy premature infants. We improved the overall depth of infant gut proteome measurement, withmore » an increase in the number of identified low abundance proteins, and observed greater than twofold improvement in metrics for microbial protein identifications and quantifications with a relatively high rank correlation to control. We further showed the substantial enhancement of this approach for extensively interpreting microbial functional categories between infants by affording more detailed and confident identified categories. This approach provided an avenue for in-depth measurement in the microbial component of infant fecal samples and thus comprehensive characterization of infant gut microbiome functionality.« less

Fecal-indicator bacteria in streams alonga gradient of residential development

USGS Publications Warehouse

Frenzel, Steven A.; Couvillion, Charles S.

2002-01-01

Fecal-indicator bacteria were sampled at 14 stream sites in Anchorage, Alaska, USA, as part of a study to determine the effects of urbanization on water quality. Population density in the subbasins sampled ranged from zero to 1,750 persons per square kilometer. Higher concentrations of fecal-coliform, E. coli, and enterococci bacteria were measured at the most urbanized sites. Although fecal-indicator bacteria concentrations were higher in summer than in winter, seasonal differences in bacteria concentrations generally were not significant. Areas served by sewer systems had significantly higher fecal-indicator bacteria concentrations than did areas served by septic systems. The areas served by sewer systems also had storm drains that discharged directly to the streams, whereas storm sewers were not present in the areas served by septic systems. Fecal-indicator bacteria concentrations were highly variable over a two-day period of stable streamflow, which may have implications for testing of compliance to water-quality standards.

Fecal indicators and zoonotic pathogens in household drinking water taps fed from rainwater tanks in Southeast Queensland, Australia.

PubMed

Ahmed, W; Hodgers, L; Sidhu, J P S; Toze, S

2012-01-01

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.

Fecal Indicators and Zoonotic Pathogens in Household Drinking Water Taps Fed from Rainwater Tanks in Southeast Queensland, Australia

PubMed Central

Hodgers, L.; Sidhu, J. P. S.; Toze, S.

2012-01-01

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks. PMID:22020514

A reliable method of analyzing dietaries of mycophagous small mammals

Treesearch

W. Colgan; A.B. Carey; James M. Trappe

1997-01-01

Two methods of analyzing the dietaries of mycophagous small mammals were compared. Fecal pellets were collected from 11 northern flying squirrels and 12 Townsend's chipmunks, all caught live. In 1 method, pellets from each individual were examined microscopically; in the other, samples from 3 or 4 individuals from each species were pooled and the number of slides...

Influence of manure age and sunlight on the community structure of cattle fecal bacteria as revealed by Illumina sequencing

NASA Astrophysics Data System (ADS)

Wong, K.; Shaw, T. I.; Oladeinde, A.; Molina, M.

2013-12-01

Fecal pollution of environmental waters is a major concern for the general public because exposure to fecal-associated pathogens can have severe impacts on human health. Stream and river impairment due to fecal pollution is largely the result of agricultural activities in the United States. In the last few years, numerous metagenomic studies utilized next generation sequencing to develop microbial community profiles by massively sequencing the 16sRNA hypervariable region. This technology supports the application of water quality assessment such as pathogen detection and fecal source tracking. The bacteria communities of samples in these studies were determined when they were freshly collected; therefore, little is known about how feces age or how environmental stress influences the microbial ecology of fecal materials. In this study we monitored bacteria community changes in cattle feces for 57 days after excretion (day 0, 2, 4 8, 15, 22, 29, 43, 57) by sequencing the 16s variable region 4, using Illumnia MiSeq. Twelve cattle feces were studied; half of the samples were directly exposed to sunlight (unshaded) and half were shaded. Results indicate that the relative abundance (RA) profile in both shaded and unshaded samples rapidly changed from day 0 to 15, but stabilized from day 22 to 57. Firmcutes were the most abundant phylum (~40%) at day 0, but were reduced to <10% by day 57. The RA of Proteobacteria was only 1% at day 0, but increased to ~50% by day 57in both shaded and unshaded samples. By the end of the study, shaded and unshaded samples had a similar RA of Firmcutes and Proteobacteria but the RA of Bacteroidetes and Actinobacteria was, respectively, about 7% lower and 10% higher for unshaded samples. UV intensity, moisture, and temperature were significantly different between shaded and unshaded plots, indicating that these environmental stresses could influence the structure of fecal bacteria community in the natural environment. According to the rarefaction curve analysis, richness of bacteria diversity in feces decreased as time progressed. Some pathogens such as Campylobacter were detected only at the beginning, meaning they substantially decayed during the course of our study. Overall, this study indicated: (1) sunlight can influence the community structure and (2) after excretion the fecal bacteria diversity can be significantly changed over time. Future studies should therefore use not only the microbial signature of fresh but also moderately aged fecal samples to develop more accurate community profiles for fecal source tracking.

SPECIFICITY AND SENSITIVITY OF FECAL BACTEROIDETES HUMAN-SPECIFIC PRIMERS WITH FECAL AND WASTEWATER SAMPLES FROM THE U.S. MIDWEST AND NORTHEAST REGIONS

EPA Science Inventory

Numerous watersheds throughout the United States are impaired due to fecal contamination. Fecal Bacteroidetes is a group of anaerobic bacteria present in high concentrations in animal feces that has shown promise as a microbial source tracking indicator of human and othe...

Detection of Shiga-Like Toxin (stx1 and stx2), Intimin (eaeA), and Enterohemorrhagic Escherichia coli (EHEC) Hemolysin (EHEC hlyA) Genes in Animal Feces by Multiplex PCR

PubMed Central

Fagan, Peter K.; Hornitzky, Michael A.; Bettelheim, Karl A.; Djordjevic, Steven P.

1999-01-01

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 and stx2), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors. PMID:9925634

A combined electrochemical-irradiation treatment of highly colored and polluted industrial wastewater

NASA Astrophysics Data System (ADS)

Barrera-Díaz, C.; Ureña-Nuñez, F.; Campos, E.; Palomar-Pardavé, M.; Romero-Romo, M.

2003-07-01

This study reports on the attainment of optimal conditions for two electrolytic methods to treat wastewater: namely, electrocoagulation and particle destabilization of a highly polluted industrial wastewater, and electrochemically induced oxidation induced by in situ generation of Fenton's reactive. Additionally, a combined method that consisted of electrochemical treatment plus γ-irradiation was carried out. A typical composition of the industrial effluent treated was COD 3400 mg/l, color 3750 Pt/Co units, and fecal coliforms 21000 MPN/ml. The best removal efficiency was obtained with electrochemical oxidation induced in situ , that resulted in the reduction of 78% for the COD, 86% color and 99.9% fecal coliforms removal. A treatment sequence was designed and carried out, such that after both electrochemical processes, a γ-irradiation technique was used to complete the procedure. The samples were irradiated with various doses in an ALC γ-cell unit provided with a Co-60 source. The removal efficiency obtained was 95% for the COD values, 90% color and 99.9% for fecal coliforms.

Administration of Bifidobacterium breve PS12929 and Lactobacillus salivarius PS12934, two strains isolated from human milk, to very low and extremely low birth weight preterm infants: a pilot study.

PubMed

Moles, Laura; Escribano, Esperanza; de Andrés, Javier; Montes, María Teresa; Rodríguez, Juan M; Jiménez, Esther; Sáenz de Pipaón, Miguel; Espinosa-Martos, Irene

2015-01-01

The preterm infant gut has been described as immature and colonized by an aberrant microbiota. Therefore, the use of probiotics is an attractive practice in hospitals to try to reduce morbidity and mortality in this population. The objective of this pilot study was to elucidate if administration of two probiotic strains isolated from human milk to preterm infants led to their presence in feces. In addition, the evolution of a wide spectrum of immunological compounds, including the inflammatory biomarker calprotectin, in both blood and fecal samples was also assessed. For this purpose, five preterm infants received two daily doses (~10(9) CFU) of a 1:1 mixture of Bifidobacterium breve PS12929 and Lactobacillus salivarius PS12934. Bacterial growth was detected by culture-dependent techniques in all the fecal samples. The phylum Firmicutes dominated in nearly all fecal samples while L. salivarius PS12934 was detected in all the infants at numerous sample collection points and B. breve PS12929 appeared in five fecal samples. Finally, a noticeable decrease in the fecal calprotectin levels was observed along time.

Ground-water-quality data for selected wells in the Beaver Creek watershed, West Tennessee

USGS Publications Warehouse

Williams, S.D.

1996-01-01

In 1993 the U.S. Geological Survey, in cooperation with the Tennessee Department of Environment and Conservation (TDEC), began an investigation of the quality of ground water in the Beaver Creek watershed in West Tennessee. A total of 408 water samples were collected from 91 wells during 5 sampling periods in 1994. Water samples were analyzed for selected water-quality properties, fecal coliform and streptococci bacteria, nutrients, and major inorganic constituents. Selected well- construction data and information on potential sources of contamination were also collected for the 91 wells sampled. Nitrate concentrations (measured as NO3) ranged from a detection limit of 0.1 to 91 milligrams per liter (mg/L). Nitrate concentrations exceeding 13 mg/L were detected in 71 of the samples collected. Nitrate concentrations in water samples collected from three wells exceeded the TDEC primary drinking water standard of 44 mg/L for nitrate (measured as NO3). Nitrite (measured as NO2), ammonium (measured as NH4), and orthophosphate (measured as PO4) concentrations in samples were generally less than 0.1 mg/L (detection limit). Fecal coliform bacteria were detected in 33 of the 408 water samples collected. Samples from 21 of the 91 wells contained fecal coliform bacteria during one or more of the five sampling periods. Fecal streptococci bacteria were detected in 123 of the 408 samples. Samples from 59 wells contained fecal streptococci bacteria during one or more of the five sampling periods.

Validation of Salivary Immunoassays for Waterborne Infections

EPA Science Inventory

Assessments of the health outcomes associated with exposure to fecally-contaminated water and inadequate sanitation and hygiene (WASH) currently rely upon self-reported symptoms and invasive collection of blood and stool samples. However, these methods are limited in their abilit...

Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

PubMed Central

Ryu, Hodon; Griffith, John F.; Khan, Izhar U. H.; Hill, Stephen; Edge, Thomas A.; Toledo-Hernandez, Carlos; Gonzalez-Nieves, Joel

2012-01-01

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters. PMID:22226950

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

PubMed

Durant, Jean-Francois; Irenge, Leonid M; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean-Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean-Luc

2012-12-07

Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

Molecular Determination of Fasciola Spp. Isolates from Domestic Ruminants Fecal Samples in the Northwest of Iran

PubMed Central

IMANI BARAN, Abbas; CHERAGHI SARAY, Habib; KATIRAEE, Farzad

2017-01-01

Background: Fasciola species are the main causes for fascioliasis with great financial losses and are among the most important food/water-borne parasites worldwide. The basic proceedings such as epidemiology and effective control of fascioliasis rely mainly on precise identification of Fasciola species. The present study was conducted to determine the Fasciola species in ruminant fecal samples from East Azerbaijan Province in Iran. Methods: Overall, 2012 fecal samples were collected and processed initially for microscopic examination of Fasciola eggs in 2014–15. Then, recovered eggs were subjected to molecular identification. A fragment of 618 bp of the 28S rRNA gene pertaining to Fasciola genus was amplified under PCR. The amplified fragment was restricted by fast digest Ava II enzyme in order to a Restriction Fragment Length Polymorphism. Results: Based on microscopic examination, 72 samples were infected, from which, 10 and 62 cases pertained to cattle and sheep samples respectively. Based on RFLP, the PCR products restricted by the Ava II restriction enzyme produced 529 bp fragments only. According to the positive controls, all restriction patterns were related to Fasciola hepatica, while no restriction patterns were linked to F. gigantica. Conclusion: Based on PCR-RFLP, F. hepatica was dominant species in animals of the studied areas and no evidence of F. gigantica was observed. Therefore, further field studies to verify these results are suggested. PMID:28761485

Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets

USGS Publications Warehouse

Schill, W.B.; Mathes, M.V.

2008-01-01

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 101 to 108 copies/??l. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 ?? 107 copies/g) to thatfound in human feces (1.1 ?? 107 copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 ?? 104 copies/100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/100 mL water.

Human Fecal Source Identification: Real-Time Quantitative PCR Method Standardization

EPA Science Inventory

Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus).

PubMed

Stoops, M A; MacKinnon, K M; Roth, T L

2012-12-01

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations. Copyright © 2012 Elsevier Inc. All rights reserved.

National Risk Management Research Laboratory (NRMRL) Microbial Research

EPA Science Inventory

Experimental design: Three host-specific PCR assays were tested against fecal and water samples. Host-specificity assays were performed against targeted and nontargeted fecal sources. Detection limits were performed against diluted fecal and water DNA extracts. Groundwater an...

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

PubMed

Raith, Meredith R; Kelty, Catherine A; Griffith, John F; Schriewer, Alexander; Wuertz, Stefan; Mieszkin, Sophie; Gourmelon, Michele; Reischer, Georg H; Farnleitner, Andreas H; Ervin, Jared S; Holden, Patricia A; Ebentier, Darcy L; Jay, Jennifer A; Wang, Dan; Boehm, Alexandria B; Aw, Tiong Gim; Rose, Joan B; Balleste, E; Meijer, W G; Sivaganesan, Mano; Shanks, Orin C

2013-11-15

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation. Published by Elsevier Ltd.

Effect of selected gastrointestinal parasites and viral agents on fecal S100A12 concentrations in puppies as a potential comparative model.

PubMed

Heilmann, Romy M; Grellet, Aurélien; Grützner, Niels; Cranford, Shannon M; Suchodolski, Jan S; Chastant-Maillard, Sylvie; Steiner, Jörg M

2018-04-17

Previous data suggest that fecal S100A12 has clinical utility as a biomarker of chronic gastrointestinal inflammation (idiopathic inflammatory bowel disease) in both people and dogs, but the effect of gastrointestinal pathogens on fecal S100A12 concentrations is largely unknown. The role of S100A12 in parasite and viral infections is also difficult to study in traditional animal models due to the lack of S100A12 expression in rodents. Thus, the aim of this study was to evaluate fecal S100A12 concentrations in a cohort of puppies with intestinal parasites (Cystoisospora spp., Toxocara canis, Giardia sp.) and viral agents that are frequently encountered and known to cause gastrointestinal signs in dogs (coronavirus, parvovirus) as a comparative model. Spot fecal samples were collected from 307 puppies [median age (range): 7 (4-13) weeks; 29 different breeds] in French breeding kennels, and fecal scores (semiquantitative system; scores 1-13) were assigned. Fecal samples were tested for Cystoisospora spp. (C. canis and C. ohioensis), Toxocara canis, Giardia sp., as well as canine coronavirus (CCV) and parvovirus (CPV). S100A12 concentrations were measured in all fecal samples using an in-house radioimmunoassay. Statistical analyses were performed using non-parametric 2-group or multiple-group comparisons, non-parametric correlation analysis, association testing between nominal variables, and construction of a multivariate mixed model. Fecal S100A12 concentrations ranged from < 24-14,363 ng/g. Univariate analysis only showed increased fecal S100A12 concentrations in dogs shedding Cystoisospora spp. (P = 0.0384) and in dogs infected with parvovirus (P = 0.0277), whereas dogs infected with coronavirus had decreased fecal S100A12 concentrations (P = 0.0345). However, shedding of any single enteropathogen did not affect fecal S100A12 concentrations in multivariate analysis (all P > 0.05) in this study. Only fecal score and breed size had an effect on fecal S100A12 concentrations in multivariate analysis (P < 0.0001). An infection with any single enteropathogen tested in this study is unlikely to alter fecal S100A12 concentrations, and these preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation.

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

PubMed Central

Fisher, Jenny C.; Eren, A. Murat; Green, Hyatt C.; Shanks, Orin C.; Morrison, Hilary G.; Vineis, Joseph H.; Sogin, Mitchell L.

2015-01-01

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, 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 family Lachnospiraceae to 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. PMID:26231648

Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

PubMed Central

Lamendella, Regina; Li, Kent C.; Oerther, Daniel

2013-01-01

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental waters. In order to address this, we studied the diversity of 9,340 partial (>600 bp in length) Bacteroidales 16S rRNA gene sequences from 13 fecal sources and nine feces-contaminated watersheds. The compositions of major Bacteroidales populations were analyzed to determine which host and environmental sequences were contributing to each group. This information allowed us to identify populations which were both exclusive to swine fecal sources and detected in swine-contaminated waters. Phylogenetic and diversity analyses revealed that some markers previously believed to be highly specific to swine populations are shared by multiple hosts, potentially explaining the cross-amplification signals obtained with nontargeted hosts. These data suggest that while many Bacteroidales populations are cosmopolitan, others exhibit a preferential host distribution and may be able to survive different environmental conditions. This study further demonstrates the importance of elucidating the diversity patterns of targeted bacterial groups to develop more inclusive fecal source tracking applications. PMID:23160126

Rectal nitric oxide and fecal calprotectin in inflammatory bowel disease.

PubMed

Reinders, Claudia A; Jonkers, Daisy; Janson, Emmellie A; Stockbrügger, Reinhold W; Stobberingh, Ellen E; Hellström, Per M; Lundberg, Jon O

2007-10-01

The assessment of intestinal inflammation in patients with inflammatory bowel disease (IBD) remains a difficult challenge. Both rectal nitric oxide (NO) and fecal calprotectin can be measured using non-invasive methods and are emerging as promising inflammatory markers in IBD. In this study the aim was to compare calprotectin and NO levels in IBD patients. Rectal NO was measured tonometrically in 23 healthy volunteers and 32 patients with IBD. In addition, we collected stool samples from all subjects for measurement of fecal calprotectin and nitrate/nitrite (NO metabolites). Patients with IBD had greatly increased NO and calprotectin levels compared to healthy volunteers (p <0.001). In addition, the nitrate levels were slightly increased in IBD patients. A weak correlation was found between rectal NO levels, disease activity and number of loose stools in IBD patients (Spearman's rho 0.37 and 0.51, respectively; p <0.05). Fecal calprotectin correlated only with age (Spearman's rho 0.51; p <0.01). However, no correlation was found between NO and calprotectin. Both rectal NO and fecal calprotectin are greatly increased during bowel inflammation, but they may reflect different parts of the inflammatory process. Future studies will elucidate the clinical usefulness of these two markers.

Vegetable Contamination by the Fecal Bacteria of Poultry Manure: Case Study of Gardening Sites in Southern Benin

PubMed Central

Atidégla, Séraphin C.; Huat, Joël; Agbossou, Euloge K.; Saint-Macary, Hervé; Glèlè Kakai, Romain

2016-01-01

A study was conducted in southern Benin to assess the contamination of vegetables by fecal coliforms, Escherichia coli, and fecal streptococci as one consequence of the intensification of vegetable cropping through fertilization with poultry manure. For this purpose, on-farm trials were conducted in 2009 and 2010 at Yodo-Condji and Ayi-Guinnou with three replications and four fertilization treatments including poultry manure and three vegetable crops (leafy eggplant, tomato, and carrot). Sampling, laboratory analyses, and counts of fecal bacteria in the samples were performed in different cropping seasons. Whatever the fertilization treatment, the logs of mean fecal bacteria count per g of fresh vegetables were variable but higher than AFNOR criteria. The counts ranged from 8 to 10 fecal coliforms, from 5 to 8 fecal streptococci, and from 2 to 6 Escherichia coli, whereas AFNOR criteria are, respectively, 0, 1, and 0. The long traditional use of poultry manure and its use during the study helped obtain this high population of fecal pathogens. Results confirmed that the contamination of vegetables by fecal bacteria is mainly due to the use of poultry manure. The use of properly composted poultry manure with innovative cropping techniques should help reduce the number and incidence of pathogens. PMID:27069914

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples.

PubMed

Stark, D; Al-Qassab, S E; Barratt, J L N; Stanley, K; Roberts, T; Marriott, D; Harkness, J; Ellis, J T

2011-01-01

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

HUMAN FECAL SOURCE IDENTIFICATION: REAL-TIME QUANTITATIVE PCR METHOD STANDARDIZATION - abstract

EPA Science Inventory

Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe

PubMed Central

Simmering, Rainer; Kleessen, Brigitta; Blaut, Michael

1999-01-01

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 × 107 to 2.0 × 109 cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA. PMID:10427069

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

PubMed

Fan, Lihua; Shuai, Jiangbing; Zeng, Ruoxue; Mo, Hongfei; Wang, Suhua; Zhang, Xiaofeng; He, Yongqiang

2017-12-01

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples

PubMed Central

Rahman, S. M. Mazidur; Song, Hyun Beom; Jin, Yan; Oh, Jin-Kyoung; Lim, Min Kyung; Hong, Sung-Tae

2017-01-01

Background Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. Methodology/Principle findings The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1–99.2) of sensitivity and 100% (95% CI, 92.9–100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. Conclusions To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis. PMID:28991924

Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples.

PubMed

Rahman, S M Mazidur; Song, Hyun Beom; Jin, Yan; Oh, Jin-Kyoung; Lim, Min Kyung; Hong, Sung-Tae; Choi, Min-Ho

2017-10-01

Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2) of sensitivity and 100% (95% CI, 92.9-100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

Ape Conservation Physiology: Fecal Glucocorticoid Responses in Wild Pongo pygmaeus morio following Human Visitation

PubMed Central

Muehlenbein, Michael P.; Ancrenaz, Marc; Sakong, Rosman; Ambu, Laurentius; Prall, Sean; Fuller, Grace; Raghanti, Mary Ann

2012-01-01

Nature-based tourism can generate important revenue to support conservation of biodiversity. However, constant exposure to tourists and subsequent chronic activation of stress responses can produce pathological effects, including impaired cognition, growth, reproduction, and immunity in the same animals we are interested in protecting. Utilizing fecal samples (N = 53) from 2 wild habituated orangutans (Pongo pygmaeus morio) (in addition to 26 fecal samples from 4 wild unhabituated orangutans) in the Lower Kinabatangan Wildlife Sanctuary of Sabah, Malaysian Borneo, we predicted that i) fecal glucocorticoid metabolite concentrations would be elevated on the day after tourist visitation (indicative of normal stress response to exposure to tourists on the previous day) compared to samples taken before or during tourist visitation in wild, habituated orangutans, and ii) that samples collected from habituated animals would have lower fecal glucocorticoid metabolites than unhabituated animals not used for tourism. Among the habituated animals used for tourism, fecal glucocorticoid metabolite levels were significantly elevated in samples collected the day after tourist visitation (indicative of elevated cortisol production on the previous day during tourist visitation). Fecal glucocorticoid metabolite levels were also lower in the habituated animals compared to their age-matched unhabituated counterparts. We conclude that the habituated animals used for this singular ecotourism project are not chronically stressed, unlike other species/populations with documented permanent alterations in stress responses. Animal temperament, species, the presence of coping/escape mechanisms, social confounders, and variation in amount of tourism may explain differences among previous experiments. Acute alterations in glucocorticoid measures in wildlife exposed to tourism must be interpreted conservatively. While permanently altered stress responses can be detrimental, preliminary results in these wild habituated orangutans suggest that low levels of predictable disturbance can likely result in low physiological impact on these animals. PMID:22438916

Ape conservation physiology: fecal glucocorticoid responses in wild Pongo pygmaeus morio following human visitation.

PubMed

Muehlenbein, Michael P; Ancrenaz, Marc; Sakong, Rosman; Ambu, Laurentius; Prall, Sean; Fuller, Grace; Raghanti, Mary Ann

2012-01-01

Nature-based tourism can generate important revenue to support conservation of biodiversity. However, constant exposure to tourists and subsequent chronic activation of stress responses can produce pathological effects, including impaired cognition, growth, reproduction, and immunity in the same animals we are interested in protecting. Utilizing fecal samples (N = 53) from 2 wild habituated orangutans (Pongo pygmaeus morio) (in addition to 26 fecal samples from 4 wild unhabituated orangutans) in the Lower Kinabatangan Wildlife Sanctuary of Sabah, Malaysian Borneo, we predicted that i) fecal glucocorticoid metabolite concentrations would be elevated on the day after tourist visitation (indicative of normal stress response to exposure to tourists on the previous day) compared to samples taken before or during tourist visitation in wild, habituated orangutans, and ii) that samples collected from habituated animals would have lower fecal glucocorticoid metabolites than unhabituated animals not used for tourism. Among the habituated animals used for tourism, fecal glucocorticoid metabolite levels were significantly elevated in samples collected the day after tourist visitation (indicative of elevated cortisol production on the previous day during tourist visitation). Fecal glucocorticoid metabolite levels were also lower in the habituated animals compared to their age-matched unhabituated counterparts. We conclude that the habituated animals used for this singular ecotourism project are not chronically stressed, unlike other species/populations with documented permanent alterations in stress responses. Animal temperament, species, the presence of coping/escape mechanisms, social confounders, and variation in amount of tourism may explain differences among previous experiments. Acute alterations in glucocorticoid measures in wildlife exposed to tourism must be interpreted conservatively. While permanently altered stress responses can be detrimental, preliminary results in these wild habituated orangutans suggest that low levels of predictable disturbance can likely result in low physiological impact on these animals.

Seed germination from lowland tapir (Tapirus terrestris) fecal samples collected during the dry season in the Northern Brazilian Amazon.

PubMed

Barcelos, Adriana Renata; Bobrowiec, Paulo Estefano D; Sanaiotti, Tânia Margarete; Gribel, Rogério

2013-03-01

This study evaluated the potential of lowland tapirs as seed dispersers in the northern Brazilian Amazon. The study analyzed the viability of seeds after passage through the gut. Fecal samples were collected from 6 different vegetation physiognomies in Viruá National Park during the dry season. The samples were then kept in a greenhouse for 16 months to allow the seeds to germinate. The seedling species were identified and classified according to the type of fruit, plant habit, seed size and type of ingestion. Of the 111 fecal samples, 94 (84.7%) had viable seeds of 75 species. Melastomataceae was the most frequent family with viable seeds in the fecal samples (69.1% of samples, N= 18 species). The data suggest that the importance of the lowland tapirs as dispersers is not restricted to the species consumed actively by frugivory but also extends to species accidentally consumed during browsing. The occurrence of both large and small viable seeds in the fecal samples as well as a number of large drupes, which probably cannot be transported via endozoochory by any other animal species, provide evidence of the ecological importance of lowland tapirs to the dynamics of the forest-campinarana vegetation mosaic in the region. © 2012 Wiley Publishing Asia Pty Ltd, ISZS and IOZ/CAS.

The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing.

PubMed

Van Donkersgoed, J; Graham, T; Gannon, V

1999-05-01

Fecal samples collected from cattle at processing during a 1-year period were tested for verotoxins (VT1, VT2), Escherichia coli O157:H7, and Salmonella. Verotoxins were detected in 42.6% (95% CI, 39.8% to 45.4%), E. coli O157:H7 in 7.5% (95% CI, 6.1% to 9.1%), and Salmonella in 0.08% (95% CI, 0.004% to 0.5%) of the fecal samples. In yearling cattle, the median within-lot prevalence (percentage of positive samples within a lot) was 40% (range, 0% to 100%) for verotoxins and 0% for E. coli O157:H7 (range, 0% to 100%) and Salmonella (range, 0% to 17%). One or more fecal samples were positive for verotoxins in 80.4% (95% CI, 72.8% to 86.4%) of the lots of yearling cattle, whereas E. coli O157:H7 were detected in 33.6% (95% CI, 26.0% to 42.0%) of the lots. In cull cows, the median within-lot prevalence was 50% (range, 0% to 100%) for verotoxins and 0% (range, 0% to 100%) for E. coli O157:H7 and Salmonella (range, 0% to 0%). Verotoxins were detected in one or more fecal samples from 78.0% (95% CI, 70.4% to 84.2%) of the lots of cull cows, whereas E. coli O157:H7 were detected in only 6.0% (95% CI, 3.0% to 11.4%) of the lots of cull cows. The prevalence of verotoxins in fecal samples was lower in yearling cattle than in cull cows, whereas the prevalence of E. coli O157:H7 in fecal samples was higher in yearling cattle than in cull cows. The prevalence of E. coli O157:H7 in fecal samples was highest in the summer months. Rumen fill, body condition score, sex, type of cattle (dairy, beef), and distance travelled to the plant were not associated with the fecal prevalence of verotoxins or E. coli O157:H7. The prevalence of verotoxins in fecal samples of cull cows was associated with the source of the cattle. It was highest in cows from the auction market (52%) and farm/ranch (47%) and lowest in cows from the feedlot (31%). In rumen samples, the prevalence of verotoxins was 6.4% (95% CI, 4.2% to 9.4%), and it was 0.8% (95% CI, 0.2% to 2.3%) for E. coli O157:H7, and 0.3% (95% CI, 0.007% to 1.5%) for Salmonella.

Minimally processed vegetable salads: microbial quality evaluation.

PubMed

Fröder, Hans; Martins, Cecília Geraldes; De Souza, Katia Leani Oliveira; Landgraf, Mariza; Franco, Bernadette D G M; Destro, Maria Teresa

2007-05-01

The increasing demand for fresh fruits and vegetables and for convenience foods is causing an expansion of the market share for minimally processed vegetables. Among the more common pathogenic microorganisms that can be transmitted to humans by these products are Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella. The aim of this study was to evaluate the microbial quality of a selection of minimally processed vegetables. A total of 181 samples of minimally processed leafy salads were collected from retailers in the city of Sao Paulo, Brazil. Counts of total coliforms, fecal coliforms, Enterobacteriaceae, psychrotrophic microorganisms, and Salmonella were conducted for 133 samples. L. monocytogenes was assessed in 181 samples using the BAX System and by plating the enrichment broth onto Palcam and Oxford agars. Suspected Listeria colonies were submitted to classical biochemical tests. Populations of psychrotrophic microorganisms >10(6) CFU/g were found in 51% of the 133 samples, and Enterobacteriaceae populations between 10(5) and 106 CFU/g were found in 42% of the samples. Fecal coliform concentrations higher than 10(2) CFU/g (Brazilian standard) were found in 97 (73%) of the samples, and Salmonella was detected in 4 (3%) of the samples. Two of the Salmonella-positive samples had <10(2) CFU/g concentrations of fecal coliforms. L. monocytogenes was detected in only 1 (0.6%) of the 181 samples examined. This positive sample was simultaneously detected by both methods. The other Listeria species identified by plating were L. welshimeri (one sample of curly lettuce) and L. innocua (2 samples of watercress). The results indicate that minimally processed vegetables had poor microbiological quality, and these products could be a vehicle for pathogens such as Salmonella and L. monocytogenes.

Laboratory Testing of Donors and Stool Samples for Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

PubMed Central

Neish, Emma M.; Miller, Nancy S.; Dhere, Tanvi; Burd, Eileen M.; Carpentieri, Cynthia; Sitchenko, Kaitlin L.

2017-01-01

ABSTRACT Fecal microbiota transplantation is an efficacious and inexpensive therapy for recurrent Clostridium difficile infection, yet its safety is thought to depend on appropriate fecal donor screening. FDA guidance for regulation of this procedure is in flux, but screening and manufacture of fecal material from asymptomatic donors present many challenges to clinical laboratories. This minireview summarizes FDA regulatory changes, principles of donor selection, and recommended laboratory screening practices for fecal microbiota transplantation. PMID:28077694

Fecal shedding of non-O157 serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle vaccinated with an Escherichia coli O157:H7 SRP vaccine or fed a Lactobacillus-based direct-fed microbial.

PubMed

Cernicchiaro, N; Renter, D G; Cull, C A; Paddock, Z D; Shi, X; Nagaraja, T G

2014-05-01

The objectives of this study were to determine whether fecal shedding of non-O157 Shiga toxin-producing Escherichia coli (STEC) in feedlot cattle was affected by the use of an E. coli O157:H7 vaccine or a direct-fed microbial (DFM) and whether the shedding of a particular non-O157 STEC serogroup within feces was associated with shedding of O157 or other non-O157 STEC serogroups. A total of 17,148 cattle in 40 pens were randomized to receive one, both, or neither (control) of the two interventions: a vaccine based on the siderophore receptor and porin proteins (E. coli SRP vaccine, two doses) and a DFM product (low-dose Bovamine). Fresh fecal samples (30 samples per pen) were collected weekly from pen floors for four consecutive weeks beginning approximately 56 days after study allocation. DNA extracted from enriched samples was tested for STEC O157 and non-O157 serogroups O26, O45, O103, O111, O121, and O145 and for four major virulence genes (stx1, stx2, eae, and ehxA) using an 11-gene multiplex PCR assay. Generalized linear mixed models were used to analyze the effects of treatments and make within-sample comparisons of the presence of O-serogroup-specific genes. Results of cumulative prevalence measures indicated that O157 (14.6%), O26 (10.5%), and O103 (10.3%) were the most prevalent STEC O serogroups. However, the vaccine, DFM, or both had no significant effect (P > 0.05) on fecal prevalence of the six non-O157 STEC serogroups in feedlot cattle. Within-sample comparisons of the presence of STEC serogroup-specific genes indicated that fecal shedding of E. coli O157 in cattle was associated with an increased probability (P < 0.05) of fecal shedding of STEC O26, O45, O103, and O121. Our study revealed that neither the E. coli O157:H7 vaccine, which reduced STEC O157 fecal shedding, nor the DFM significantly affected fecal shedding of non-O157 STEC serogroups, despite the fact that the most prevalent non-O157 STEC serogroups tended to occur concurrently with O157 STEC strains within fecal samples.

COMPARISON OF THE RECOVERIES OF ESCHERICHIA COLI AND TOTAL COLIFORMS FROM DRINKING WATER BY THE MI AGAR METHOD AND THE U.S. ENVIRONMENTAL PROTECTION AGENCY-APPROVED MEMBRANE FILTER METHOD

EPA Science Inventory

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. c...

Occurrence and Persistence of Bacterial Pathogens and Indicator Organisms in Beach Sand along the California Coast

PubMed Central

Yamahara, Kevan M.; Sassoubre, Lauren M.; Goodwin, Kelly D.

2012-01-01

This report documents the presence of fecal indicators and bacterial pathogens in sand at 53 California marine beaches using both culture-dependent and -independent (PCR and quantitative PCR [QPCR]) methods. Fecal indicator bacteria were widespread in California beach sand, with Escherichia coli and enterococci detected at 68% and 94% of the beaches surveyed, respectively. Somatic coliphages and a Bacteroidales human-specific fecal marker were detected at 43% and 13% of the beaches, respectively. Dry sand samples from almost 30% of the beaches contained at least one of the following pathogens: Salmonella spp., Campylobacter spp., Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), which were detected at 15%, 13%, 14%, and 3% of tested beaches, respectively. Fecal indicators and pathogens were poorly correlated to one another and to land cover. Sands were dry at the time of collection, and those with relatively high moisture tended to have higher concentrations or a more frequent occurrence of both indicators and pathogens. Using culture-dependent assays, fecal indicators decayed faster than pathogens in microcosm experiments using unaltered beach sand seeded with sewage and assessed by culture-dependent assays. The following order of persistence was observed (listed from most to least persistent): Campylobacter > Salmonella > somatic coliphages > enterococci > E. coli > F+ phages. In contrast, pathogens decayed faster than fecal indicators in culture-independent assays: enterococci > Bacteroidales human-specific marker > Salmonella > Campylobacter. Microcosm experiments demonstrated that both indicators and pathogens were mobilized by wetting with seawater. Decay rates measured by QPCR were lower than those measured with culture-dependent methods. Enterococcal persistence and possible growth were observed for wetted microcosms relative to unwetted controls. PMID:22247142

[Isolation and identification of cow-origin Cryptosporidium isolates in Hefei].

PubMed

Sun, Tao; Liu, Wei; Wang, Ju-Hua; Xue, Xiu-Heng; Zhao, Chang-Cheng; Li, Pei-Ying

2011-12-01

To isolate cow-origin Cryptosporidium in Hefei, and identify its species. 285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp., the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software. Five fecal samples were positive by morphological methods with an infection rate of 1.8% (5/285). Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 microm x 6.13 microm and 7.58 microm x 6.20 microm, and a shape index of 1.20 and 1.22, respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp, respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni (DQ989573), and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates (AY954892 and DQ989576), and they were placed into the same clade. The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

Longitudinal Study of Fecal Shedding of Escherichia coli O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover

PubMed Central

LeJeune, J. T.; Besser, T. E.; Rice, D. H.; Berg, J. L.; Stilborn, R. P.; Hancock, D. D.

2004-01-01

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms. PMID:14711666

Coliform Contamination of Peri-urban Grown Vegetables and Potential Public Health Risks: Evidence from Kumasi, Ghana.

PubMed

Abass, Kabila; Ganle, John Kuumuori; Adaborna, Eric

2016-04-01

Peri-urban vegetable farming in Ghana is an important livelihood activity for an increasing number of people. However, increasing quality and public health concerns have been raised, partly because freshwater availability for irrigation purposes is a major constraint. This paper investigated on-farm vegetable contamination and potential health risks using samples of lettuce, spring onions and cabbage randomly selected from 18 vegetable farms in peri-urban Kumasi, Ghana. Vegetable samples were tested for total coliform, fecal coliform, Escherichia coli and Salmonella spp. bacteria contamination using the Most Probable Number method. Results show high contamination levels of total and fecal coliforms, and Escherichia coli bacteria in all 18 vegetable samples. The mean total coliform/100 ml concentration for spring onions, lettuce and cabbage were 9.15 × 10(9), 4.7 × 10(7) and 8.3 × 10(7) respectively. The mean fecal coliform concentration for spring onions, lettuce and cabbage were also 1.5 × 10(8), 4.15 × 10(7) and 2.15 × 10(7) respectively, while the mean Escherichia coli bacteria contamination for spring onions, lettuce and cabbage were 1.4 × 10(8), 2.2 × 10(7) and 3.2 × 10(7) respectively. The level of total coliform, fecal coliform and Escherichia coli bacteria contamination in all the vegetable samples however declined as the distance between the main water source (Wiwi River) and farms increases. Nonetheless, all contamination levels were well above acceptable standards, and could therefore pose serious public health risks to consumers. Increased education and supervision of farmers, as well as public health and food hygiene education of consumers, are critical to reducing on-farm vegetable contamination and the health risks associated with consumption of such vegetables.

Identifying fecal sources in a selected catchment reach using multiple source-tracking tools

USGS Publications Warehouse

Vogel, J.R.; Stoeckel, D.M.; Lamendella, R.; Zelt, R.B.; Santo, Domingo J.W.; Walker, S.R.; Oerther, D.B.

2007-01-01

Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools - Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers - were evaluated in a selected reach of Plum Creek in sooth-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to lowing surface water. ?? ASA, CSSA, SSSA.

Noninvasive genetics provides insights into the population size and genetic diversity of an Amur tiger population in China.

PubMed

Wang, Dan; Hu, Yibo; Ma, Tianxiao; Nie, Yonggang; Xie, Yan; Wei, Fuwen

2016-01-01

Understanding population size and genetic diversity is critical for effective conservation of endangered species. The Amur tiger (Panthera tigris altaica) is the largest felid and a flagship species for wildlife conservation. Due to habitat loss and human activities, available habitat and population size are continuously shrinking. However, little is known about the true population size and genetic diversity of wild tiger populations in China. In this study, we collected 55 fecal samples and 1 hair sample to investigate the population size and genetic diversity of wild Amur tigers in Hunchun National Nature Reserve, Jilin Province, China. From the samples, we determined that 23 fecal samples and 1 hair sample were from 7 Amur tigers: 2 males, 4 females and 1 individual of unknown sex. Interestingly, 2 fecal samples that were presumed to be from tigers were from Amur leopards, highlighting the significant advantages of noninvasive genetics over traditional methods in studying rare and elusive animals. Analyses from this sample suggested that the genetic diversity of wild Amur tigers is much lower than that of Bengal tigers, consistent with previous findings. Furthermore, the genetic diversity of this Hunchun population in China was lower than that of the adjoining subpopulation in southwest Primorye Russia, likely due to sampling bias. Considering the small population size and relatively low genetic diversity, it is urgent to protect this endangered local subpopulation in China. © 2015 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods.

PubMed

Gyawali, P; Ahmed, W; Sidhu, J P S; Nery, S V; Clements, A C; Traub, R; McCarthy, J S; Llewellyn, S; Jagals, P; Toze, S

2016-09-01

In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79-80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 10(5) ± 6.4 × 10(5) and 6.3 × 10(5) ± 4.7 × 10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 10(5) ± 1.5 × 10(4) (determined by qPCR) compared to 4.9 × 10(4) ± 3.7 × 10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

Coliphages and Gastrointestinal Illness in Recreational Waters

PubMed Central

Benjamin-Chung, Jade; Arnold, Benjamin F.; Wade, Timothy J.; Schiff, Kenneth; Griffith, John F.; Dufour, Alfred P.; Weisberg, Stephen B.

2017-01-01

Background: Coliphages have been proposed as indicators of fecal contamination in recreational waters because they better mimic the persistence of pathogenic viruses in the environment and wastewater treatment than fecal indicator bacteria. We estimated the association between coliphages and gastrointestinal illness and compared it with the association with culturable enterococci. Methods: We pooled data from six prospective cohort studies that enrolled coastal beachgoers in California, Alabama, and Rhode Island. Water samples were collected and gastrointestinal illness within 10 days of the beach visit was recorded. Samples were tested for enterococci and male-specific and somatic coliphages. We estimated cumulative incidence ratios (CIR) for the association between swimming in water with detectable coliphage and gastrointestinal illness when human fecal pollution was likely present, not likely present, and under all conditions combined. The reference group was unexposed swimmers. We defined continuous and threshold-based exposures (coliphage present/absent, enterococci >35 vs. ≤35 CFU/100 ml). Results: Under all conditions combined, there was no association between gastrointestinal illness and swimming in water with detectable coliphage or enterococci. When human fecal pollution was likely present, coliphage and enterococci were associated with increased gastrointestinal illness, and there was an association between male-specific coliphage level and illness that was somewhat stronger than the association between enterococci and illness. There were no substantial differences between male-specific and somatic coliphage. Conclusions: Somatic coliphage and enterococci had similar associations with gastrointestinal illness; there was some evidence that male-specific coliphage had a stronger association with illness than enterococci in marine waters with human fecal contamination. PMID:28489717

Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial [ISRCTN10820810

PubMed Central

St-Onge, Marie-Pierre; Farnworth, Edward R; Savard, Tony; Chabot, Denise; Mafu, Akier; Jones, Peter JH

2002-01-01

Background Fermented milk products have been shown to affect serum cholesterol concentrations in humans. Kefir, a fermented milk product, has been traditionally consumed for its potential health benefits but has to date not been studied for its hypocholesterolemic properties. Methods Thirteen healthy mildly hypercholesterolemic male subjects consumed a dairy supplement in randomized crossover trial for 2 periods of 4 wk each. Subjects were blinded to the dairy supplement consumed. Blood samples were collected at baseline and after 4 wk of supplementation for measurement of plasma total, low-density lipoprotein, and high-density lipoprotein cholesterol and triglyceride concentrations, as well as fatty acid profile and cholesterol synthesis rate. Fecal samples were collected at baseline and after 2 and 4 wk of supplementation for determination of fecal short chain fatty acid level and bacterial content. Results Kefir had no effect on total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglyceride concentrations nor on cholesterol fractional synthesis rates after 4 wk of supplementation. No significant change on plasma fatty acid levels was observed with diet. However, both kefir and milk increased (p < 0.05) fecal isobutyric, isovaleric and propionic acids as well as the total amount of fecal short chain fatty acids. Kefir supplementation resulted in increased fecal bacterial content in the majority of the subjects. Conclusions Since kefir consumption did not result in lowered plasma lipid concentrations, the results of this study do not support consumption of kefir as a cholesterol-lowering agent. PMID:11825344

Influence of wet distiller's grains on prevalence of Escherichia coli O157:H7 and Salmonella in feedlot cattle and antimicrobial susceptibility of generic Escherichia coli isolates.

PubMed

Edrington, Tom S; MacDonald, Jim C; Farrow, Russell L; Callaway, Todd R; Anderson, Robin C; Nisbet, David J

2010-05-01

The current research examined the inclusion of 20% wet distiller's grains (WDG) fed with steam-flaked corn (SFC) or dry-rolled corn (DRC) in diets fed to feedlot cattle on fecal prevalence of Escherichia coli O157:H7 and Salmonella. Crossbred beef heifers (n = 272; average initial body weight (BW) = 354 kg) were blocked by BW and pen size and randomly assigned to treatment. Fecal samples from freshly voided fecal pats were collected from each pen on the day cattle shipped for slaughter (237 fecal samples: 72, 125, and 40 from cattle 132, 160, and 181 days on feed, respectively). Fecal samples were cultured quantitatively and qualitatively for the above pathogens. Populations of E. coli O157:H7 and Salmonella were generally low with very few samples containing quantifiable populations. Similarly, after enrichment, few samples were E. coli O157:H7 positive in any collection with no treatment differences (p > 0.10). More samples were Salmonella positive during the first collection with an increased (p < 0.05) prevalence observed in the SFC and DRC treatments compared with DRC + WDG treatment. No other treatment differences were observed for Salmonella. Putative fecal coliform isolates (18 per treatment; first collection) were examined for antimicrobial susceptibility, and the majority were susceptible to all of the antibiotics examined. Most of the resistance was observed in the SFC (n = 3) and DRC (n = 4) treatments, and only one isolate in each of the two WDG treatments demonstrated resistance (one antibiotic each, streptomycin and tetracycline). All multidrug resistance (2-4 antibiotics) was observed in isolates cultured from the DRC and SFC treatments (n = 2 isolates in each treatment). Results of the current research found no significant effect of feeding WDG to feedlot cattle on fecal prevalence, at time of shipment for slaughter, of E. coli O157:H7, and only modest differences (decreases) in Salmonella prevalence with no apparent affect on antimicrobial susceptibility of fecal coliform isolates.

Comparison of the Immunomagnetic Separation/Adenosine Triphosphate Rapid Method and the Modified mTEC Membrane-Filtration Method for Enumeration of Escherichia coli

USGS Publications Warehouse

Brady, Amie M.G.; Bushon, Rebecca N.; Bertke, Erin E.

2009-01-01

Water quality at beaches is monitored for fecal indicator bacteria by traditional, culture-based methods that can take 18 to 24 hours to obtain results. A rapid detection method that provides estimated concentrations of fecal indicator bacteria within 1 hour from the start of sample processing would allow beach managers to post advisories or close the beach when the conditions are actually considered unsafe instead of a day later, when conditions may have changed. A rapid method that couples immunomagnetic separation with adenosine triphosphate detection (IMS/ATP rapid method) was evaluated through monitoring of Escherichia coli (E. coli) at three Lake Erie beaches in Ohio (Edgewater and Villa Angela in Cleveland and Huntington in Bay Village). Beach water samples were collected between 4 and 5 days per week during the recreational seasons (May through September) of 2006 and 2007. Composite samples were created in the lab from two point samples collected at each beach and were shown to be comparable substitutes for analysis of two individual samples. E. coli concentrations in composite samples, as determined by the culture-based method, ranged from 4 to 24,000 colony-forming units per 100 milliliters during this study across all beaches. Turbidity also was measured for each sample and ranged from 0.8 to 260 neophelometric turbidity ratio units. Environmental variables were noted at the time of sampling, including number of birds at the beach and wave height. Rainfall amounts were measured at National Weather Service stations at local airports. Turbidity, rainfall, and wave height were significantly related to the culture-based method results each year and for both years combined at each beach. The number of birds at the beach was significantly related to the culture-based method results only at Edgewater during 2006 and during both years combined. Results of the IMS/ATP method were compared to results of the culture-based method for samples by year for each beach. The IMS/ATP method underwent several changes and refinements during the first year, including changes in reagents and antibodies and alterations to the method protocol. Because of the changes in the method, results from the two years of study could not be combined. Kendall's tau correlation coefficients for relations between the IMS/ATP and culture-based methods were significant except for samples collected during 2006 at Edgewater and for samples collected during 2007 at Villa Angela. Further, relations were stronger for samples collected in 2006 than for those collected in 2007, except at Edgewater where the reverse was observed. The 2007 dataset was examined to identify possible reasons for the observed difference in significance of relations by year. By dividing the 2007 data set into groups as a function of sampling date, relations (Kendall's tau) between methods were observed to be stronger for samples collected earlier in the season than for those collected later in the season. At Edgewater and Villa Angela, there were more birds at the beach at time of sampling later in the season compared to earlier in the season. (The number of birds was not examined at Huntington.) Also, more wet days (when rainfall during the 24 hours prior to sampling was greater than 0.05 inch) were sampled later in the season compared to earlier in the season. Differences in the dominant fecal source may explain the change in the relations between the culture-based and IMS/ATP methods.

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

USGS Publications Warehouse

Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

2007-01-01

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

Canine fecal contamination in a metropolitan area (Milan, north-western Italy): prevalence of intestinal parasites and evaluation of health risks.

PubMed

Zanzani, Sergio Aurelio; Di Cerbo, Anna Rita; Gazzonis, Alessia Libera; Genchi, Marco; Rinaldi, Laura; Musella, Vincenzo; Cringoli, Giuseppe; Manfredi, Maria Teresa

2014-01-01

Intestinal parasites of dogs represent a serious threat to human health due to their zoonotic potential. Thus, metropolitan areas presenting high concentrations of pets and urban fecal contamination on public areas are at sanitary risk. Major aim of this survey was to determine prevalence of zoonotic parasites in dog fecal samples collected from public soil of Milan (north-western Italy). Differences in parasites prevalence distribution were explored by a geographical information system- (GIS-) based approach, and risk factors (human density, sizes of green parks, and dog areas) were considered. The metropolitan area was divided into 157 rectangular subareas and sampling was performed following a 1-kilometer straight transect. A total of 463 fecal samples were analyzed using centrifugation-flotation technique and ELISA to detect Giardia and Cryptosporidium coproantigens. A widespread fecal contamination of soil was highlighted, being fecal samples found in 86.8% of the subareas considered. The overall prevalence of intestinal parasites was 16.63%. Zoonotic parasites were found, such as Trichuris vulpis (3.67%), Toxocara canis (1.72%), Strongyloides stercoralis (0.86%), Ancylostomatidae (0.43%), and Dipylidium caninum (0.43%). Giardia duodenalis was the most prevalent zoonotic protozoa (11.06%), followed by Cryptosporidium (1.10%). Faeces from subareas characterized by broad green areas showed to be particularly prone to infection.

Canine Fecal Contamination in a Metropolitan Area (Milan, North-Western Italy): Prevalence of Intestinal Parasites and Evaluation of Health Risks

PubMed Central

Zanzani, Sergio Aurelio; Di Cerbo, Anna Rita; Gazzonis, Alessia Libera; Genchi, Marco; Rinaldi, Laura; Musella, Vincenzo; Cringoli, Giuseppe

2014-01-01

Intestinal parasites of dogs represent a serious threat to human health due to their zoonotic potential. Thus, metropolitan areas presenting high concentrations of pets and urban fecal contamination on public areas are at sanitary risk. Major aim of this survey was to determine prevalence of zoonotic parasites in dog fecal samples collected from public soil of Milan (north-western Italy). Differences in parasites prevalence distribution were explored by a geographical information system- (GIS-) based approach, and risk factors (human density, sizes of green parks, and dog areas) were considered. The metropolitan area was divided into 157 rectangular subareas and sampling was performed following a 1-kilometer straight transect. A total of 463 fecal samples were analyzed using centrifugation-flotation technique and ELISA to detect Giardia and Cryptosporidium coproantigens. A widespread fecal contamination of soil was highlighted, being fecal samples found in 86.8% of the subareas considered. The overall prevalence of intestinal parasites was 16.63%. Zoonotic parasites were found, such as Trichuris vulpis (3.67%), Toxocara canis (1.72%), Strongyloides stercoralis (0.86%), Ancylostomatidae (0.43%), and Dipylidium caninum (0.43%). Giardia duodenalis was the most prevalent zoonotic protozoa (11.06%), followed by Cryptosporidium (1.10%). Faeces from subareas characterized by broad green areas showed to be particularly prone to infection. PMID:25478583

Intestinal blood loss as an aggravating factor of iron deficiency in infants aged 9 to 12 months fed whole cow's milk.

PubMed

Fernandes, Sandra Maria Rodrigues; de Morais, Mauro Batista; Amancio, Olga Maria Silverio

2008-02-01

To verify the occurrence of occult intestinal blood loss and iron deficiency in infants aged 9 to 12 months. A consecutive sample of 98 infants of the Pediatric Public Health Primary Care Unit in the town of Arapongas, Parana State, Brazil was involved in this cross-sectional study. Dietary history, hemoglobin, serum iron, transferrin saturation, ferritin, and an occult fecal blood loss investigation, by the immune chromatographic method specific for human hemoglobin were performed. Presence of occult intestinal blood occurred in 8/23 of the breast-fed (plus complementary feed) infants and in 30/64 of the infants who were fed with cow's milk (plus complementary feed) (P=0.449). The comparison of body iron indicators in accordance to positive or negative occult fecal blood, did not show any significant difference in the 23 breast-fed infants. Serum ferritin (median=4.2 ng/mL) was significantly lower (P=0.004) in infants who received whole cow's milk and had positive occult fecal blood, than in those infants who received whole cow's milk but were without occult fecal blood (median=12.1 ng/mL). In breast-fed infants with negative occult fecal blood, iron deficiency severity is not greater than in those with positive results. In infants fed whole cow's milk, occult fecal blood loss is an aggravating factor of iron deficiency.

Plasma Septin9 versus Fecal Immunochemical Testing for Colorectal Cancer Screening: A Prospective Multicenter Study

PubMed Central

Johnson, David A.; Barclay, Robert L.; Mergener, Klaus; Weiss, Gunter; König, Thomas; Beck, Jürgen; Potter, Nicholas T.

2014-01-01

Background Screening improves outcomes related to colorectal cancer (CRC); however, suboptimal participation for available screening tests limits the full benefits of screening. Non-invasive screening using a blood based assay may potentially help reach the unscreened population. Objective To compare the performance of a new Septin9 DNA methylation based blood test with a fecal immunochemical test (FIT) for CRC screening. Design: In this trial, fecal and blood samples were obtained from enrolled patients. To compare test sensitivity for CRC, patients with screening identified colorectal cancer (n = 102) were enrolled and provided samples prior to surgery. To compare test specificity patients were enrolled prospectively (n = 199) and provided samples prior to bowel preparation for screening colonoscopy. Measurements Plasma and fecal samples were analyzed using the Epi proColon and OC Fit-Check tests respectively. Results For all samples, sensitivity for CRC detection was 73.3% (95% CI 63.9–80.9%) and 68.0% (95% CI 58.2–76.5%) for Septin9 and FIT, respectively. Specificity of the Epi proColon test was 81.5% (95% CI 75.5–86.3%) compared with 97.4% (95% CI 94.1–98.9%) for FIT. For paired samples, the sensitivity of the Epi proColon test (72.2% –95% CI 62.5–80.1%) was shown to be statistically non-inferior to FIT (68.0%–95% CI 58.2–76.5%). When test results for Epi proColon and FIT were combined, CRC detection was 88.7% at a specificity of 78.8%. Conclusions At a sensitivity of 72%, the Epi proColon test is non- inferior to FIT for CRC detection, although at a lower specificity. With negative predictive values of 99.8%, both methods are identical in confirming the absence of CRC. Trial Registration ClinicalTrials.gov NCT01580540 PMID:24901436

Water quality and sources of fecal coliform bacteria in the Meduxnekeag River, Houlton, Maine

USGS Publications Warehouse

Culbertson, Charles W.; Huntington, Thomas G.; Stoeckel, Donald M.; Caldwell, James M.; O'Donnell, Cara

2014-01-01

In response to bacterial contamination in the Meduxnekeag River and the desire to manage the watershed to reduce contaminant sources, the Houlton Band of Maliseet Indians (HBMI) and the U.S. Geological Survey began a cooperative effort to establish a baseline of water-quality data that can be used in future studies and to indicate potential sources of nutrient and bacterial contamination. This study was conducted during the summer of 2005 in the Meduxnekeag River Basin near Houlton, Maine. Continuously recorded specific conductance can be a good indicator for water quality. Specific conductance increased downstream from the town of Houlton, between runoff events, and decreased sharply following major runoff events. Collections of discrete samples during the summer of 2005 indicated seasonal positive concentration-discharge relations for total phosphorus and total nitrogen; these results indicate that storm runoff may mobilize and transport these nutrients from the terrestrial environment to the river. Data collected by the HBMI on fecal coliform bacteria indicated that bacterial contamination enters the Meduxnekeag River from multiple paths including tributaries and surface drains (ditches) in developed areas in Houlton, Maine. The Houlton wastewater treatment discharge was not an important source of bacterial contamination. Bacteroidales-based tests for general fecal contamination (Bac32 marker) were predominantly positive in samples that had excessive fecal contamination as indicated by Enterococci density greater than 104 colony-forming units per 100 millilters. Of the 22 samples tested for Bacteroidales-based markers of human-associated fecal contamination (HF134 and HF183), 8 were positive. Of the 22 samples tested for Bacteroidales-based markers of ruminant-associated fecal contamination (CF128 and CF193), 7 were positive. Human fecal contamination was detected consistently at two sites (surface drains in urban areas in the town of Houlton) and occasionally detected at one site (Moose Brook) but was not detected at other sites. Fecal contamination (as indicated by fecal coliform density) apparently is localized under normal flow conditions with the highest levels restricted to drains in urban areas and to a lesser extent B Stream, Pearce Brook, and Big Brook, all tributaries to the main stem of the Meduxnekeag River. Coliphage were enumerated as an alternate indicator of fecal contamination with the intent of typing the virus into host-associated classes (human or ruminant), as was done for Enterococci; however, insufficient coliphage were isolated to provide more than preliminary indications. In spite of low coliphage enumeration, the preliminary results strengthen the conclusion that the Enterococci data correctly indicated the samples that contained human and ruminant fecal contamination. The finding that contamination was in many of the tributaries following storms in mid-July indicates that storm runoff likely carries fecal contaminants to more locations than runoff under lower flow conditions.

Microbiological quality of Puget Sound Basin streams and identification of contaminant sources

USGS Publications Warehouse

Embrey, S.S.

2001-01-01

Fecal coliforms, Escherichia coli, enterococci, and somatic coliphages were detected in samples from 31 sites on streams draining urban and agricultural regions of the Puget Sound Basin Lowlands. Densities of bacteria in 48 and 71 percent of the samples exceeded U.S. Environmental Protection Agency's freshwater recreation criteria for Escherichia coli and enterococci, respectively, and 81 percent exceeded Washington State fecal coliform standards. Male-specific coliphages were detected in samples from 15 sites. Male-specific F+RNA coliphages isolated from samples taken at South Fork Thornton and Longfellow Creeks were serotyped as Group II, implicating humans as potential contaminant sources. These two sites are located in residential, urban areas. F+RNA coliphages in samples from 10 other sites, mostly in agricultural or rural areas, were serotyped as Group I, implicating non-human animals as likely sources. Chemicals common to wastewater, including fecal sterols, were detected in samples from several urban streams, and also implicate humans, at least in part, as possible sources of fecal bacteria and viruses to the streams.

Fluoroquinolones and qnr genes in sediment, water, soil, and human fecal flora in an environment polluted by manufacturing discharges.

PubMed

Rutgersson, Carolin; Fick, Jerker; Marathe, Nachiket; Kristiansson, Erik; Janzon, Anders; Angelin, Martin; Johansson, Anders; Shouche, Yogesh; Flach, Carl-Fredrik; Larsson, D G Joakim

2014-07-15

There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.

Salmonella Prevalence and Antimicrobial Susceptibility from the National Animal Health Monitoring System Sheep 2011 Study.

PubMed

Dargatz, David A; Marshall, Katherine L; Fedorka-Cray, Paula J; Erdman, Matthew M; Kopral, Christine A

2015-12-01

Salmonella is a major cause of foodborne illness and can cause clinical disease in animals. Understanding the on-farm ecology of Salmonella will be helpful in decreasing the risk of foodborne transmission. An objective of this study was to determine the prevalence of Salmonella among fecal samples collected on sheep operations in the United States. Another objective was to compare the use of composite fecal samples with fecal samples collected from individual sheep as a tool for screening sheep flocks for Salmonella. Sheep fecal samples (individual and composite) were collected on operations in 22 states. Salmonella isolates were characterized with regard to species, serotype, and antimicrobial susceptibility profile. Most operations (72.1%) had at least one positive sample and overall 26.9% of samples were positive. The percentage of positive samples varied by animal age class. Composite and individual samples gave similar results. The majority of the isolates (94%) were Salmonella enterica subspecies diarizonae serotype 61:-:1,5,7. Nearly all of the isolates (91.2%) tested for antimicrobial susceptibility were susceptible to all antimicrobials in the panel. The findings suggest that salmonellae typically associated with foodborne disease transmission are infrequently found on sheep operations in the United States.

Evaluation of fecal elastase and serum cholecystokinin in dogs with a false positive fecal elastase test.

PubMed

Steiner, J M; Rehfeld, J F; Pantchev, N

2010-01-01

An assay for the measurement of pancreatic elastase in dog feces has been introduced. The goal of this study was to evaluate the rate of false-positive fecal-elastase test results in dogs with suspected exocrine pancreatic insufficiency (EPI) and to assess serum cholecystokinin (CCK) concentrations in dogs with a false positive fecal elastase test result. Twenty-six fecal and serum samples from dogs suspected of EPI, for which samples had been submitted to a commercial laboratory (Vet Med Labor) for analysis. Prospective study. Serum trypsin-like immunoreactivity (TLI) was measured in 26 dogs with a decreased fecal elastase concentration of <10 microg/g feces. Serum CCK concentrations were measured in 21 of these dogs. Of 26 dogs with a decreased fecal elastase concentration, 6 (23%) had serum TLI concentrations within or above the reference range. Serum CCK concentrations were significantly higher in dogs with a true positive fecal elastase test result (median: 1.1 pmol/L; range: 0.1-3.3 pmol/L) than in those with a false positive fecal elastase test result (median: 0.1 pmol/L; range: 0.1-0.9 pmol/L; P value = .0163). The rate of false positive fecal elastase test results was high in this group of dogs, suggesting that diagnosis of EPI must be confirmed by other means. The decreased CCK concentration in dogs with a false positive fecal elastase test result could suggest that false positive results are because of decreased stimulation of exocrine pancreatic function caused by other conditions.

Integral strategy for evaluation of fecal indicator performance in bird-influenced saline inland waters.

PubMed

Kirschner, Alexander K T; Zechmeister, Thomas C; Kavka, Gerhard G; Beiwl, Christian; Herzig, Alois; Mach, Robert L; Farnleitner, Andreas H

2004-12-01

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive Escherichia coli and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that E. coli and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems.

Integral Strategy for Evaluation of Fecal Indicator Performance in Bird-Influenced Saline Inland Waters

PubMed Central

Kirschner, Alexander K. T.; Zechmeister, Thomas C.; Kavka, Gerhard G.; Beiwl, Christian; Herzig, Alois; Mach, Robert L.; Farnleitner, Andreas H.

2004-01-01

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive Escherichia coli and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that E. coli and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems. PMID:15574941

Eukaryotic viruses in wastewater samples from the United States

USGS Publications Warehouse

Symonds, E.M.; Griffin, Dale W.; Breitbart, M.

2009-01-01

Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Comparison of a modified phenol/chloroform and commercial-kit methods for extracting DNA from horse fecal material.

PubMed

Janabi, Ali H D; Kerkhof, Lee J; McGuinness, Lora R; Biddle, Amy S; McKeever, Kenneth H

2016-10-01

There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P<0.05). In contrast, Firmicutes had greater relative abundance and more unique species in Ph/Chl+C3 extracts than in MoBio-K (P<0.05). The other major bacterial phyla were equally abundant in samples using both extraction methods. Alpha diversity and Shannon Weaver indices showed greater evenness of bacterial distribution in Ph/Chl+C3 compared with MoBio-K (P<0.05), but there was no difference in the OTU richness. Principle coordinate analysis (PCoA) indicated a distinct separation between the two methods (P<0.05) and tighter clustering (less variability) in Ph/Chl+C3 than in MoBio-K. These results suggest that the Ph/Chl+C3 may be preferred for research to identify specific Firmicutes taxa such as Clostridium, and Bacillus. However; MoBio-K may be a better choice for projects focusing on Bacteroidetes abundance. The Ph/Chl+C3 method required less time, but has some safety concerns associated with exposure and disposal of phenol and chloroform. While the MoBio-K may be better choice for researchers with less access to safety equipment like a fume hood. Copyright © 2016 Elsevier B.V. All rights reserved.

State of the art molecular markers for fecal pollution source tracking in water.

PubMed

Roslev, Peter; Bukh, Annette S

2011-03-01

Most environmental waters are susceptible to fecal contamination from animal and/or human pollution sources. To attenuate or eliminate such contamination, it is often critical that the pollution sources are rapidly and correctly identified. Fecal pollution source tracking (FST) is a promising research area that aims to identify the origin(s) of fecal pollution in water. This mini-review focuses on the potentials and limitations of library independent molecular markers that are exclusively or strongly associated with fecal pollution from humans and different animals. Fecal-source-associated molecular markers include nucleic acid sequences from prokaryotes and viruses associated with specific biological hosts, but also sequences such as mitochondrial DNA retrieved directly from humans and animals. However, some fecal-source-associated markers may not be absolutely specific for a given source type, and apparent specificity and frequency established in early studies are sometimes compromised by new studies suggesting variation in specificity and abundance on a regional, global and/or temporal scale. It is therefore recommended that FST studies are based on carefully selected arrays of markers, and that identification of human and animal contributions are based on a multi-marker toolkit with several markers for each source category. Furthermore, future FST studies should benefit from increased knowledge regarding sampling strategies and temporal and spatial variability of marker ratios. It will also be important to obtain a better understanding of marker persistence and the quantitative relationship between marker abundance and the relative contribution from individual fecal pollution source types. A combination of enhanced pathogen screening methods, and validated quantitative source tracking techniques could then contribute significantly to future management of environmental water quality including improved microbial risk assessment.

Bacterial indicator occurrence and the use of an F+ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida

USGS Publications Warehouse

Griffin, Dale W.; Stokes, Rodger; Rose, J.B.; Paul, J.H.

2000-01-01

A microbiological water quality study of Homosassa Springs State Wildlife Park (HSSWP) and surrounding areas was undertaken. Samples were collected in November of 1997 (seven sites) and again in November of 1998 (nine sites). Fecal bacterial concentrations (total and fecal coliforms, Clostridium perfringens, and enterococci) were measured as relative indicators of fecal contamination. F+-specific coliphage genotyping was performed to determine the source of fecal contamination at the study sites. Bacterial levels were considerably higher at most sites in the 1997 sampling compared to the 1998 sampling, probably because of the greater rainfall that year. In November of 1997, 2 of the 7 sites were in violation of all indicator standards and guidance levels. In November of 1998, 1 of 9 sites was in violation of all indicator standard and guidance levels. The highest concentrations of all fecal indicators were found at a station downstream of the animal holding pens in HSSWP. The lowest levels of indicators were found at the Homosassa Main Spring vent. Levels of fecal indicators downstream of HSSWP (near the point of confluence with the river) were equivalent to those found in the Southeastern Fork and areas upstream of the park influences. F+ specific RNA coliphage analysis indicated that fecal contamination at all sites that tested positive was from animal sources (mammals and birds). These results suggest that animal (indigenous and those in HSSWP) and not human sources influenced microbial water quality in the area of Homosassa River covered by this study.

IDENTIFICATION OF CHICKEN-SPECIFIC FECAL MICROBIAL SEQUENCES USING A METAGENOMIC APPROACH

EPA Science Inventory

In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ between different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (C-P) and between chicken fecal DNA and an ...

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

PubMed

Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

2003-02-01

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Effects of green tea consumption on human fecal microbiota with special reference to Bifidobacterium species.

PubMed

Jin, Jong-Sik; Touyama, Mutsumi; Hisada, Takayoshi; Benno, Yoshimi

2012-11-01

Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10-day green-tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer-restriction enzyme systems for HIM and by using a real-time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter-individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter-species transition, but by an intra-species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species. © 2012 The Societies and Wiley Publishing Asia Pty Ltd.

OCCURRENCE OF INTRINSIC VANCOMYCIN RESISTANT ENTEROCOCCI IN ANIMAL FECES

EPA Science Inventory

A survey was conducted to determine the occurrence of vancomycin resistant enterococci (VRE) in animal and human fecal samples. Fecal samples from 14 animal species and humans were analyzed by quantitative culture for enterococci and VRE. Over 800 VRE isolates were characterize...

DNA from fecal immunochemical test can replace stool for detection of colonic lesions using a microbiota-based model.

PubMed

Baxter, Nielson T; Koumpouras, Charles C; Rogers, Mary A M; Ruffin, Mack T; Schloss, Patrick D

2016-11-14

There is a significant demand for colorectal cancer (CRC) screening methods that are noninvasive, inexpensive, and capable of accurately detecting early stage tumors. It has been shown that models based on the gut microbiota can complement the fecal occult blood test and fecal immunochemical test (FIT). However, a barrier to microbiota-based screening is the need to collect and store a patient's stool sample. Using stool samples collected from 404 patients, we tested whether the residual buffer containing resuspended feces in FIT cartridges could be used in place of intact stool samples. We found that the bacterial DNA isolated from FIT cartridges largely recapitulated the community structure and membership of patients' stool microbiota and that the abundance of bacteria associated with CRC were conserved. We also found that models for detecting CRC that were generated using bacterial abundances from FIT cartridges were equally predictive as models generated using bacterial abundances from stool. These findings demonstrate the potential for using residual buffer from FIT cartridges in place of stool for microbiota-based screening for CRC. This may reduce the need to collect and process separate stool samples and may facilitate combining FIT and microbiota-based biomarkers into a single test. Additionally, FIT cartridges could constitute a novel data source for studying the role of the microbiome in cancer and other diseases.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has provided recommended beach advisory values in its 2012 recreational water quality criteria (RWQC) for states wishing to use quantitative polymerase chain reaction (qPCR) for the monitoring of Enterococcus fecal indicator bacteria...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Detection of low-concentration host mRNA transcripts in Malawian children at risk for environmental enteropathy

USDA-ARS?s Scientific Manuscript database

Transcriptomic analysis of fecal samples is an emerging method for the diagnosis of gastrointestinal pathology because it is noninvasive and requires minute volumes of analyte; however, detection of mRNA in low copy numbers in human stool is challenging. Our objective was to develop a method for det...

Viral metagenomic analysis of feces of wild small carnivores

PubMed Central

2014-01-01

Background Recent studies have clearly demonstrated the enormous virus diversity that exists among wild animals. This exemplifies the required expansion of our knowledge of the virus diversity present in wildlife, as well as the potential transmission of these viruses to domestic animals or humans. Methods In the present study we evaluated the viral diversity of fecal samples (n = 42) collected from 10 different species of wild small carnivores inhabiting the northern part of Spain using random PCR in combination with next-generation sequencing. Samples were collected from American mink (Neovison vison), European mink (Mustela lutreola), European polecat (Mustela putorius), European pine marten (Martes martes), stone marten (Martes foina), Eurasian otter (Lutra lutra) and Eurasian badger (Meles meles) of the family of Mustelidae; common genet (Genetta genetta) of the family of Viverridae; red fox (Vulpes vulpes) of the family of Canidae and European wild cat (Felis silvestris) of the family of Felidae. Results A number of sequences of possible novel viruses or virus variants were detected, including a theilovirus, phleboviruses, an amdovirus, a kobuvirus and picobirnaviruses. Conclusions Using random PCR in combination with next generation sequencing, sequences of various novel viruses or virus variants were detected in fecal samples collected from Spanish carnivores. Detected novel viruses highlight the viral diversity that is present in fecal material of wild carnivores. PMID:24886057

Antimicrobial Use and Resistance in Swine Waste Treatment Systems▿

PubMed Central

Jindal, Archana; Kocherginskaya, Svetlana; Mehboob, Asma; Robert, Matthew; Mackie, Roderick I.; Raskin, Lutgarde; Zilles, Julie L.

2006-01-01

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLSB), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLSB resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLSB resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further. PMID:17041160

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.

PubMed

Sun, Kui; Xing, Weiwei; Yu, Xinling; Fu, Wenliang; Wang, Yuanyuan; Zou, Minji; Luo, Zhihong; Xu, Donggang

2016-08-31

With the continuous decline in prevalence and intensity of Schistosoma japonicum infection in China, more accurate and sensitive methods suitable for field detection become much needed for schistosomiasis control. Here, a novel rapid and visual detection method based on the combination of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) was developed to detect S. japonicum DNA in fecal samples. The LFD-RPA assay targeting SjR2 could detect 5 fg S. japonicum DNA, which was identical to qPCR and real-time RPA assay, and showed no cross-reaction with other parasites. The detection could be finished within 15-20 min at a wide temperature range (25-45 °C), and the results could be visualized by naked eye. The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons, and compared with that of Enzyme-linked immunosorbent assay (ELSIA) and Indirect Hemagglutination Assay (IHA). The LFD-RPA assay showed 92.68 % sensitivity, 100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test (k = 0.947, Z = 6.36, P < 0.001), whereas ELISA showed 85.71 % sensitivity, 93.55 % specificity, and substantial diagnostic agreement (k = 0.793, Z = 5.31, P < 0.001), and IHA showed 78.57 % sensitivity, 83.87 % specificity, and moderate diagnostic agreement (k = 0.600, Z = 4.05, P < 0.001), indicating that the LFD-RPA was much better than the traditional methods. The LFD-RPA assay established by us is a sensitive, specific, rapid and convenient method for the diagnosis of schistosomiasis, and shows a great potency in field application.

Evaluation of methods to sample fecal indicator bacteria in foreshore sand and pore water at freshwater beaches.

PubMed

Vogel, Laura J; Edge, Thomas A; O'Carroll, Denis M; Solo-Gabriele, Helena M; Kushnir, Caitlin S E; Robinson, Clare E

2017-09-15

Fecal indicator bacteria (FIB) are known to accumulate in foreshore beach sand and pore water (referred to as foreshore reservoir) where they act as a non-point source for contaminating adjacent surface waters. While guidelines exist for sampling surface waters at recreational beaches, there is no widely-accepted method to collect sand/sediment or pore water samples for FIB enumeration. The effect of different sampling strategies in quantifying the abundance of FIB in the foreshore reservoir is unclear. Sampling was conducted at six freshwater beaches with different sand types to evaluate sampling methods for characterizing the abundance of E. coli in the foreshore reservoir as well as the partitioning of E. coli between different components in the foreshore reservoir (pore water, saturated sand, unsaturated sand). Methods were evaluated for collection of pore water (drive point, shovel, and careful excavation), unsaturated sand (top 1 cm, top 5 cm), and saturated sand (sediment core, shovel, and careful excavation). Ankle-depth surface water samples were also collected for comparison. Pore water sampled with a shovel resulted in the highest observed E. coli concentrations (only statistically significant at fine sand beaches) and lowest variability compared to other sampling methods. Collection of the top 1 cm of unsaturated sand resulted in higher and more variable concentrations than the top 5 cm of sand. There were no statistical differences in E. coli concentrations when using different methods to sample the saturated sand. Overall, the unsaturated sand had the highest amount of E. coli when compared to saturated sand and pore water (considered on a bulk volumetric basis). The findings presented will help determine the appropriate sampling strategy for characterizing FIB abundance in the foreshore reservoir as a means of predicting its potential impact on nearshore surface water quality and public health risk. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sample handling factors affecting the enumeration of lactobacilli and cellulolytic bacteria in equine feces

USDA-ARS?s Scientific Manuscript database

The objectives were to compare media types and evaluate the effects of fecal storage time and temperature on the enumeration of cellulolytic bacteria and lactobacilli from horses. Fecal samples were collected from horses (n = 3) and transported to the lab (CO2, 37 ºC, 0.5 h). The samples were assign...

Evidence of Avian and Possum Fecal Contamination in Rainwater Tanks as Determined by Microbial Source Tracking Approaches

PubMed Central

Hamilton, K. A.; Gyawali, P.; Toze, S.; Haas, C. N.

2016-01-01

ABSTRACT Avian and possum fecal droppings may negatively impact roof-harvested rainwater (RHRW) water quality due to the presence of zoonotic pathogens. This study was aimed at evaluating the performance characteristics of a possum feces-associated (PSM) marker by screening 210 fecal and wastewater samples from possums (n = 20) and a range of nonpossum hosts (n = 190) in Southeast Queensland, Australia. The host sensitivity and specificity of the PSM marker were 0.90 and 0.95 (maximum value, 1.00), respectively. The mean concentrations of the GFD marker in possum fecal DNA samples (8.8 × 107 gene copies per g of feces) were two orders of magnitude higher than those in the nonpossum fecal DNA samples (5.0 × 105 gene copies per g of feces). The host sensitivity, specificity, and concentrations of the avian feces-associated GFD marker were reported in our recent study (W. Ahmed, V. J. Harwood, K. Nguyen, S. Young, K. Hamilton, and S. Toze, Water Res 88:613–622, 2016, http://dx.doi.org/10.1016/j.watres.2015.10.050). The utility of the GFD and PSM markers was evaluated by testing a large number of tank water samples (n = 134) from the Brisbane and Currumbin areas. GFD and PSM markers were detected in 39 of 134 (29%) and 11 of 134 (8%) tank water samples, respectively. The GFD marker concentrations in PCR-positive samples ranged from 3.7 × 102 to 8.5 × 105 gene copies per liter, whereas the concentrations of the PSM marker ranged from 2.0 × 103 to 6.8 × 103 gene copies per liter of water. The results of this study suggest the presence of fecal contamination in tank water samples from avian and possum hosts. This study has established an association between the degradation of microbial tank water quality and avian and possum feces. Based on the results, we recommend disinfection of tank water, especially for tanks designated for potable use. IMPORTANCE The use of roof-harvested rainwater (RHRW) for domestic purposes is a globally accepted practice. The presence of pathogens in rainwater tanks has been reported by several studies, supporting the necessity for the management of potential health risks. The sources of fecal pollution in rainwater tanks are unknown. However, the application of microbial source tracking (MST) markers has the potential to identify the sources of fecal contamination in a rainwater tank. In this study, we provide evidence of avian and possum fecal contamination in tank water samples using molecular markers. This study established a potential link between the degradation of the microbial quality of tank water and avian and possum feces. PMID:27208100

Quantification of Human and Animal Viruses to Differentiate the Origin of the Fecal Contamination Present in Environmental Samples

PubMed Central

Bofill-Mas, Sílvia; Rusiñol, Marta; Fernandez-Cassi, Xavier; Carratalà, Anna; Hundesa, Ayalkibet

2013-01-01

Many different viruses are excreted by humans and animals and are frequently detected in fecal contaminated waters causing public health concerns. Classical bacterial indicator such as E. coli and enterococci could fail to predict the risk for waterborne pathogens such as viruses. Moreover, the presence and levels of bacterial indicators do not always correlate with the presence and concentration of viruses, especially when these indicators are present in low concentrations. Our research group has proposed new viral indicators and methodologies for determining the presence of fecal pollution in environmental samples as well as for tracing the origin of this fecal contamination (microbial source tracking). In this paper, we examine to what extent have these indicators been applied by the scientific community. Recently, quantitative assays for quantification of poultry and ovine viruses have also been described. Overall, quantification by qPCR of human adenoviruses and human polyomavirus JC, porcine adenoviruses, bovine polyomaviruses, chicken/turkey parvoviruses, and ovine polyomaviruses is suggested as a toolbox for the identification of human, porcine, bovine, poultry, and ovine fecal pollution in environmental samples. PMID:23762826

DETECTION OF INTRINSIC VANCOMYCIN RESISTANT ENTEROCOCCI IN ANIMAL AND HUMAN FECES

EPA Science Inventory

A survey was conducted to determine the occurrence of vancomycin resistant enterococci (VRE) in animal and human fecal samples. Fecal samples from 14 animal species and humans were analyzed by quantitative culture for enterococci and VRE. Over 800 VRE isolates were characterize...

Evaluation of Bovine Feces-Associated Microbial Source Tracking Markers and Their Correlations with Fecal Indicators and Zoonotic Pathogens in a Brisbane, Australia, Reservoir

PubMed Central

Sritharan, T.; Palmer, A.; Sidhu, J. P. S.; Toze, S.

2013-01-01

This study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral [bovine adenovirus (B-AVs)] microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB), Campylobacter spp., Escherichia coli O157, and Salmonella spp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for the Campylobacter 16S rRNA and E. coli O157 rfbE genes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens. PMID:23417003

EVALUATION OF THE USE OF DIFFERENT ANTIBIOTICS IN THE DIRECT VIABLE COUNT METHOD TO DETECT FECAL ENTEROCOCCI

EPA Science Inventory

The detection of fecal pollution is performed via culturing methods in spite of the fact that culturable counts can severely underestimate the densities of fecal microorganisms. One approach that has been used to enumerate bacteria is the direct viable count method (DVC). The ob...

Fecal coliform and Escherichia coli bacteria in the St. Croix National Scenic Riverway, Summer 1999

USGS Publications Warehouse

Kroening, Sharon E.

1999-01-01

Fecal coliform and Escherichia coli (E. coli) concentrations were determined in the St. Croix National Scenic Riverway to assess whether pathogenic organisms pose a potential problem for recreational use. Samples were collected from May through September 1999 at 22 locations on the St. Croix and Namekagon Rivers. No concentrations exceeded water-quality criteria or standards set by the U.S. Environmental Protection Agency or the states of Minnesota and Wisconsin. Maximum fecal coliform and E. coli concentrations were measured in the St. Croix River at St. Croix Falls, Wisconsin. Median fecal coliform and E. coli concentrations were greater in the St. Croix River near Woodland Corner, Wisconsin, and at Marine on St. Croix, Minnesota than at other locations sampled. There were no consistent short-term variations in fecal coliform or E. coli concentrations during the summer period or any significant relations between concentrations and stream discharge, based on these results.

Utility of Microbial Source-Tracking Markers for Assessing Fecal Contamination in the Portage River Watershed, Northwestern Ohio, 2008

USGS Publications Warehouse

Kephart, Christopher M.; Bushon, Rebecca N.

2010-01-01

An influx of concentrated animal feeding operations in northwest Ohio has prompted local agencies to examine the effects of these industrial farms on water quality in the upper Portage River watershed. The utility of microbial source-tracking (MST) tools as a means of characterizing sources of fecal contamination in the watershed was evaluated. From 2007 to 2008, scientists with the U.S. Geological Survey, Bowling Green State University, and the Wood County Health Department collected and analyzed 17 environmental samples and 13 fecal source samples for Bacteroides-based host-associated DNA markers. At many of the environmental sites tested, MST marker results corroborated the presumptive fecal contamination sources. Results from this demonstration study support the utility of using MST with host-specific molecular markers to characterize the sources of fecal contamination in the Portage River watershed.

Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

PubMed Central

Garrido, Joseba M.; Molina, Elena; Geijo, María V.; Elguezabal, Natalia; Vázquez, Patricia; Juste, Ramón A.

2014-01-01

The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs. PMID:24727272

Specific detection of Echinococcus spp. from the Tibetan fox (Vulpes ferrilata) and the red fox (V. vulpes) using copro-DNA PCR analysis.

PubMed

Jiang, Weibin; Liu, Nan; Zhang, Gaotian; Renqing, Pengcuo; Xie, Fei; Li, Tiaoying; Wang, Zhenghuan; Wang, Xiaoming

2012-10-01

There are three Echinococcus species, Echinococcus granulosus, E. multilocularis, and E. shiquicus, which are distributed on the vast area of pastureland on the eastern Tibetan plateau in China. Tibetan foxes (Vulpes ferrilata) have been determined to be the main wild definitive host of E. multilocularis and E. shiquicus, but little information is available on the prevalence of these two parasites in Tibetan foxes. Consequently, the copro-prevalence of these parasites in foxes from the eastern Tibetan plateau was evaluated in this study. For each copro-DNA sample extracted from fox feces, a 133-bp segment of EgG1 Hae III was used to screen for infection with E. granulosus. Multiplex nested polymerase chain reaction (PCR) analysis was used to target an 874-bp segment of the mitochondrial COI gene to distinguish E. multilocularis and E. shiquicus. Among 184 fecal samples, 120 were from Tibetan foxes and six from red foxes (Vulpes vulpes). Of the fecal samples from Tibetan foxes, 74 (giving a copro-prevalence of 62%) showed the presence of Echinococcus spp.: 23 (19%) were found to contain E. multilocularis, 32 (27%) E. shiquicus, and 19 (16%) showed mixed infection with both E. multilocularis and E. shiquicus. Two fecal samples from red foxes were found to be infected with E. multilocularis. No fox feces were found to be infected with E. granulosus. Tests on zinc finger protein genes and a 105-bp fragment of the Sry gene found no significant difference in the prevalence of the two parasites between sexes. The efficiency of our multiplex nested PCR methods were compared with previous polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) methods and some problems associated with the copro-PCR were discussed.

Comparison of the Microbial Community Structures of Untreated Wastewaters from Different Geographic Locales

PubMed Central

Shanks, Orin C.; Newton, Ryan J.; Kelty, Catherine A.; Huse, Susan M.; Sogin, Mitchell L.

2013-01-01

Microbial sewage communities consist of a combination of human fecal microorganisms and nonfecal microorganisms, which may be residents of urban sewer infrastructure or flowthrough originating from gray water or rainwater inputs. Together, these different microorganism sources form an identifiable community structure that may serve as a signature for sewage discharges and as candidates for alternative indicators specific for human fecal pollution. However, the structure and variability of this community across geographic space remains uncharacterized. We used massively parallel 454 pyrosequencing of the V6 region in 16S rRNA genes to profile microbial communities from 13 untreated sewage influent samples collected from a wide range of geographic locations in the United States. We obtained a total of 380,175 high-quality sequences for sequence-based clustering, taxonomic analyses, and profile comparisons. The sewage profile included a discernible core human fecal signature made up of several abundant taxonomic groups within Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. DNA sequences were also classified into fecal, sewage infrastructure (i.e., nonfecal), and transient groups based on data comparisons with fecal samples. Across all sewage samples, an estimated 12.1% of sequences were fecal in origin, while 81.4% were consistently associated with the sewage infrastructure. The composition of feces-derived operational taxonomic units remained congruent across all sewage samples regardless of geographic locale; however, the sewage infrastructure community composition varied among cities, with city latitude best explaining this variation. Together, these results suggest that untreated sewage microbial communities harbor a core group of fecal bacteria across geographically dispersed wastewater sewage lines and that ambient water quality indicators targeting these select core microorganisms may perform well across the United States. PMID:23435885

Fecal-indicator bacteria concentrations in the Illinois River between Hennepin and Peoria, Illinois: 2007-08

USGS Publications Warehouse

Dupre, David H.; Hortness, Jon E.; Terrio, Paul J.; Sharpe, Jennifer B.

2012-01-01

The Illinois Environmental Protection Agency has designated portions of the Illinois River in Peoria, Woodford, and Tazewell Counties, Illinois, as impaired owing to the presence of fecal coliform bacteria. The U.S. Geological Survey, in cooperation with the Tri-County Regional Planning Commission, examined the water quality in the Illinois River and major tributaries within a 47-mile reach between Peoria and Hennepin, Ill., during water year 2008 (October 2007–September 2008). Investigations included synoptic (snapshot) sampling at multiple locations in a 1-day period: once in October 2007 during lower streamflow conditions, and again in June 2008 during higher streamflow conditions. Five locations in the study area were monitored for the entire year at monthly or more frequent intervals. Two indicator bacteria were analyzed in each water sample: fecal coliform and Escherichia coli (E. coli). Streamflow information from previously established monitoring locations in the study area was used in the analysis. Correlation analyses were used to characterize the relation between the two fecal-indicator bacteria and the relation of either indicator to streamflow. Concentrations of the two measured fecal-indicator bacteria correlated well for all samples analyzed (r = 0.94, p E. coli: rho = -0.43, p = 0.0157). The correlation between fecal indicators and streamflow in tributaries or in the Illinois River at Hennepin was found to be statistically significant, yet moderate in strength with coefficient values ranging from r = 0.4 to 0.6. Indirect observations from the June 2008 higher flow synoptic event may indicate continued effects from combined storm and sanitary sewers in the vicinity of the Illinois River near Peoria, Ill., contributing to observed single-sample exceedance of the State criterion for fecal coliform.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

PubMed

Chandler, J C; Pérez-Méndez, A; Paar, J; Doolittle, M M; Bisha, B; Goodridge, L D

2017-01-01

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (10 6 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water. Copyright © 2016 Elsevier B.V. All rights reserved.

FECAL CORTISOL LEVELS IN SCIMITAR-HORNED ORYX, ORYX DAMMAH, REVEALS DIFFERENCES BETWEEN CAPTIVE ENVIRONMENTS.

PubMed

Pauling, Cassandra D; Lankford, Scott E; Jackson, Victoria L

2017-12-01

Due to the intensive management of the scimitar-horned oryx, Oryx dammah, involving both captivity and reintroductions, understanding the stress associated with environmental situations this endangered species might experience would be particularly helpful. Fecal cortisol levels were measured across seasons, between captive management programs, and among varying reproductive states in animals held at Fossil Rim Wildlife Center (FRWC) and Kansas City Zoo (KCZ). A total of 72 samples were collected from FRWC and 69 samples were collected from KCZ. The herd size and sex ratio changed for both locations in the middle of sampling due to translocations and birth. The herd sizes ranged from 25 to 28 individuals at FRWC and 22 to 24 individuals at KCZ. An ELISA was optimized and utilized to investigate fecal cortisol variances across seasons, captive management programs, and among varying reproductive states. Fecal cortisol levels ranged from 68.9 to 668.7 pg/g throughout this study, but key differences were found in response to reproductive status, environmental conditions, and social status. The highest level of fecal cortisol measured (668.7 pg/g) was in a pregnant female 9 days prior to parturition. During winter months, an increase in fecal cortisol levels occurred in both herds (FRWC 160.3 pg/g to 335.1 pg/g and KCZ 118.8 pg/g to 505.0 pg/g). In addition, when intact males were held together in an enclosure, the dominant males had lower fecal cortisol levels compared with submissive males during three of the four sampling periods. Understanding how these data relate to the physiologic stress response will require further study, but these results can be utilized to help establish expected fecal cortisol ranges in multiple environments and can aid current captive scimitar-horned oryx management programs, as well as future reintroduction efforts.

Evaluation of Traditional and Contemporary Methods for Detecting Syphacia obvelata and Aspiculuris tetraptera in Laboratory Mice

PubMed Central

Gerwin, Philip M; Arbona, Rodolfo J Ricart; Riedel, Elyn R; Lepherd, Michelle L; Henderson, Ken S; Lipman, Neil S

2017-01-01

There is no consensus regarding the best practice for detecting murine pinworm infections. Initially, we evaluated 7 fecal concentration methods by using feces containing Aspiculuris tetraptera (AT) eggs (n = 20 samples per method). Sodium nitrate flotation, sodium nitrate centrifugation, Sheather sugar centrifugation, and zinc sulfate centrifugation detected eggs in 100% of samples; zinc sulfate flotation and water sedimentation detected eggs in 90%. All had better detection rates than Sheather sugar flotation (50%). To determine optimal detection methods, Swiss Webster mice were exposed to Syphacia obvelata (SO; n = 60) or AT (n = 60). We compared the following methods at days 0, 30, and 90, beginning 21 or 28 d after SO and AT exposure, respectively: fecal concentration (AT only), anal tape test (SO only), direct examination of intestinal contents (cecum and colon), Swiss roll histology (cecum and colon), and PCR analysis (pooled fur swab and feces). Detection rates for SO-exposed mice were: PCR analysis, 45%; Swiss roll histology, 30%; intestinal content exam, 27%; and tape test, 27%. The SO detection rate for PCR analysis was significantly greater than that for the tape test. Detection rates for AT-exposed mice were: intestinal content exam, 53%; PCR analysis, 33%; fecal flotation, 22%; and Swiss roll histology, 17%. The AT detection rate of PCR analysis combined with intestinal content examination was greater than for PCR analysis only and the AT detection rate of intestinal content examination was greater than for Swiss roll histology. Combining PCR analysis with intestinal content examination detected 100% of infected animals. No single test detected all positive animals. We recommend combining PCR analysis with intestinal content examination for optimal pinworm detection. PMID:28905712

DYNAMICS OF AQUATIC FECAL CONTAMINATION, FECAL SOURCE IDENTIFICATION, AND CORRELATION OF BACTEROIDALES HOST-SPECIFIC MARKERS DETECTION WITH FECAL PATHOGENS

EPA Science Inventory

Fecal pollution impairs the health and productivity of coastal waters and causes human disease. PCR of host-specific 16S rDNA sequences from anaerobic Bacteroidales bacteria offers a promising method of tracking fecal contamination and identifying its source(s). Before Bacteroida...

Sewage reflects the microbiomes of human populations.

PubMed

Newton, Ryan J; McLellan, Sandra L; Dila, Deborah K; Vineis, Joseph H; Morrison, Hilary G; Eren, A Murat; Sogin, Mitchell L

2015-02-24

Molecular characterizations of the gut microbiome from individual human stool samples have identified community patterns that correlate with age, disease, diet, and other human characteristics, but resources for marker gene studies that consider microbiome trends among human populations scale with the number of individuals sampled from each population. As an alternative strategy for sampling populations, we examined whether sewage accurately reflects the microbial community of a mixture of stool samples. We used oligotyping of high-throughput 16S rRNA gene sequence data to compare the bacterial distribution in a stool data set to a sewage influent data set from 71 U.S. cities. On average, only 15% of sewage sample sequence reads were attributed to human fecal origin, but sewage recaptured most (97%) human fecal oligotypes. The most common oligotypes in stool matched the most common and abundant in sewage. After informatically separating sequences of human fecal origin, sewage samples exhibited ~3× greater diversity than stool samples. Comparisons among municipal sewage communities revealed the ubiquitous and abundant occurrence of 27 human fecal oligotypes, representing an apparent core set of organisms in U.S. populations. The fecal community variability among U.S. populations was significantly lower than among individuals. It clustered into three primary community structures distinguished by oligotypes from either: Bacteroidaceae, Prevotellaceae, or Lachnospiraceae/Ruminococcaceae. These distribution patterns reflected human population variation and predicted whether samples represented lean or obese populations with 81 to 89% accuracy. Our findings demonstrate that sewage represents the fecal microbial community of human populations and captures population-level traits of the human microbiome. The gut microbiota serves important functions in healthy humans. Numerous projects aim to define a healthy gut microbiome and its association with health states. However, financial considerations and privacy concerns limit the number of individuals who can be screened. By analyzing sewage from 71 cities, we demonstrate that geographically distributed U.S. populations share a small set of bacteria whose members represent various common community states within U.S. adults. Cities were differentiated by their sewage bacterial communities, and the community structures were good predictors of a city's estimated level of obesity. Our approach demonstrates the use of sewage as a means to sample the fecal microbiota from millions of people and its potential to elucidate microbiome patterns associated with human demographics. Copyright © 2015 Newton et al.

Genetic diversity and antimicrobial resistance among isolates of Escherichia coli O157: H7 from feces and hides of super-shedders and low-shedding pen-mates in two commercial beef feedlots

PubMed Central

2012-01-01

Background Cattle shedding at least 104 CFU Escherichia coli O157:H7/g feces are described as super-shedders and have been shown to increase transmission of E. coli O157:H7 to other cattle in feedlots. This study investigated relationships among fecal isolates from super-shedders (n = 162), perineal hide swab isolates (PS) from super-shedders (n = 137) and fecal isolates from low-shedder (< 104 CFU/g feces) pen-mates (n = 496) using pulsed-field gel electrophoresis (PFGE). A subsample of these fecal isolates (n = 474) was tested for antimicrobial resistance. Isolates of E. coli O157:H7 were obtained from cattle in pens (avg. 181 head) at 2 commercial feedlots in southern Alberta with each steer sampled at entry to the feedlot and prior to slaughter. Results Only 1 steer maintained super-shedder status at both samplings, although approximately 30% of super-shedders in sampling 1 had low-shedder status at sampling 2. A total of 85 restriction endonuclease digestion clusters (REPC; 90% or greater similarity) and 86 unique isolates (< 90% similarity) were detected, with the predominant REPC (30% of isolates) being isolated from cattle in all feedlot pens, although it was not associated with shedding status (super- or low-shedder; P = 0.94). Only 2/21 super-shedders had fecal isolates in the same REPC at both samplings. Fecal and PS isolates from individual super-shedders generally belonged to different REPCs, although fecal isolates of E. coli O157:H7 from super- and low-shedders showed greater similarity (P < 0.001) than those from PS. For 77% of super-shedders, PFGE profiles of super-shedder fecal and PS isolates were distinct from all low-shedder fecal isolates collected in the same pen. A low level of antimicrobial resistance (3.7%) was detected and prevalence of antimicrobial resistance did not differ among super- and low-shedder isolates (P = 0.69), although all super-shedder isolates with antimicrobial resistance (n = 3) were resistant to multiple antimicrobials. Conclusions Super-shedders did not have increased antimicrobial resistance compared to low-shedder pen mates. Our data demonstrated that PFGE profiles of individual super-shedders varied over time and that only 1/162 steers remained a super-shedder at 2 samplings. In these two commercial feedlots, PFGE subtypes of E. coli O157:H7 from fecal isolates of super- and low-shedders were frequently different as were subtypes of fecal and perineal hide isolates from super-shedders. PMID:23014060

Water quality, weather and environmental factors associated with fecal indicator organism density in beach sand at two recreational marine beaches.

PubMed

Heaney, Christopher D; Exum, Natalie G; Dufour, Alfred P; Brenner, Kristen P; Haugland, Richard A; Chern, Eunice; Schwab, Kellogg J; Love, David C; Serre, Marc L; Noble, Rachel; Wade, Timothy J

2014-11-01

Recent studies showing an association between fecal indicator organisms (FIOs) in sand and gastrointestinal (GI) illness among beachgoers with sand contact have important public health implications because of the large numbers of people who recreate at beaches and engage in sand contact activities. Yet, factors that influence fecal pollution in beach sand remain unclear. During the 2007 National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study, sand samples were collected at three locations (60 m apart) on weekend days (Sat, Sun) and holidays between June and September at two marine beaches - Fairhope Beach, AL and Goddard Beach, RI - with nearby publicly-owned treatment works (POTWs) outfalls. F(+) coliphage, enterococci, Bacteroidales, fecal Bacteroides spp., and Clostridium spp. were measured in sand using culture and qPCR-based calibrator-cell equivalent methods. Water samples were also collected on the same days, times and transects as the 144 sand samples and were assayed using the same FIO measurements. Weather and environmental data were collected at the time of sample collection. Mean FIO concentrations in sand varied over time, but not space. Enterococci CFU and CCE densities in sand were not correlated, although other FIOs in sand were. The strongest correlation between FIO density in sand and water was fecal Bacteroides CCE, followed by enterococci CFU, Clostridium spp. CCE, and Bacteroidales CCE. Overall, the factors associated with FIO concentrations in sand were related to the sand-water interface (i.e., sand-wetting) and included daily average densities of FIOs in water, rainfall, and wave height. Targeted monitoring that focuses on daily trends of sand FIO variability, combined with information about specific water quality, weather, and environmental factors may inform beach monitoring and management decisions to reduce microbial burdens in beach sand. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Copyright © 2014 Elsevier B.V. All rights reserved.

IDENTIFICATION OF SOURCES OF FECAL POLLUTION IN ENVIRONMENTAL WATERS

EPA Science Inventory

A number of Microbial Source Tracking (MST) methods are currently used to determine the origin of fecal pollution impacting environmental waters. MST is based on the assumption that given the appropriate method and indicator organism, the source of fecal microbial pollution can ...

CULTURE-INDEPENDENT MOLECULAR METHODS FOR FECAL SOURCE IDENTIFICATION

EPA Science Inventory

Fecal contamination is widespread in the waterways of the United States. Both to correct the problem, and to estimate public health risk, it is necessary to identify the source of the contamination. Several culture-independent molecular methods for fecal source identification hav...

Development of Cross-Assembly Phage PCR-Based Methods for Human Fecal Source Identification

EPA Science Inventory

Technologies that can characterize human fecal pollution in environmental waters offer many advantages over traditional general indicator approaches. However, many human-associated methods cross-react with non-human animal sources and lack suitable sensitivity for fecal source id...

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.

PubMed

Wu, Gary D; Lewis, James D; Hoffmann, Christian; Chen, Ying-Yu; Knight, Rob; Bittinger, Kyle; Hwang, Jennifer; Chen, Jun; Berkowsky, Ronald; Nessel, Lisa; Li, Hongzhe; Bushman, Frederic D

2010-07-30

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.

Fecal indicators in sand, sand contact, and risk of enteric illness among beachgoers

PubMed Central

Heaney, Christopher D.; Sams, Elizabeth; Dufour, Alfred P.; Brenner, Kristen P.; Haugland, Richard A.; Chern, Eunice; Wing, Steve; Marshall, Stephen; Love, David C.; Serre, Marc; Noble, Rachel; Wade, Timothy J.

2011-01-01

Background Beach sand can harbor fecal indicator organisms and pathogens, but enteric illness risk associated with sand contact remains unclear. Methods In 2007, visitors at two recreational marine beaches were asked on the day of their visit about sand contact. Ten to 12 days later, participants answered questions about health symptoms since the visit. F+ coliphage, Enterococcus, Bacteroidales, fecal Bacteroides, and Clostridium spp. in wet sand were measured using culture and molecular methods. Results We analyzed 144 wet sand samples and completed 4,999 interviews. Adjusted odds ratios (aORs) were computed, comparing those in the highest tertile of fecal indicator exposure with those who reported no sand contact. Among those digging in sand compared with those not digging in sand, a molecular measure of Enterococcus spp. (calibrator cell equivalents/g) in sand was positively associated with gastrointestinal (GI) illness (aOR = 2.0 [95% confidence interval (CI) = 1.2–3.2]) and diarrhea (2.4 [1.4–4.2]). Among those buried in sand, point estimates were greater for GI illness (3.3 [1.3–7.9]) and diarrhea (4.9 [1.8–13]). Positive associations were also observed for culture-based Enterococcus (colony-forming units/g) with GI illness (aOR digging = 1.7 [1.1–2.7]) and diarrhea (2.1 [1.3–3.4]). Associations were not found among non-swimmers with sand exposure. Conclusions We observed a positive relationship between sand contact activities and enteric illness as a function of concentrations of fecal microbial pollution in beach sand. PMID:22157306

Application of 1H NMR spectroscopy-based metabonomics to feces of cervical cancer patients with radiation-induced acute intestinal symptoms.

PubMed

Chai, Yanlan; Wang, Juan; Wang, Tao; Yang, Yunyi; Su, Jin; Shi, Fan; Wang, Jiquan; Zhou, Xi; He, Bin; Ma, Hailin; Liu, Zi

2015-11-01

Radiation-induced acute intestinal symptoms (RIAISs) are a common complication of radiotherapy for cervical cancer. The aim of this study was to use (1)H nuclear magnetic resonance ((1)H NMR) combined with chemometric analysis to develop a metabolic profile of patients with RIAISs. Fecal samples were collected from 66 patients with cervical cancer before and after pelvic radiotherapy. After radiotherapy, RIAISs occurred in eleven patients. We selected another 11 patients from participants without RIAISs whose age, stage, histological type and treatment methods are matched with RIAIS patients as the control group. (1)H NMR spectroscopy combined with multivariate pattern recognition analysis was used to generate metabolic profile data, as well as to establish a RIAIS-specific metabolic phenotype. Orthogonal partial least-squares discriminant analysis was used to distinguish samples between the pre- and post-radiotherapy RIAIS patients and between RIAIS patients and controls. Fecal samples from RIAIS patients after pelvic radiotherapy were characterized by increased concentrations of α-ketobutyrate, valine, uracil, tyrosine, trimethylamine N-oxide, phenylalanine, lysine, isoleucine, glutamine, creatinine, creatine, bile acids, aminohippurate, and alanine, accompanied by reduced concentrations of α-glucose, n-butyrate, methylamine, and ethanol relative to samples from RIAIS patients before pelvic radiotherapy, while in RIAIS patients relative to controls, trimethylamine, n-butyrate, fumarate and acetate were down-regulated and valine, TMAO, taurine, phenylalanine, lactate, isoleucine and creatinine were up-regulated. We obtained the metabolic profile of RIAIS patients from fecal samples using NMR-based metabonomics. This profile has the potential to be developed into a novel clinical tool for RIAIS diagnosis or therapeutic monitoring, and could contribute to an improved understanding of the disease mechanism. However, because of the limitations of methods, technique, bacterial contamination of feces and small sample size, further research and verification are needed. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Metabarcoding of Fecal Samples to Determine Herbivore Diets: A Case Study of the Endangered Pacific Pocket Mouse

PubMed Central

Adams, Cynthia R.; Kohn, Joshua R.; Fisher, Robert N.; Brehme, Cheryl S.

2016-01-01

Understanding the diet of an endangered species illuminates the animal’s ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4–14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants. PMID:27851756

Metabarcoding of Fecal Samples to Determine Herbivore Diets: A Case Study of the Endangered Pacific Pocket Mouse.

PubMed

Iwanowicz, Deborah D; Vandergast, Amy G; Cornman, Robert S; Adams, Cynthia R; Kohn, Joshua R; Fisher, Robert N; Brehme, Cheryl S

2016-01-01

Understanding the diet of an endangered species illuminates the animal's ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4-14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

PubMed Central

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Metabarcoding of fecal samples to determine herbivore diets: A case study of the endangered Pacific pocket mouse

USGS Publications Warehouse

Iwanowicz, Deborah; Vandergast, Amy; Cornman, Robert S.; Adams, Cynthia; Kohn, Joshua R.; Fisher, Robert N.; Brehme, Cheryl S.

2016-01-01

Understanding the diet of an endangered species illuminates the animal’s ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4–14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

A comparison of fecal percent dry matter and number of Cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves.

PubMed

Bellosa, Mary L; Nydam, Daryl V; Liotta, Janice L; Zambriski, Jennifer A; Linden, Thomas C; Bowman, Dwight D

2011-04-01

Evaluation of dairy calf feces is often used in research and for clinical decision making to assess severity of diarrhea. However, this has not been validated for agreement between dry matter content and observed fecal consistency. Therefore, a comparison of observed fecal consistency score to fecal percent dry matter and Cryptosporidium parvum oocyst shedding was performed to assess the accuracy of observational scoring as a measure of diarrhea and its association with number of oocysts shed. Fecal samples from 20 dairy calves experimentally infected with C. parvum oocysts were collected daily post-infection and scored on a scale from 1 to 4, with 1 being normal feces to 4 being severe diarrhea. An aliquot of each sample was analyzed for percent dry matter and Cryptosporidium oocyst counts by using immunofluorescent microscopy. Fecal consistency scores of 1, 2, 3, and 4 had median percent dry matter of 20.9, 16.3, 9.6, and 5.8, respectively. Using percent dry matter assessed by fecal consistency scoring were significantly different from each other (P < 0.001). A higher fecal consistency score also was associated with a greater number of Cryptosporidium oocysts shed (P < 0 .0001). Scores of 1, 2, 3, and 4 had median oocyst counts of 0, 0, 1.3 × 10⁶, and 2.8 × 10⁶, respectively. These results suggest that observational scoring is a useful proxy to assess diarrhea in dairy calves.

Inactivation of fecal bacteria in drinking water by solar heating.

PubMed

Joyce, T M; McGuigan, K G; Elmore-Meegan, M; Conroy, R M

1996-02-01

We report simulations of the thermal effect of strong equatorial sunshine on water samples contaminated with high populations of fecal coliforms. Water samples, heavily contaminated with a wild-type strain of Escherichia coli (starting population = 20 x 10(5) CFU/ml), are heated to those temperatures recorded for 2-liter samples stored in transparent plastic bottles and exposed to full Kenyan sunshine (maximum water temperature, 55 degrees C). The samples are completely disinfected within 7 h, and no viable E. coli organisms are detected at either the end of the experiment or a further 12 h later, showing that no bacterial recovery has occurred. The feasibility of employing solar disinfection for highly turbid, fecally contaminated water is discussed.

The Fecal Microbiota Profile and Bronchiolitis in Infants

PubMed Central

Linnemann, Rachel W.; Mansbach, Jonathan M.; Ajami, Nadim J.; Espinola, Janice A.; Petrosino, Joseph F.; Piedra, Pedro A.; Stevenson, Michelle D.; Sullivan, Ashley F.; Thompson, Amy D.; Camargo, Carlos A.

2016-01-01

BACKGROUND: Little is known about the association of gut microbiota, a potentially modifiable factor, with bronchiolitis in infants. We aimed to determine the association of fecal microbiota with bronchiolitis in infants. METHODS: We conducted a case–control study. As a part of multicenter prospective study, we collected stool samples from 40 infants hospitalized with bronchiolitis. We concurrently enrolled 115 age-matched healthy controls. By applying 16S rRNA gene sequencing and an unbiased clustering approach to these 155 fecal samples, we identified microbiota profiles and determined the association of microbiota profiles with likelihood of bronchiolitis. RESULTS: Overall, the median age was 3 months, 55% were male, and 54% were non-Hispanic white. Unbiased clustering of fecal microbiota identified 4 distinct profiles: Escherichia-dominant profile (30%), Bifidobacterium-dominant profile (21%), Enterobacter/Veillonella-dominant profile (22%), and Bacteroides-dominant profile (28%). The proportion of bronchiolitis was lowest in infants with the Enterobacter/Veillonella-dominant profile (15%) and highest in the Bacteroides-dominant profile (44%), corresponding to an odds ratio of 4.59 (95% confidence interval, 1.58–15.5; P = .008). In the multivariable model, the significant association between the Bacteroides-dominant profile and a greater likelihood of bronchiolitis persisted (odds ratio for comparison with the Enterobacter/Veillonella-dominant profile, 4.24; 95% confidence interval, 1.56–12.0; P = .005). In contrast, the likelihood of bronchiolitis in infants with the Escherichia-dominant or Bifidobacterium-dominant profile was not significantly different compared with those with the Enterobacter/Veillonella-dominant profile. CONCLUSIONS: In this case–control study, we identified 4 distinct fecal microbiota profiles in infants. The Bacteroides-dominant profile was associated with a higher likelihood of bronchiolitis. PMID:27354456

Comparative effect of direct-fed microbials on fecal shedding of Escherichia coli O157:H7 and Salmonella in naturally infected feedlot cattle.

PubMed

Tabe, Ebot S; Oloya, James; Doetkott, Dawn K; Bauer, Marc L; Gibbs, Penelope S; Khaitsa, Margaret L

2008-03-01

The effect of direct-fed microbials (DFM) on fecal shedding of Escherichia coli O157:H7 and Salmonella in naturally infected feedlot cattle was evaluated in a clinical trial involving 138 feedlot steers. Following standard laboratory methods, fecal samples collected from steers were evaluated for change in the detectable levels of E. coli O157:H7 and Salmonella shed in feces after DFM treatment. Sampling of steers was carried out every 3 weeks for 84 days. A significant reduction (32%) in fecal shedding of E. coli O157:H7 (P < 0.001), but not Salmonella (P = 0.24), was observed among the treatment steers compared with the control group during finishing. The probability of recovery of E. coli O157:H7 from the feces of treated and control steers was 34.0 and 66.0%, respectively. Steers placed on DFM supplement were almost three times less likely to shed E. coli O157:H7 (odds ratio, 0.36; 95% confidence interval, 0.25 to 0.53; P < 0.001) in their feces as opposed to their control counterparts. The probability of recovery of Salmonella from the feces of the control (14.0%) and the treated (11.3%) steers was similar. However, the DFM significantly reduced probability of new infections with Salmonella among DFM-treated cattle compared with controls (nontreated ones). It appears that DFM as applied in our study are capable of significantly reducing fecal shedding of E. coli O157:H7 in naturally infected cattle but not Salmonella. The factors responsible for the observed difference in the effects of DFM on E. coli O157:H7 and Salmonella warrants further investigation.

Chemical and biological tracers to determine groundwater flow in karstic aquifer, Yucatan Peninsula

NASA Astrophysics Data System (ADS)

Lenczewski, M.; Leal-Bautista, R. M.; McLain, J. E.

2013-05-01

Little is known about the extent of pollution in groundwater in the Yucatan Peninsula; however current population growth, both from international tourism and Mexican nationals increases the potential for wastewater release of a vast array of contaminants including personal care products, pharmaceuticals (Rx), and pathogenic microorganisms. Pathogens and Rx in groundwater can persist and can be particularly acute in this region where high permeability of the karst bedrock and the lack of top soil permit the rapid transport of contaminants into groundwater aquifers. The objective of this research is to develop and utilize novel biological and chemical source tracking methods to distinguish between different sources of anthropogenic pollution in degraded groundwater. Although several methods have been used successfully to track fecal contamination sources in small scale studies, little is known about their spatial limitations, as source tracking studies rarely include sample collection over a wide geographical area and with different sources of water. In addition, although source tracking methods to distinguish human from animal fecal contamination are widely available, this work has developed source tracking distinguish between separate human populations is highly unique. To achieve this objective, we collected water samples from a series of drinking wells, cenotes (sinkholes), wastewater treatment plants, and injection wells across the Yucatan Peninsula and examine potential source tracers within the collected water samples. The result suggests that groundwater sources impacted by tourist vs. local populations contain different chemical stressors. This work has developed a more detailed understanding of the presence and persistence of personal care products, pharmaceuticals, and fecal indicators in a karstic system; such understanding will be a vital component for the protection Mexican groundwater and human health. Quantification of different pollution sources within groundwater samples identified point sources of pollution, identify potential remediation strategies, and contribute to an improved understanding of the environmental impact of tourism and tourism-generated waste products on this groundwater-dependent ecosystem.

Computational Approaches for Predicting Nonlinear Interactions of Chemical Mixtures in Biological Systems

DTIC Science & Technology

2009-12-01

exposure. Feces samples were collected at 24hrs and 48hrs. No fecal matter was present at 6hrs. Urine samples were collected at 6hrs, 24hrs and 48hrs...atmosphere sampling technique (Tremblay et al, in press) to characterize the inhalation exposure chamber for JP-8 and S- 8 exposures at Loma Linda Veterans... samples were collected for 1 min at 400 mL/min and quantified using thermal desorption gas chromatography mass spectrometry (TDS-GC/MS). The method was

RAPID DETECTION METHOD FOR E.COLI, ENTEROCOCCI AND BACTEROIDES IN RECREATIONAL WATER

EPA Science Inventory

Current methodology for determining fecal contamination of drinking water sources and recreational waters rely on the time-consuming process of bacterial multiplication and require at least 24 hours from the time of sampling to the possible determination that the water is unsafe ...

Detection of human and animal sources of pollution by microbial and chemical methods

USDA-ARS?s Scientific Manuscript database

A multi-indicator approach comprising Enterococcus, bacterial source tracking (BST), and sterol analysis was tested for pollution source identification. Fecal contamination was detected in 100% of surface water sites tested. Enterococcus faecium was the dominant species in aged litter samples from p...

THE EMPACT BEACHES PROJECT - A STUDY OF THE PARAMETERS THAT AFFECT MICROBIOLOGICAL MONITORING

EPA Science Inventory

The current U. S. Environmental Protection Agency (EPA) water sample collection method for recreational water was suggested by the Federal Water Pollution Control Agency in 1968 as part of the fecal coliform guideline. Although improvements have been made in the indicator bacter...

Effects of field storage method on E. coli concentrations measured in storm water runoff

USDA-ARS?s Scientific Manuscript database

Storm water runoff is increasingly assessed for fecal indicator organisms (e.g., Escherichia coli, E. coli) and its impact on contact recreation. Concurrently, use of autosamplers along with logistic, economic, technical, and personnel barriers are challenging conventional protocols for sample hold...

Development and Testing of Novel Canine Fecal Source-Identification Assays

EPA Science Inventory

The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. Recent method comparison studies have shown that available Bacteroidales 16S rRNA-based methods for the detection of canine fecal ...

BLENDING BIOSOLIDS SAMPLES MAKES A DIFFERENCE IN ORGANISM RECOVERY, PRINTED IN WATER ENVIRONMENT LABORATORY SOLUTIONS, VOL 8, NO. 3, PGS 10-14, PUBLISHED BY WATER ENVIRONMENT FEDERATION, 2001

EPA Science Inventory

Current federal regulations (40 CFR 503) require enumeration of fecal coliform or Salmoella prior to land application of Class A biosolids. This regulation specifies use of enumeration methods included in "Standard Methods for the Examination of Water and Wastewater 18th Edi...

Animal Feces Contribute to Domestic Fecal Contamination: Evidence from E. coli Measured in Water, Hands, Food, Flies, and Soil in Bangladesh.

PubMed

Ercumen, Ayse; Pickering, Amy J; Kwong, Laura H; Arnold, Benjamin F; Parvez, Sarker Masud; Alam, Mahfuja; Sen, Debashis; Islam, Sharmin; Kullmann, Craig; Chase, Claire; Ahmed, Rokeya; Unicomb, Leanne; Luby, Stephen P; Colford, John M

2017-08-01

Fecal-oral pathogens are transmitted through complex, environmentally mediated pathways. Sanitation interventions that isolate human feces from the environment may reduce transmission but have shown limited impact on environmental contamination. We conducted a study in rural Bangladesh to (1) quantify domestic fecal contamination in settings with high on-site sanitation coverage; (2) determine how domestic animals affect fecal contamination; and (3) assess how each environmental pathway affects others. We collected water, hand rinse, food, soil, and fly samples from 608 households. We analyzed samples with IDEXX Quantitray for the most probable number (MPN) of E. coli. We detected E. coli in source water (25%), stored water (77%), child hands (43%), food (58%), flies (50%), ponds (97%), and soil (95%). Soil had >120 000 mean MPN E. coli per gram. In compounds with vs without animals, E. coli was higher by 0.54 log 10 in soil, 0.40 log 10 in stored water and 0.61 log 10 in food (p < 0.05). E. coli in stored water and food increased with increasing E. coli in soil, ponds, source water and hands. We provide empirical evidence of fecal transmission in the domestic environment despite on-site sanitation. Animal feces contribute to fecal contamination, and fecal indicator bacteria do not strictly indicate human fecal contamination when animals are present.

Animal Feces Contribute to Domestic Fecal Contamination: Evidence from E. coli Measured in Water, Hands, Food, Flies, and Soil in Bangladesh

PubMed Central

2017-01-01

Fecal-oral pathogens are transmitted through complex, environmentally mediated pathways. Sanitation interventions that isolate human feces from the environment may reduce transmission but have shown limited impact on environmental contamination. We conducted a study in rural Bangladesh to (1) quantify domestic fecal contamination in settings with high on-site sanitation coverage; (2) determine how domestic animals affect fecal contamination; and (3) assess how each environmental pathway affects others. We collected water, hand rinse, food, soil, and fly samples from 608 households. We analyzed samples with IDEXX Quantitray for the most probable number (MPN) of E. coli. We detected E. coli in source water (25%), stored water (77%), child hands (43%), food (58%), flies (50%), ponds (97%), and soil (95%). Soil had >120 000 mean MPN E. coli per gram. In compounds with vs without animals, E. coli was higher by 0.54 log10 in soil, 0.40 log10 in stored water and 0.61 log10 in food (p < 0.05). E. coli in stored water and food increased with increasing E. coli in soil, ponds, source water and hands. We provide empirical evidence of fecal transmission in the domestic environment despite on-site sanitation. Animal feces contribute to fecal contamination, and fecal indicator bacteria do not strictly indicate human fecal contamination when animals are present. PMID:28686435

Comparing community and specialty provider-based recruitment in a randomized clinical trial: clinical trial in fecal incontinence.

PubMed

Whitebird, Robin R; Bliss, Donna Zimmaro; Savik, Kay; Lowry, Ann; Jung, Hans-Joachim G

2010-12-01

Recruitment of participants to clinical trials remains a significant challenge, especially for research addressing topics of a sensitive nature such as fecal incontinence (FI). In the Fiber Study, a randomized controlled trial on symptom management for FI, we successfully enrolled 189 community-living adults through collaborations with specialty-based and community-based settings, each employing methods tailored to the organizational characteristics of their site. Results show that using the two settings increased racial and ethnic diversity of the sample and inclusion of informal caregivers. There were no differential effects on enrollment, final eligibility, or completion of protocol by site. Strategic collaborations with complementary sites can achieve sample recruitment goals for clinical trials on topics that are sensitive or known to be underreported. Copyright © 2010 Wiley Periodicals, Inc.

Association between thermal environment and Salmonella in fecal samples from dairy cattle in midwestern United States

PubMed Central

Likavec, Tasha; Pires, Alda F.A.; Funk, Julie A.

2016-01-01

The objective of this study was to describe the association between thermal measures in the barn environment (pen temperature and humidity) and fecal shedding of Salmonella in dairy cattle. A repeated cross-sectional study was conducted within a commercial dairy herd located in the midwestern United States. Five pooled fecal samples were collected monthly from each pen for 9 mo and submitted for microbiological culture. Negative binomial regression methods were used to test the association [incidence rate ratio (IRR)] between Salmonella pen status (the count of Salmonella-positive pools) and thermal environmental parameters [average temperature and temperature humidity index (THI)] for 3 time periods (48 h, 72 h, and 1 wk) before fecal sampling. Salmonella was cultured from 10.8% [39/360; 95% confidence interval (CI): 7.8% to 14.5%] of pooled samples. The highest proportion of positive pools occurred in August. The IRR ranged from 1.26 (95% CI: 1.15 to 1.39, THI 1 wk) to 4.5 (95% CI: 2.13 to 9.51, heat exposure 1 wk) across all thermal parameters and lag time periods measured. For example, the incidence rate of Salmonella-positive pools increased by 54% for every 5°C increment in average temperature (IRR = 1.54; 95% CI: 1.29 to 1.85) and 29% for every 5-unit increase in THI (IRR = 1.29; 95% CI: 1.16 to 1.42) during the 72 h before sampling. The incidence rate ratio for pens exposed to higher temperatures (> 25°C) was 4.5 times (95% CI: 2.13 to 9.51) the incidence rate ratio for pens exposed to temperatures < 25°C in the 72 h before sampling. Likewise, the incidence rate ratio for pens exposed to THI > 70 was 4.23 times greater (95% CI: 2.1 to 8.28) than when the THI was < 70 in the 72 h before sampling. An association was found between the thermal environment and Salmonella shedding in dairy cattle. Further research is warranted in order to fully understand the component risks associated with the summer season and increased Salmonella shedding. PMID:27408330

Source tracking swine fecal waste in surface water proximal to swine concentrated animal feeding operations

PubMed Central

Heaney, Christopher D.; Myers, Kevin; Wing, Steve; Hall, Devon; Baron, Dothula; Stewart, Jill R.

2015-01-01

Swine farming has gone through many changes in the last few decades, resulting in operations with a high animal density known as confined animal feeding operations (CAFOs). These operations produce a large quantity of fecal waste whose environmental impacts are not well understood. The purpose of this study was to investigate microbial water quality in surface waters proximal to swine CAFOs including microbial source tracking of fecal microbes specific to swine. For one year, surface water samples at up- and downstream sites proximal to swine CAFO lagoon waste land application sites were tested for fecal indicator bacteria (fecal coliforms, Escherichia coli and Enterococcus) and candidate swine-specific microbial source-tracking (MST) markers (Bacteroidales Pig-1-Bac, Pig-2-Bac, and Pig-Bac-2, and methanogen P23-2). Testing of 187 samples showed high fecal indicator bacteria concentrations at both up- and downstream sites. Overall, 40%, 23%, and 61% of samples exceeded state and federal recreational water quality guidelines for fecal coliforms, E. coli, and Enterococcus, respectively. Pig-1-Bac and Pig-2-Bac showed the highest specificity to swine fecal wastes and were 2.47 (95% confidence interval [CI] = 1.03, 5.94) and 2.30 times (95% CI = 0.90, 5.88) as prevalent proximal down- than proximal upstream of swine CAFOs, respectively. Pig-1-Bac and Pig-2-Bac were also 2.87 (95% CI = 1.21, 6.80) and 3.36 (95% CI = 1.34, 8.41) times as prevalent when 48 hour antecedent rainfall was greater than versus less than the mean, respectively. Results suggest diffuse and overall poor sanitary quality of surface waters where swine CAFO density is high. Pig-1-Bac and Pig-2-Bac are useful for tracking off-site conveyance of swine fecal wastes into surface waters proximal to and downstream of swine CAFOs and during rain events. PMID:25600418

Source tracking swine fecal waste in surface water proximal to swine concentrated animal feeding operations.

PubMed

Heaney, Christopher D; Myers, Kevin; Wing, Steve; Hall, Devon; Baron, Dothula; Stewart, Jill R

2015-04-01

Swine farming has gone through many changes in the last few decades, resulting in operations with a high animal density known as confined animal feeding operations (CAFOs). These operations produce a large quantity of fecal waste whose environmental impacts are not well understood. The purpose of this study was to investigate microbial water quality in surface waters proximal to swine CAFOs including microbial source tracking of fecal microbes specific to swine. For one year, surface water samples at up- and downstream sites proximal to swine CAFO lagoon waste land application sites were tested for fecal indicator bacteria (fecal coliforms, Escherichia coli and Enterococcus) and candidate swine-specific microbial source-tracking (MST) markers (Bacteroidales Pig-1-Bac, Pig-2-Bac, and Pig-Bac-2, and methanogen P23-2). Testing of 187 samples showed high fecal indicator bacteria concentrations at both up- and downstream sites. Overall, 40%, 23%, and 61% of samples exceeded state and federal recreational water quality guidelines for fecal coliforms, E. coli, and Enterococcus, respectively. Pig-1-Bac and Pig-2-Bac showed the highest specificity to swine fecal wastes and were 2.47 (95% confidence interval [CI]=1.03, 5.94) and 2.30 times (95% CI=0.90, 5.88) as prevalent proximal down- than proximal upstream of swine CAFOs, respectively. Pig-1-Bac and Pig-2-Bac were also 2.87 (95% CI=1.21, 6.80) and 3.36 (95% CI=1.34, 8.41) times as prevalent when 48 hour antecedent rainfall was greater than versus less than the mean, respectively. Results suggest diffuse and overall poor sanitary quality of surface waters where swine CAFO density is high. Pig-1-Bac and Pig-2-Bac are useful for tracking off-site conveyance of swine fecal wastes into surface waters proximal to and downstream of swine CAFOs and during rain events. Copyright © 2014 Elsevier B.V. All rights reserved.

Fecal Leukocytes in Children Infected with Diarrheagenic Escherichia coli▿

PubMed Central

Mercado, Erik H.; Ochoa, Theresa J.; Ecker, Lucie; Cabello, Martin; Durand, David; Barletta, Francesca; Molina, Margarita; Gil, Ana I.; Huicho, Luis; Lanata, Claudio F.; Cleary, Thomas G.

2011-01-01

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response. PMID:21325554

Use of Spatial Sampling and Microbial Source-Tracking Tools for Understanding Fecal Contamination at Two Lake Erie Beaches

USGS Publications Warehouse

Francy, Donna S.; Bertke, Erin E.; Finnegan, Dennis P.; Kephart, Christopher M.; Sheets, Rodney A.; Rhoades, John; Stumpe, Lester

2006-01-01

Source-tracking tools were used to identify potential sources of fecal contamination at two Lake Erie bathing beaches: an urban beach (Edgewater in Cleveland, Ohio) and a beach in a small city (Lakeshore in Ashtabula, Ohio). These tools included identifying spatial patterns of Escherichia coli (E. coli) concentrations in each area, determining weather patterns that caused elevated E. coli, and applying microbial source tracking (MST) techniques to specific sites. Three MST methods were used during this study: multiple antibiotic resistance (MAR) indexing of E. coli isolates and the presence of human-specific genetic markers within two types of bacteria, the genus Bacteroides and the species Enterococcus faecium. At Edgewater, sampling for E. coli was done during 2003-05 at bathing-area sites, at nearshore lake sites, and in shallow ground water in foreshore and backshore areas. Spatial sampling at nearshore lake sites showed that fecal contamination was most likely of local origin; E. coli concentrations near the mouths of rivers and outfalls remote to the beach were elevated (greater than 235 colony-forming units per 100 milliliters (CFU/100 mL)) but decreased along transport pathways to the beach. In addition, E. coli concentrations were generally highest in bathing-area samples collected at 1- and 2-foot water depths, midrange at 3-foot depths, and lowest in nearshore lake samples typically collected 150 feet from the shoreline. Elevated E. coli concentrations at bathing-area sites were generally associated with increased wave heights and rainfall, but not always. E. coli concentrations were often elevated in shallow ground-water samples, especially in samples collected less than 10 feet from the edge of water (near foreshore area). The interaction of shallow ground water and waves may be a mechanism of E. coli storage and accumulation in foreshore sands. Infiltration of bird feces through sand with surface water from rainfall and high waves may be concentrating E. coli in shallow ground water in foreshore and backshore sands. At Lakeshore, sampling for E. coli was done at bathing-area, nearshore lake, and parking-lot sites during 2004-05. Low concentrations of E. coli at nearshore lake sites furthest from the shoreline indicated that fecal contamination was most likely of local origin. High concentrations of E. coli in water and bed sediments at several nearshore lake sites showed that contamination was emanating from several points along the shoreline during wet and dry weather, including the boat ramp, an area near the pond drainage, and parking-lot sediments. Physical evidence confirmed that runoff from the parking lot leads to degradation of water quality at the beach. MST samples were collected to help interpret spatial findings and determine whether sources of fecal contamination were from wastewater or bird feces and if a human-specific marker was present. MAR indices were useful in distinguishing between bird feces and wastewater sources because they were about 10 times higher in the latter. The results from MAR indices agreed with results from the two human-specific markers in some but not all of the samples tested. Bacteroides and enterococci human-specific markers were found on one day at Edgewater and two days at Lakeshore. On three days at Edgewater and two days at Lakeshore, the MAR index indicated a mixed source, but neither marker was found in bathing-water samples; this may be because bacterial indicator concentrations were too low to detect a marker. Multiple tools are needed to help identify sources of fecal contamination at coastal beaches. Spatial sampling identified patterns in E. coli concentrations and yielded information on the physical pathways of contamination. MST methods provided information on whether the source was likely of human or nonhuman origin only; however, MST did not provide information on the pathways of contamination.

STANDARDIZATION AND VALIDATION OF METHODS FOR ENUMERATION OF FECAL COLIFORM AND SALMONELLA IN BIOSOLIDS

EPA Science Inventory

Current federal regulations required monitoring for fecal coliforms or Salmonella in biosolids destined for land application. Methods used for analysis of fecal coliforms and Salmonella were reviewed and a standard protocol was developed. The protocols were then...

STANDARDIZATION AND VALIDATION OF METHODS FOR ENUMERATION OF FECAL COLIFORM AND SALMONELLA IN BIOSOLIDS

EPA Science Inventory

Current federal regulations require monitoring for fecal coliforms or Salmonella in biosolids destined for land application. Methods used for analysis of fecal coliforms and Salmonella were reviewed and a standard protocol was developed. The protocols were then evaluated by testi...

Improving the understanding of schistosomiasis among adolescents in endemic areas in Brazil: A comparison of educational methods.

PubMed

Gazzinelli, Maria Flávia; Lobato, Lucas; Andrade, Gisele; Matoso, Leonardo Ferreira; Diemert, David J; Gazzinelli, Andréa

2016-10-01

To evaluate the effectiveness of two teaching strategies, both guided by the concept of dialogicity, on adolescents' knowledge about schistosomiasis and adherence to diagnostic fecal testing. Two teaching strategies related to schistosomiasis were developed, an educational video and group conversation, which were tested in two groups of students aged 10-15 years old. Before and after the intervention, a questionnaire was applied to assess participants' knowledge about schistosomiasis and, after the intervention, two fecal samples were requested from each participant. Comparisons were performed by paired t- and McNemar tests. Both strategies resulted in statistically significant improvements in knowledge between the pre- and post-tests. Students who watched the video had a higher return rate of fecal samples and percentage of correct questionnaire answers, mainly on questions about schistosomiasis infection. Teaching strategies based on dialogue favored the construction of concepts about schistosomiasis that can influence the adoption of positives attitudes related to health. Using teaching strategies based on the concept of dialogicity can favor the increase of knowledge of school age children about schistosomiasis and can influence behavioral change related to health. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Bluephage: A rapid method for the detection of somatic coliphages used as indicators of fecal pollution in water.

PubMed

Muniesa, M; Ballesté, E; Imamovic, L; Pascual-Benito, M; Toribio-Avedillo, D; Lucena, F; Blanch, A R; Jofre, J

2018-01-01

The use of somatic coliphages as indicators of fecal and viral pollution in water and food has great potential due to the reliability, reproducibility, speed and cost effectiveness of methods for their detection. Indeed, several countries already use this approach in their water management policies. Although standardized protocols for somatic coliphage detection are available, user-friendly commercial kits would facilitate their routine implementation in laboratories. The new method presented here allows detection of up to 1 somatic coliphage in under 3.5 h, well within one working day. The method is based on a modified Escherichia coli strain with knocked-out uidB and uidC genes, which encode the transport of glucuronic acid inside cells, and overexpressing uidA, which encodes the enzyme β-glucuronidase. The enzyme accumulated in the bacterial cells only has contact with its substrate after cell lysis, such as that caused by phages, since the strain cannot internalize the substrate. When the enzyme is released into the medium, which contains a chromogen analogous to glucuronic acid, it produces a change of color from yellow to dark blue. This microbiological method for the determination of fecal pollution via the detection of culturable microorganisms can be applied to diverse sample types and volumes for qualitative (presence/absence) and quantitative analysis and is the fastest reported to date. Copyright © 2017 Elsevier Ltd. All rights reserved.

Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica

USGS Publications Warehouse

Lisle, J.T.; Smith, J.J.; Edwards, D.D.; McFeters, G.A.

2004-01-01

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.

Development and validation of a highly sensitive urine-based test to identify patients with colonic adenomatous polyps.

PubMed

Wang, Haili; Tso, Victor; Wong, Clarence; Sadowski, Dan; Fedorak, Richard N

2014-03-20

Adenomatous polyps are precursors of colorectal cancer; their detection and removal is the goal of colon cancer screening programs. However, fecal-based methods identify patients with adenomatous polyps with low levels of sensitivity. The aim or this study was to develop a highly accurate, prototypic, proof-of-concept, spot urine-based diagnostic test using metabolomic technology to distinguish persons with adenomatous polyps from those without polyps. Prospective urine and stool samples were collected from 876 participants undergoing colonoscopy examination in a colon cancer screening program, from April 2008 to October 2009 at the University of Alberta. Colonoscopy reference standard identified 633 participants with no colonic polyps and 243 with colonic adenomatous polyps. One-dimensional nuclear magnetic resonance spectra of urine metabolites were analyzed to define a diagnostic metabolomic profile for colonic adenomas. A urine metabolomic diagnostic test for colonic adenomatous polyps was established using 67% of the samples (un-blinded training set) and validated using the other 33% of the samples (blinded testing set). The urine metabolomic diagnostic test's specificity and sensitivity were compared with those of fecal-based tests. Using a two-component, orthogonal, partial least-squares model of the metabolomic profile, the un-blinded training set identified patients with colonic adenomatous polyps with 88.9% sensitivity and 50.2% specificity. Validation using the blinded testing set confirmed sensitivity and specificity values of 82.7% and 51.2%, respectively. Sensitivities of fecal-based tests to identify colonic adenomas ranged from 2.5 to 11.9%. We describe a proof-of-concept spot urine-based metabolomic diagnostic test that identifies patients with colonic adenomatous polyps with a greater level of sensitivity (83%) than fecal-based tests.

Contribution of Colonic Fermentation and Fecal Water Toxicity to the Pathophysiology of Lactose-Intolerance

PubMed Central

Windey, Karen; Houben, Els; Deroover, Lise; Verbeke, Kristin

2015-01-01

Whether or not abdominal symptoms occur in subjects with small intestinal lactose malabsorption might depend on differences in colonic fermentation. To evaluate this hypothesis, we collected fecal samples from subjects with lactose malabsorption with abdominal complaints (LM-IT, n = 11) and without abdominal complaints (LM-T, n = 8) and subjects with normal lactose digestion (NLD, n = 15). Lactose malabsorption was diagnosed using a 13C-lactose breath test. Colonic fermentation was characterized in fecal samples at baseline and after incubation with lactose for 3 h, 6 h and 24 h through a metabolomics approach using gas chromatography-mass spectrometry (GC-MS). Fecal water cytotoxicity was analyzed using a colorimetric assay. Fecal water cytotoxicity was not different between the three groups (Kruskall-Wallis p = 0.164). Cluster analysis of the metabolite patterns revealed separate clusters for NLD, LM-T and LM-IT samples at baseline and after 24 h incubation with lactose. Levels of 5-methyl-2-furancarboxaldehyde were significantly higher in LM-IT and LM-T compared to NLD whereas those of an unidentified aldehyde were significantly higher in LM-IT compared to LM-T and NLD. Incubation with lactose increased short chain fatty acid (SCFA) concentrations more in LM-IT and LM-T compared to NLD. In conclusion, fermentation patterns were clearly different in NLD, LM-IT and LM-T, but not related to differences in fecal water cytotoxicity. PMID:26371036

Contribution of Colonic Fermentation and Fecal Water Toxicity to the Pathophysiology of Lactose-Intolerance.

PubMed

Windey, Karen; Houben, Els; Deroover, Lise; Verbeke, Kristin

2015-09-08

Whether or not abdominal symptoms occur in subjects with small intestinal lactose malabsorption might depend on differences in colonic fermentation. To evaluate this hypothesis, we collected fecal samples from subjects with lactose malabsorption with abdominal complaints (LM-IT, n = 11) and without abdominal complaints (LM-T, n = 8) and subjects with normal lactose digestion (NLD, n = 15). Lactose malabsorption was diagnosed using a (13)C-lactose breath test. Colonic fermentation was characterized in fecal samples at baseline and after incubation with lactose for 3 h, 6 h and 24 h through a metabolomics approach using gas chromatography-mass spectrometry (GC-MS). Fecal water cytotoxicity was analyzed using a colorimetric assay. Fecal water cytotoxicity was not different between the three groups (Kruskall-Wallis p = 0.164). Cluster analysis of the metabolite patterns revealed separate clusters for NLD, LM-T and LM-IT samples at baseline and after 24 h incubation with lactose. Levels of 5-methyl-2-furancarboxaldehyde were significantly higher in LM-IT and LM-T compared to NLD whereas those of an unidentified aldehyde were significantly higher in LM-IT compared to LM-T and NLD. Incubation with lactose increased short chain fatty acid (SCFA) concentrations more in LM-IT and LM-T compared to NLD. In conclusion, fermentation patterns were clearly different in NLD, LM-IT and LM-T, but not related to differences in fecal water cytotoxicity.

Diagnostic stages of the parasites of the Florida manatee, Trichechus manatus latirostris.

PubMed

Bando, Monica; Larkin, Iskande V; Wright, Scott D; Greiner, Ellis C

2014-02-01

Limited information is available on diagnostic stages of parasites in Florida manatees (Trichechus manatus latirostris). We examined 67 fecal samples from captive and wild manatees to define the diagnostic stages of the parasite fauna known to occur in Florida manatees. Parasite eggs were freshly extracted ex utero from identified mature helminths and subsequently characterized, illustrated, and matched to those isolated from fecal samples. In addition, coccidian oocysts in the fecal samples were identified. These diagnostic stages included eggs from 5 species of trematodes (Chiorchis fabaceus, Chiorchis groschafti, Pulmonicola cochleotrema, Moniligerum blairi, and Nudacotyle undicola), 1 nematode (Heterocheilus tunicatus), and oocysts of 2 coccidians (Eimeria manatus and Eimeria nodulosa).

Molecular assays for targeting human and bovine enteric viruses in coastal waters and their application for library-independent source tracking

USGS Publications Warehouse

Fong, T.-T.; Griffin, Dale W.; Lipp, E.K.

2005-01-01

Rapid population growth and urban development along waterways and coastal areas have led to decreasing water quality. To examine the effects of upstream anthropogenic activities on microbiological water quality, methods for source-specific testing are required. In this study, molecular assays targeting human enteroviruses (HEV), bovine enteroviruses (BEV), and human adenoviruses (HAdV) were developed and used to identify major sources of fecal contamination in the lower Altamaha River, Georgia. Two-liter grab samples were collected monthly from five tidally influenced stations between July and December 2002. Samples were analyzed by reverse transcription- and nested-PCR. PCR results were confirmed by dot blot hybridization. Eleven and 17 of the 30 surface water samples tested positive for HAdV and HEV, respectively. Two-thirds of the samples tested positive for either HEV or HAdV, and the viruses occurred simultaneously in 26% of samples. BEV were detected in 11 of 30 surface water samples. Binary logistic regression analysis showed that the presence of both human and bovine enteric viruses was not significantly related to either fecal coliform or total coliform levels. The presence of these viruses was directly related to dissolved oxygen and streamflow but inversely related to water temperature, rainfall in the 30 days preceding sampling, and chlorophyll-?? concentrations. The stringent host specificity of enteric viruses makes them good library-independent indicators for identification of water pollution sources. Viral pathogen detection by PCR is a highly sensitive and easy-to-use tool for rapid assessment of water quality and fecal contamination when public health risk characterization is not necessary. Copyright ?? 2005, American Society for Microbiology. All Rights Reserved.

Development of a method which discriminates between endogenous and exogenous beta-boldenone.

PubMed

Blokland, M H; van Rossum, H J; Sterk, S S; van Ginkel, L A; Stephany, R W

2007-03-14

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

Effects of Saccharomyces boulardii on fecal short-chain fatty acids and microflora in patients on long-term total enteral nutrition

PubMed Central

Schneider, Stéphane M; Girard-Pipau, Fernand; Filippi, Jérôme; Hébuterne, Xavier; Moyse, Dominique; Hinojosa, Gustavo Calle; Pompei, Anne; Rampal, Patrick

2005-01-01

AIM: To assess the effects of Sb on fecal flora and short-chain fatty acids (SCFA) in patients on long-term TEN. METHODS: Ten patients (3 females, 7 males, 59±5.5 years), on TEN for a median of 13 mo (1-125), and 15 healthy volunteers (4 females, 11 males, 32±2.0 years) received Sb (0.5 g bid PO) for 6 d. Two stool samples were taken before, on the last 2 d and 9-10 d after treatment, for SCFA measurement and for culture and bacterial identification. Values (mean±SE) were compared using sign tests and ANOVA. RESULTS: Fecal butyrate levels were lower in patients (10.1±2.9 mmol/kg) than in controls (19.2±2.9, P = 0.02). Treatment with Sb increased total fecal SCFA levels in patients (150.2±27.2vs 107.5?8.2 mmol/kg, P = 0.02) but not in controls (129.0±28.6 vs 113.0±15.2 mmol/kg, NS). At the end of treatment with Sb, patients had higher fecal butyrate (16.0±4.4 vs 10.1 [2.9] mmol/kg, P = 0.004). Total SCFAs remained high 9 d after treatment was discon-tinued. Before the treatment, the anaerobe to aerobe ratio was lower in patients compared to controls (2.4±2.3 vs 69.8±1.8, P = 0.003). There were no significant changes in the fecal flora of TEN patients. CONCLUSION: Sb-induced increase of fecal SCFA concentrations (especially butyrate) may explain the preventive effects of this yeast on TEN-induced diarrhea. PMID:16273644

Survival, transport, and sources of fecal bacteria in streams and survival in land-applied poultry litter in the upper Shoal Creek basin, southwestern Missouri, 2001-2002

USGS Publications Warehouse

Schumacher, John G.

2003-01-01

Densities of fecal coliform bacteria along a 5.7-mi (mile) reach of Shoal Creek extending upstream from State Highway 97 (site 3) to State Highway W (site 2) and in two tributaries along this reach exceeded the Missouri Department of Natural Resources (MDNR) standard of 200 col/100 mL (colonies per 100 milliliters) for whole-body contact recreation. A combination of techniques was used in this report to provide information on the source, transport, and survival of fecal bacteria along this reach of Shoal Creek. Results of water-quality samples collected during dye-trace and seepage studies indicated that at summer low base-flow conditions, pastured cattle likely were a substantial source of fecal bacteria in Shoal Creek at the MDNR monitoring site (site 3) at State Highway 97. Using repeat element Polymerase Chain Reaction (rep-PCR), cattle were the presumptive source of about 50 percent of the Escherichia coli (E. coli) isolates in water samples from site 3. Cattle, horses, and humans were the most common presumptive source of E. coli isolates at sites further upstream. Poultry was identified by rep-PCR as a major source of E. coli in Pogue Creek, a tributary in the upper part of the study area. Results of the rep-PCR were in general agreement with the detection and distribution of trace concentrations of organic compounds commonly associated with human wastewater, such as caffeine, the antimicrobial agent triclosan, and the pharmaceutical compounds acetaminophen and thiabendazole (a common cattle anthelmintic). Significant inputs of fecal bacteria to Shoal Creek occurred along a 1.6-mi reach of Shoal Creek immediately upstream from site 3. During a 36-hour period in July 2001, average densities of fecal coliform and E. coli bacteria increased from less than or equal to 500 col/100 mL upstream from this stream reach (sample site 2c) to 2,100 and 1,400 col/100 mL, respectively, at the MDNR sampling site. Fecal bacteria densities exhibited diurnal variability at all five sampling sites along the 5.7-mi study reach of Shoal Creek, but the trends at successive downstream sites were out of phase and could not be explained by simple advection and dispersion. At base-flow conditions, the travel time of bacteria in Shoal Creek along the 5.7-mi reach between State Highway W (site 2) and the MDNR sampling site (site 3) was about 26 hours. Substantial dispersion and dilution occurs along the upper 4.1 mi of this reach because of inflows from a number of springs and tributaries and the presence of several long pools and channel meanders. Minimal dispersion and dilution occurs along the 1.6-mi reach immediately upstream from the MDNR sampling site. Measurements of fecal bacteria decay in Shoal Creek during July 2001 indicated that about 8 percent of fecal coliform and E. coli bacteria decay each hour with an average first-order decay constant of 0.084 h-1 (per hour). Results of field test plots indicated that substantial numbers of fecal bacteria present in poul try litter can survive in fields for as much as 8 weeks after the application of the litter to the land surface. Median densities of fecal coliform and E. coli in slurry-water samples collected from fields increased from less than 60 col/100 mL before the application of turkey and broiler litter, to as large as 420,000 and 290,000 col/100 mL after the application of litter. Bacteria densities in the test plots generally decreased in a exponential manner over time with decay rates ranging from 0.085 to 0.185 d-1 (per day) for fecal coliform to between 0.100 and 0.250 d-1 for E. coli. The apparent survival of significant numbers of fecal bacteria on fields where poultry litter has been applied indicates that runoff from these fields is a potential source of fecal bacteria to vicinity streams for many weeks following litter application.

Transcriptional changes detected in fecal RNA of neonatal dairy calves undergoing a mild diarrhea are associated with inflammatory biomarkers

PubMed Central

Rosa, Fernanda; Busato, Sebastiano; Avaroma, Fatima C.; Linville, Kali; Trevisi, Erminio; Bionaz, Massimo

2018-01-01

After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiological changes. In particular, maturation of the gastrointestinal tract in a new environment loaded with potential pathogens, which can predispose neonatal calves to develop diarrhea, and is a major cause of morbidity and mortality during the first 4 wks of life. We aimed to investigate the inflammatory adaptations at a transcriptomic level in the gastrointestinal (GI) tract to a mild diarrhea in neonatal dairy calves using RNA isolated from fresh fecal samples. Eight newborn Jersey male calves were used from birth to 5 wks of age and housed in individual pens. After birth, calves received 1.9 L of colostrum from their respective dams. Calves had ad-libitum access to water and starter grain (22% CP) and were fed twice daily a total of 5.6 L pasteurized whole milk. Starter intake, body weight (BW), fecal score, withers height (WH), and rectal temperature (RT) were recorded throughout the experiment. Blood samples were collected weekly for metabolic and inflammatory profiling from wk 0 to wk 5. Fresh fecal samples were collected weekly and immediately flash frozen until RNA was extracted using a Trizol-based method, and subsequently, an RT-qPCR analysis was performed. Orthogonal contrasts were used to evaluate linear or quadratic effects over time. Starter intake, BW, and WH increased over time. Fecal score was greatest (2.6 ± 0.3) during wk 2. The concentrations of IL-6, ceruloplasmin, and haptoglobin had a positive quadratic effect with maximal concentrations during wk 2, which corresponded to the maximal fecal score observed during the same time. The concentration of serum amyloid A decreased over time. The mRNA expression of the proinflammatory related genes TLR4, TNFA, IL8, and IL1B had a positive quadratic effect of time. A time effect was observed for the cell membrane sodium-dependent glucose transporter SLC5A1, for the major carbohydrate facilitated transporter SLC2A2, and water transport function AQP3, where SLC5A1 and AQP3 had a negative quadratic effect over time. Our data support the use of the fecal RNA as a noninvasive tool to investigate intestinal transcriptomic profiling of dairy calves experiencing diarrhea, which would be advantageous for future research including nutritional effects and health conditions. PMID:29373601

Transcriptional changes detected in fecal RNA of neonatal dairy calves undergoing a mild diarrhea are associated with inflammatory biomarkers.

PubMed

Rosa, Fernanda; Busato, Sebastiano; Avaroma, Fatima C; Linville, Kali; Trevisi, Erminio; Osorio, Johan S; Bionaz, Massimo

2018-01-01

After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiological changes. In particular, maturation of the gastrointestinal tract in a new environment loaded with potential pathogens, which can predispose neonatal calves to develop diarrhea, and is a major cause of morbidity and mortality during the first 4 wks of life. We aimed to investigate the inflammatory adaptations at a transcriptomic level in the gastrointestinal (GI) tract to a mild diarrhea in neonatal dairy calves using RNA isolated from fresh fecal samples. Eight newborn Jersey male calves were used from birth to 5 wks of age and housed in individual pens. After birth, calves received 1.9 L of colostrum from their respective dams. Calves had ad-libitum access to water and starter grain (22% CP) and were fed twice daily a total of 5.6 L pasteurized whole milk. Starter intake, body weight (BW), fecal score, withers height (WH), and rectal temperature (RT) were recorded throughout the experiment. Blood samples were collected weekly for metabolic and inflammatory profiling from wk 0 to wk 5. Fresh fecal samples were collected weekly and immediately flash frozen until RNA was extracted using a Trizol-based method, and subsequently, an RT-qPCR analysis was performed. Orthogonal contrasts were used to evaluate linear or quadratic effects over time. Starter intake, BW, and WH increased over time. Fecal score was greatest (2.6 ± 0.3) during wk 2. The concentrations of IL-6, ceruloplasmin, and haptoglobin had a positive quadratic effect with maximal concentrations during wk 2, which corresponded to the maximal fecal score observed during the same time. The concentration of serum amyloid A decreased over time. The mRNA expression of the proinflammatory related genes TLR4, TNFA, IL8, and IL1B had a positive quadratic effect of time. A time effect was observed for the cell membrane sodium-dependent glucose transporter SLC5A1, for the major carbohydrate facilitated transporter SLC2A2, and water transport function AQP3, where SLC5A1 and AQP3 had a negative quadratic effect over time. Our data support the use of the fecal RNA as a noninvasive tool to investigate intestinal transcriptomic profiling of dairy calves experiencing diarrhea, which would be advantageous for future research including nutritional effects and health conditions.

Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci

PubMed Central

Biswas, Priyanka Paul; Dey, Sangeeta; Sen, Aninda; Adhikari, Luna

2016-01-01

Background: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. Materials and Methods: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. Results: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. Conclusion: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms. PMID:27013840

Microbial source tracking in shellfish harvesting waters in the Gulf of Nicoya, Costa Rica.

PubMed

Symonds, E M; Young, S; Verbyla, M E; McQuaig-Ulrich, S M; Ross, E; Jiménez, J A; Harwood, V J; Breitbart, M

2017-03-15

Current microbial water quality monitoring is generally limited to culture-based measurements of fecal indicator bacteria (FIB). Given the many possible sources of fecal pollution within a watershed and extra-intestinal FIB reservoirs, it is important to determine source(s) of fecal pollution as a means to improve water quality and protect public health. The principal objective of this investigation was to characterize the microbial water quality of shellfish harvesting areas in the Gulf of Nicoya, Costa Rica during 2015. In order to achieve this objective, the specificity and sensitivity of 11 existing microbial source tracking (MST) PCR assays, associated with cows (BacCow), dogs (BacCan, DogBac), domestic wastewater (PMMoV), general avian (GFD), gulls (Gull2), horses (HorseBac, HoF), humans (HF183, HPyV), and pigs (PF), were evaluated using domestic wastewater and animal fecal samples collected from the region. The sensitivity of animal-associated assays ranged from 13 to 100%, while assay specificity ranged from 38 to 100%. The specificity of pepper mild mottle virus (PMMoV) and human polyomavirus (HPyV) was 100% for domestic wastewater, as compared to 94% specificity of the HF183 Bacteroidales marker. PMMoV was identified as a useful domestic wastewater-associated marker, with concentrations as high as 1.1 × 10 5 copies/ml and 100% sensitivity and specificity. Monthly surface water samples collected from four shellfish harvesting areas were analyzed using culture-based methods for Escherichia coli as well as molecular methods for FIB and a suite of MST markers, which were selected for their specificity in the region. While culturable E. coli results suggested possible fecal pollution during the monitoring period, the absence of human/domestic wastewater-associated markers and low FIB concentrations determined using molecular methods indicated sufficient microbial water quality for shellfish harvesting. This is the first study to our knowledge to test the performance of MST markers in Costa Rica as well as in Central America. Given the lack of wastewater treatment and the presence of secondary sources of FIB, this study highlights the importance of an MST toolbox approach to characterize water quality in tropical regions. Furthermore, it confirms and extends the geographic range of PMMoV as an effective tool for monitoring domestic wastewater pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection.

PubMed

Dorn-In, Samart; Schwaiger, Karin; Twarużek, Magdalena; Grajewski, Jan; Gottschalk, Christoph; Gareis, Manfred

2017-02-01

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Feedlot- and Pen-Level Prevalence of Enterohemorrhagic Escherichia coli in Feces of Commercial Feedlot Cattle in Two Major U.S. Cattle Feeding Areas.

PubMed

Cull, Charley A; Renter, David G; Dewsbury, Diana M; Noll, Lance W; Shridhar, Pragathi B; Ives, Samuel E; Nagaraja, Tiruvoor G; Cernicchiaro, Natalia

2017-06-01

The objective of this study was to determine feedlot- and pen-level fecal prevalence of seven enterohemorrhagic Escherichia coli (EHEC) belonging to serogroups (O26, O45, O103, O111, O121, O145, and O157, or EHEC-7) in feces of feedlot cattle in two feeding areas in the United States. Cattle pens from four commercial feedlots in each of the two major U.S. beef cattle areas were sampled. Up to 16 pen-floor fecal samples were collected from each of 4-6 pens per feedlot, monthly, for a total of three visits per feedlot, from June to August, 2014. Culture procedures including fecal enrichment in E. coli broth, immunomagnetic separation, and plating on selective media, followed by confirmation through polymerase chain reaction (PCR) testing, were conducted. Generalized linear mixed models were fitted to estimate feedlot-, pen-, and sample-level fecal prevalence of EHEC-7 and to evaluate associations between potential demographic and management risk factors with feedlot and within-pen prevalence of EHEC-7. All study feedlots and 31.0% of the study pens had at least one non-O157 EHEC-positive fecal sample, whereas 62.4% of pens tested positive for EHEC O157; sample-level prevalence estimates ranged from 0.0% for EHEC O121 to 18.7% for EHEC O157. Within-pen prevalence of EHEC O157 varied significantly by sampling month; similarly within-pen prevalence of non-O157 EHEC varied significantly by month and by the sex composition of the pen (heifer, steer, or mixed). Feedlot management factors, however, were not significantly associated with fecal prevalence of EHEC-7. Intraclass correlation coefficients for EHEC-7 models indicated that most of the variation occurred between pens, rather than within pens, or between feedlots. Hence, the potential combination of preharvest interventions and pen-level management strategies may have positive food safety impacts downstream along the beef chain.

INVESTIGATION ON CRYPTOSPORIDIUM INFECTIONS IN WILD ANIMALS IN A ZOO IN ANHUI PROVINCE.

PubMed

Gu, Youfang; Wang, Xiaolong; Zhou, Cefan; Li, Peiying; Xu, Qianming; Zhao, Changcheng; Liu, Wei; Xu, Wenlong

2016-09-01

To assess Cryptosporidium infections among wild animals in a zoo located in Anhui province, we conducted an investigation on the fecal samples collected from 44 primates, 41 herbivores, 44 carnivores and omnivores, and 103 birds in the zoo with the use of Sheather's sugar flotation technique and modified acid-fast staining. Cryptosporidium oocysts were detected in the fecal samples from six primates, two herbivores, four carnivores and omnivores, and seven birds by using Sheather's sugar flotation technique; the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 13.64, 4.88, 9.09, and 6.80%, respectively. Modified acid-fast staining detected the presence of Cryptosporidium oocysts in the fecal samples of one primate, three herbivores, 0 carnivores and omnivores, and one bird, and the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 2.27, 7.32, 0.00, and 0.97%, respectively. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) and phylogenetic analysis with the use of the neighbor-joining (NJ) method based on the aligned partial small-subunit (SSU) rRNA gene sequences showed that the protozoan pathogen isolated from primates was Cryptosporidium hominis and the pathogen isolated from camels ( Camelus dromedarius ) was Cryptosporidium andersoni. Subtyping the Cryptosporidium hominis by 60-kDa glycoprotein (GP60) gene phylogenetic analysis showed the Cryptosporidium hominis belongs to the subtype IdA and IbA.

Feasibility of the hydrogen sulfide test for the assessment of drinking water quality in post-earthquake Haiti.

PubMed

Weppelmann, Thomas A; Alam, Meer T; Widmer, Jocelyn; Morrissey, David; Rashid, Mohammed H; De Rochars, Valery M Beau; Morris, J Glenn; Ali, Afsar; Johnson, Judith A

2014-12-01

In 2010, a magnitude 7.0 earthquake struck Haiti, severely damaging the drinking and wastewater infrastructure and leaving millions homeless. Compounding this problem, the introduction of Vibrio cholerae resulted in a massive cholera outbreak that infected over 700,000 people and threatened the safety of Haiti's drinking water. To mitigate this public health crisis, non-government organizations installed thousands of wells to provide communities with safe drinking water. However, despite increased access, Haiti currently lacks the monitoring capacity to assure the microbial safety of any of its water resources. For these reasons, this study was designed to assess the feasibility of using a simple, low-cost method to detect indicators of fecal contamination of drinking water that could be implemented at the community level. Water samples from 358 sources of drinking water in the Léogâne flood basin were screened with a commercially available hydrogen sulfide test and a standard membrane method for the enumeration of thermotolerant coliforms. When compared with the gold standard method, the hydrogen sulfide test had a sensitivity of 65 % and a specificity of 93 %. While the sensitivity of the assay increased at higher fecal coliform concentrations, it never exceeded 88 %, even with fecal coliform concentrations greater than 100 colony-forming units per 100 ml. While its simplicity makes the hydrogen sulfide test attractive for assessing water quality in low-resource settings, the low sensitivity raises concerns about its use as the sole indicator of the presence or absence of fecal coliforms in individual or community water sources.

Feasibility of the Hydrogen Sulfide Test for the Assessment of Drinking Water Quality in Post-Earthquake Haiti

PubMed Central

Weppelmann, Thomas A.; Alam, Meer T.; Widmer, Jocelyn; Morrissey, David; Rashid, Mohammed H.; Beau De Rochars, Valery M.; Morris, J. Glenn; Ali, Afsar; Johnson, Judith A.

2014-01-01

In 2010 a magnitude 7.0 earthquake struck Haiti, severely damaging the drinking and waste water infrastructure and leaving millions homeless. Compounding this problem, the introduction of Vibrio cholera resulted in a massive cholera outbreak that infected over 700,000 people and threatened the safety of Haiti’s drinking water. To mitigate this public health crisis, non-government organizations installed thousands of wells to provide communities with safe drinking water. However, despite increased access, Haiti currently lacks the monitoring capacity to assure the microbial safety of any of its water resources. For these reasons, this study was designed to assess the feasibility of using a simple, low cost method to detect indicators of fecal contamination of drinking water that could be implemented at the community level. Water samples from 358 sources of drinking water in the Léogâne flood basin were screened with a commercially available hydrogen sulfide test and a standard membrane method for the enumeration of thermotolerant coliforms. When compared with the gold standard method, the hydrogen sulfide test had a sensitivity of 65% and a specificity of 93%. While the sensitivity of the assay increased at higher fecal coliform concentrations, it never exceeded 88%, even with fecal coliform concentrations greater than 100 colony forming units per 100 milliliters. While its simplicity makes the hydrogen sulfide test attractive for assessing water quality in low resource settings, the low sensitivity raises concerns about its use as the sole indicator of the presence or absence of fecal coliforms in individual or community water sources. PMID:25182685

Microbial Source Tracking Markers for Detection of Fecal Contamination in Environmental Waters: Relationships Between Pathogens and Human Health Outcomes

EPA Science Inventory

Microbial source tracking (MST) describes a suite of methods and an investigative strategy designed to identify the dominant sources of fecal pollution in environmental waters. The methods rely on the close association of certain fecal microorganisms with a particular host speci...

Evaluation of fecal indicator and pathogenic bacteria originating from swine manure applied to agricultural lands using culture-based and quantitative real-time PCR methods.

EPA Science Inventory

Fecal bacteria, including those originating from concentrated animal feeding operations, are a leading contributor to water quality impairments in agricultural areas. Rapid and reliable methods are needed that can accurately characterize fecal pollution in agricultural settings....

Evaluation of Fecal Indicator and Pathogenic Bacteria Originating from Swine Manure Applied to Agricultural Lands Using Culture-Based and Quantitative Real-Time PCR Methods

EPA Science Inventory

Fecal bacteria, including those originating from concentrated animal feeding operations, are a leading contributor to water quality impairments in agricultural areas. Rapid and reliable methods are needed that can accurately characterize fecal pollution in agricultural settings....

Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

EPA Science Inventory

Ability to distinguish between human and animal fecal pollution is important for risk assessment and watershed management, particularly in bodies of water used as sources of drinking water or for recreation. PCR-based methods were used to determine the source of fecal pollution ...

Potential for gulls to transport bacteria from human waste sites to beaches.

PubMed

Alm, Elizabeth W; Daniels-Witt, Quri R; Learman, Deric R; Ryu, Hodon; Jordan, Dustin W; Gehring, Thomas M; Santo Domingo, Jorge

2018-02-15

Contamination of recreational beaches due to fecal waste from gulls complicates beach monitoring and may pose a risk to public health. Gulls that feed at human waste sites may ingest human fecal microorganisms associated with that waste. If these gulls also visit beaches, they may serve as vectors, transporting fecal microorganisms to the beach where they may subsequently contaminate sand and water. In this study, samples collected from landfills, treated wastewater storage lagoons, and public beaches demonstrated a spatial and temporal overlap of markers for gull and human-associated microorganisms. In addition, markers for gull, fecal indicator bacteria, and the human-associated marker, HF183, were detected in gull feces and cloacae samples. Further, HF183 was detected in cloacae samples from gulls that were documented by radio-telemetry traveling between human waste sites and public beaches. This study highlights the potential for gulls that visit human waste sites to disperse human-associated microorganisms in the beach landscape. Copyright © 2017 Elsevier B.V. All rights reserved.

Fecal microbiota of lambs fed purple prairie clover (Dalea purpurea Vent.) and alfalfa (Medicago sativa).

PubMed

Huang, Qianqian; Holman, Devin B; Alexander, Trevor; Hu, Tianming; Jin, Long; Xu, Zhongjun; McAllister, Tim A; Acharya, Surya; Zhao, Guoqi; Wang, Yuxi

2018-01-01

The present study assessed the effect of purple prairie clover (PPC) and PPC condensed tannins (CT) on the fecal microbiota of lambs using high-throughput 16S rRNA gene pyrosequencing. A total of 18 individual lambs were randomly divided into three groups and fed either green chop alfalfa (Alf), a 40:60 (DM basis; Mix) mixture of Alf and PPC, or Mix supplemented with polyethylene glycol (Mix-P) for 18 days. Fecal samples were collected on days 13 through 18 using digital rectal retrieval. The DNA of fecal samples was extracted and the microbial 16S rRNA gene amplicons were sequenced using 454 pyrosequencing. Regardless of diet, the bacterial community was dominated by Firmicutes and Bacteroidetes with many sequences unclassified at the genus level. Forage type and CT had no effect on the fecal microbial composition at the phylum level or on α-diversity. Compared to the Alf diet, the Mix diet reduced the relative abundance of Akkermansia (P = 0.03) and Asteroleplasma (P = 0.05). Fecal microbial populations in Alf and Mix-P clustered separately from each other when assessed using unweighted UniFrac (P < 0.05). These results indicate that PPC CT up to 36 g/kg DM in the diet had no major effect on fecal microbial flora at the phyla level and exerted only minor effects on the genera composition of fecal microbiota in lambs.

Effects of Holding Time, Storage, and the Preservation of Samples on Sample Integrity for the Detection of Fecal Indicator Bacteria by Quantitative Polymerase Chain Reaction (qPCR)-based assays.

EPA Science Inventory

The purpose of this project was to answer questions related to storage of samples to be analyzed by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. The project was divided into two parts. The first part was to determine if filters th...

ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

EPA Science Inventory

Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

Association of Supply Type with Fecal Contamination of Source Water and Household Stored Drinking Water in Developing Countries: A Bivariate Meta-analysis

PubMed Central

Bain, Robert E.S.; Cronk, Ryan; Wright, Jim A.; Bartram, Jamie

2015-01-01

Background Access to safe drinking water is essential for health. Monitoring access to drinking water focuses on water supply type at the source, but there is limited evidence on whether quality differences at the source persist in water stored in the household. Objectives We assessed the extent of fecal contamination at the source and in household stored water (HSW) and explored the relationship between contamination at each sampling point and water supply type. Methods We performed a bivariate random-effects meta-analysis of 45 studies, identified through a systematic review, that reported either the proportion of samples free of fecal indicator bacteria and/or individual sample bacteria counts for source and HSW, disaggregated by supply type. Results Water quality deteriorated substantially between source and stored water. The mean percentage of contaminated samples (noncompliance) at the source was 46% (95% CI: 33, 60%), whereas mean noncompliance in HSW was 75% (95% CI: 64, 84%). Water supply type was significantly associated with noncompliance at the source (p < 0.001) and in HSW (p = 0.03). Source water (OR = 0.2; 95% CI: 0.1, 0.5) and HSW (OR = 0.3; 95% CI: 0.2, 0.8) from piped supplies had significantly lower odds of contamination compared with non-piped water, potentially due to residual chlorine. Conclusions Piped water is less likely to be contaminated compared with other water supply types at both the source and in HSW. A focus on upgrading water services to piped supplies may help improve safety, including for those drinking stored water. Citation Shields KF, Bain RE, Cronk R, Wright JA, Bartram J. 2015. Association of supply type with fecal contamination of source water and household stored drinking water in developing countries: a bivariate meta-analysis. Environ Health Perspect 123:1222–1231; http://dx.doi.org/10.1289/ehp.1409002 PMID:25956006

Identification of an Intestinal Microbiota Signature Associated With Severity of Irritable Bowel Syndrome.

PubMed

Tap, Julien; Derrien, Muriel; Törnblom, Hans; Brazeilles, Rémi; Cools-Portier, Stéphanie; Doré, Joël; Störsrud, Stine; Le Nevé, Boris; Öhman, Lena; Simrén, Magnus

2017-01-01

We have limited knowledge about the association between the composition of the intestinal microbiota and clinical features of irritable bowel syndrome (IBS). We collected information on the fecal and mucosa-associated microbiota of patients with IBS and evaluated whether these were associated with symptoms. We collected fecal and mucosal samples from adult patients who met the Rome III criteria for IBS at a secondary/tertiary care outpatient clinics in Sweden, as well as from healthy subjects. The exploratory set comprised 149 subjects (110 with IBS and 39 healthy subjects); 232 fecal samples and 59 mucosal biopsy samples were collected and analyzed by 16S ribosomal RNA targeted pyrosequencing. The validation set comprised 46 subjects (29 with IBS and 17 healthy subjects); 46 fecal samples, but no mucosal samples, were collected and analyzed. For each subject, we measured exhaled H 2 and CH 4 , oro-anal transit time, and the severity of psychological and gastrointestinal symptoms. Fecal methanogens were measured by quantitative polymerase chain reaction. Numerical ecology analyses and a machine learning procedure were used to analyze the data. Fecal microbiota showed covariation with mucosal adherent microbiota. By using classic approaches, we found no differences in fecal microbiota abundance or composition between patients with IBS vs healthy patients. A machine learning procedure, a computational statistical technique, allowed us to reduce the 16S ribosomal RNA data complexity into a microbial signature for severe IBS, consisting of 90 bacterial operational taxonomic units. We confirmed the robustness of the intestinal microbial signature for severe IBS in the validation set. The signature was able to discriminate between patients with severe symptoms, patients with mild/moderate symptoms, and healthy subjects. By using this intestinal microbiota signature, we found IBS symptom severity to be associated negatively with microbial richness, exhaled CH 4 , presence of methanogens, and enterotypes enriched with Clostridiales or Prevotella species. This microbiota signature could not be explained by differences in diet or use of medications. In analyzing fecal and mucosal microbiota from patients with IBS and healthy individuals, we identified an intestinal microbiota profile that is associated with the severity of IBS symptoms. NCT01252550. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area

PubMed Central

Sinigalliano, C. D.; Gidley, M. L.; Shibata, T.; Whitman, D.; Dixon, T. H.; Laws, E.; Hou, A.; Bachoon, D.; Brand, L.; Amaral-Zettler, L.; Gast, R. J.; Steward, G. F.; Nigro, O. D.; Fujioka, R.; Betancourt, W. Q.; Vithanage, G.; Mathews, J.; Fleming, L. E.; Solo-Gabriele, H. M.

2007-01-01

Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within 2 months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent 3 months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters. PMID:17488814

ESTABLISHING NORMAL FECAL FLORA IN WILD AUSTRALIAN PASSERINE BIRDS BY USE OF THE FECAL GRAM STAIN.

PubMed

Latham, Benjamin; Leishman, Alan; Martin, John; Phalen, David

2017-09-01

The purpose of this study was to determine the normal fecal bacterial and fungal flora and parasite prevalence in wild passerine birds found at the Australian Botanic Garden (Mount Annan, New South Wales). Wild passerine birds (n = 186) from 28 species were captured with mist nets. Fecal Gram stains (n = 155) were made from 26 species and analyzed for bacterial density, Gram stain morphology, and the presence of yeast. Fecal wet preparations (n = 139) were made from 24 passerine species and were analyzed for parasites. Our results showed that 81.9% of passerines sampled had bacteria present in their feces. The bacteria found were entirely Gram positive and consisted predominantly of cocci. Individuals that were caught on multiple occasions were found to have stable bacterial populations, apart from the red-browed finch (Neochmia temporalis). Insectivores had higher bacterial densities and cocci proportions than nectarivores had. Yeasts were rare in most species, with the exception of the bell miner (Manorina melanophrys) and noisy miner (Manorina melanocephala). The yeast, Macrorhabdus ornithogaster, and parasites were not observed in any fecal samples. These results will help practitioners to assess the health of Australian passerine species submitted for care or housed in zoological collections.

Relationship Between Microbiota of the Colonic Mucosa vs Feces and Symptoms, Colonic Transit, and Methane Production in Female Patients With Chronic Constipation.

PubMed

Parthasarathy, Gopanandan; Chen, Jun; Chen, Xianfeng; Chia, Nicholas; O'Connor, Helen M; Wolf, Patricia G; Gaskins, H Rex; Bharucha, Adil E

2016-02-01

In fecal samples from patients with chronic constipation, the microbiota differs from that of healthy subjects. However, the profiles of fecal microbiota only partially replicate those of the mucosal microbiota. It is not clear whether these differences are caused by variations in diet or colonic transit, or are associated with methane production (measured by breath tests). We compared the colonic mucosal and fecal microbiota in patients with chronic constipation and in healthy subjects to investigate the relationships between microbiota and other parameters. Sigmoid colonic mucosal and fecal microbiota samples were collected from 25 healthy women (controls) and 25 women with chronic constipation and evaluated by 16S ribosomal RNA gene sequencing (average, 49,186 reads/sample). We assessed associations between microbiota (overall composition and operational taxonomic units) and demographic variables, diet, constipation status, colonic transit, and methane production (measured in breath samples after oral lactulose intake). Fourteen patients with chronic constipation had slow colonic transit. The profile of the colonic mucosal microbiota differed between constipated patients and controls (P < .05). The overall composition of the colonic mucosal microbiota was associated with constipation, independent of colonic transit (P < .05), and discriminated between patients with constipation and controls with 94% accuracy. Genera from Bacteroidetes were more abundant in the colonic mucosal microbiota of patients with constipation. The profile of the fecal microbiota was associated with colonic transit before adjusting for constipation, age, body mass index, and diet; genera from Firmicutes (Faecalibacterium, Lactococcus, and Roseburia) correlated with faster colonic transit. Methane production was associated with the composition of the fecal microbiota, but not with constipation or colonic transit. After adjusting for diet and colonic transit, the profile of the microbiota in the colonic mucosa could discriminate patients with constipation from healthy individuals. The profile of the fecal microbiota was associated with colonic transit and methane production (measured in breath), but not constipation. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Optimizing the diagnostic testing of Clostridium difficile infection.

PubMed

Bouza, Emilio; Alcalá, Luis; Reigadas, Elena

2016-09-01

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and is associated with a considerable health and cost burden. However, there is still not a clear consensus on the best laboratory diagnosis approach and a wide variation of testing methods and strategies can be encountered. We aim to review the most practical aspects of CDI diagnosis providing our own view on how to optimize CDI diagnosis. Expert commentary: Laboratory diagnosis in search of C. difficile toxins should be applied to all fecal diarrheic samples reaching the microbiology laboratory in patients > 2 years old, with or without classic risk factors for CDI. Detection of toxins either directly in the fecal sample or in the bacteria isolated in culture confirm CDI in the proper clinical setting. Nuclear Acid Assay techniques (NAAT) allow to speed up the process with epidemiological and therapeutic consequences.

Chapter A7. Biological Indicators

USGS Publications Warehouse

Myers, Donna N.; Wilde, Franceska D.

2003-01-01

The National Field Manual for the Collection of Water-Quality Data (National Field Manual) provides guidelines and standard procedures for U.S. Geological Survey (USGS) personnel who collect data used to assess the quality of the Nation's surface-water and ground-water resources. This chapter of the manual includes procedures for the (1) determination of biochemical oxygen demand using a 5-day bioassay test; (2) collection, identification, and enumeration of fecal indicator bacteria; (3) collection of samples and information on two laboratory methods for fecal indicator viruses (coliphages); and (4) collection of samples for protozoan pathogens. Each chapter of the National Field Manual is published separately and revised periodically. Newly published and revised chapters are posted on the World Wide Web on the USGS page 'National Field Manual for the Collection of Water-Quality Data.' The URL for this page is http://pubs.water.usgs.gov/twri9A/ (accessed November 25, 2003).

Bacteriological water quality in the Lake Pontchartrain basin Louisiana following Hurricanes Katrina and Rita, September 2005

USGS Publications Warehouse

Stoeckel, Donald M.; Bushon, Rebecca N.; Demcheck, Dennis K.; Skrobialowski, Stanley C.; Kephart, Christopher M.; Bertke, Erin E.; Mailot, Brian E.; Mize, Scott V.; Fendick, Robert B.

2005-01-01

The U.S. Geological Survey (USGS), in collaboration with the Louisiana Department of Environmental Quality, monitored bacteriological quality of water at 22 sites in and around Lake Pontchartrain, La., for three consecutive weeks beginning September 13, 2005, following hurricanes Katrina and Rita and the associated flooding. Samples were collected and analyzed by USGS personnel from the USGS Louisiana Water Science Center and the USGS Ohio Water Microbiology Laboratory. Fecal-indicator bacteria (Escherichia coli, enterococci, and fecal coliform) concentrations ranged from the detection limit to 36,000 colony-forming units per 100 milliliters. Data are presented in tabular form and as plots of data in the context of available historical data and water-quality standards and criteria for each site sampled. Quality-control data were reviewed to ensure that methods performed as expected in a mobile laboratory setting.

Recovery of the gut microbiome following fecal microbiota transplantation.

PubMed

Seekatz, Anna M; Aas, Johannes; Gessert, Charles E; Rubin, Timothy A; Saman, Daniel M; Bakken, Johan S; Young, Vincent B

2014-06-17

Clostridium difficile infection is one of the most common health care-associated infections, and up to 40% of patients suffer from recurrence of disease following standard antibiotic therapy. Recently, fecal microbiota transplantation (FMT) has been successfully used to treat recurrent C. difficile infection. It is hypothesized that FMT aids in recovery of a microbiota capable of colonization resistance to C. difficile. However, it is not fully understood how this occurs. Here we investigated changes in the fecal microbiota structure following FMT in patients with recurrent C. difficile infection, and imputed a hypothetical functional profile based on the 16S rRNA profile using a predictive metagenomic tool. Increased relative abundance of Bacteroidetes and decreased abundance of Proteobacteria were observed following FMT. The fecal microbiota of recipients following transplantation was more diverse and more similar to the donor profile than the microbiota prior to transplantation. Additionally, we observed differences in the imputed metagenomic profile. In particular, amino acid transport systems were overrepresented in samples collected prior to transplantation. These results suggest that functional changes accompany microbial structural changes following this therapy. Further identification of the specific community members and functions that promote colonization resistance may aid in the development of improved treatment methods for C. difficile infection. Within the last decade, Clostridium difficile infection has surpassed other bacterial infections to become the leading cause of nosocomial infections. Antibiotic use, which disrupts the gut microbiota and its capability in providing colonization resistance against C. difficile, is a known risk factor in C. difficile infection. In particular, recurrent C. difficile remains difficult to treat with standard antibiotic therapy. Fecal microbiota transplantation (FMT) has provided a successful treatment method for some patients with recurrent C. difficile infection, but its mechanism and long-term effects remain unknown. Our results provide insight into the structural and potential metabolic changes that occur following FMT, which may aid in the development of new treatment methods for C. difficile infection. Copyright © 2014 Seekatz et al.

Increasing Efficiency of Fecal Coliform Testing Through EPA-Approved Alternate Method Colilert*-18

NASA Technical Reports Server (NTRS)

Cornwell, Brian

2017-01-01

The 21 SM 9221 E multiple-tube fermentation method for fecal coliform analysis requires a large time and reagent investment for the performing laboratory. In late 2010, the EPA approved an alternative procedure for the determination of fecal coliforms designated as Colilert*-18. However, as of late 2016, only two VELAP-certified laboratories in the Commonwealth of Virginia have been certified in this method.

Mesquite seed density in fecal samples of Raramuri Criollo vs. Angus x Hereford cows grazing Chihuahuan Desert Rangeland

USDA-ARS?s Scientific Manuscript database

This study was part of a larger project investigating breed-related differences in feeding habits of Raramuri Criollo (RC) versus Angus x Hereford (AH) cows. Seed densities in fecal samples collected in July and August 2015 were analyzed to compare presumed mesquite bean consumption of RC and AH cow...

A microhistological technique for analysis of food habits of mycophagous rodents.

Treesearch

Patrick W. McIntire; Andrew B. Carey

1989-01-01

We present a technique, based on microhistological analysis of fecal pellets, for quantifying the diets of forest rodents. This technique provides for the simultaneous recording of fungal spores and vascular plant material. Fecal samples should be freeze dried, weighed, and rehydrated with distilled water. We recommend a minimum sampling intensity of 50 fields of view...

Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

EPA Science Inventory

A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

Fiber source and inclusion level affects characteristics of excreta from growing pigs

PubMed Central

Ndou, Saymore Petros; Bakare, Archibold Garikayi

2018-01-01

Objective The objective of the study was to determine the influence of varying fibrous diets on fecal characteristics of growing pigs. Methods A total of 104 pigs (initial weight 18±2.0 kg) were used in the study. They were housed in individual pens and fed on diets containing maize cob, grass hay, lucerne hay, maize stover, and sunflower husk. These fibers were included at 0, 80, 160, 240, 320 and 400 g/kg. Fecal and urine samples were collected. Results Fecal output was largest amongst pigs fed on diets containing grass hay and maize stover (p<0.05). Nitrogen content was highest in feces from pigs fed on sunflower husk (p< 0.05). Pigs fed on diets containing maize stover and maize cobs produced the largest concentrations of short chain fatty acids. Acetate concentration was high in feces of pigs fed maize stover than those fed grass hay and lucerne hay (p<0.05). As the level of fiber inclusion increased, fecal consistency and nitrogen content increased linearly (p<0.05). Urea nitrogen decreased as the inclusion level increased across all the fibers (p<0.05), with maize cobs containing the largest content of urea nitrogen. As dietary fiber content increased, fecal nitrogen content also increased (p<0.05). Conclusion It was concluded that different fiber sources influence fecal characteristics, thereby having different implications on pig waste management. It is vital to monitor fiber inclusion thresholds so as to easily manage environmental pollutants such as butyrate that contribute to odors. PMID:26954189

Screening of nursing home residents for colonization with carbapenem-resistant Enterobacteriaceae admitted to acute care hospitals: Incidence and risk factors.

PubMed

Cunha, Cheston B; Kassakian, Steven Z; Chan, Ryan; Tenover, Fred C; Ziakas, Panos; Chapin, Kimberle C; Mermel, Leonard A

2016-02-01

There are increasing reports of multidrug-resistant gram-negative bacilli in nursing homes and acute care hospitals. We performed a point prevalence survey to detect fecal carriage of gram-negative bacteria carrying carbapenem resistance genes or which were otherwise resistant to carbapenem antibiotics among 500 consecutive admissions from local nursing homes to 2 hospitals in Providence, Rhode Island. We performed a case-control study to identify risk factors associated with carriage of carbapenem-resistant Enterobacteriaceae (CRE). There were 404 patients with 500 hospital admissions during which they had rectal swab samples cultured. Fecal carriage of any carbapenem-resistant or carbapenemase- producing gram-negative bacteria was found in 23 (4.6%) of the 500 hospital admissions, including 7 CRE (1.4%), 2 (0.4%) of which were Klebsiella pneumoniae carbapenemase (ie, blaKPC) producing (CPE) Citrobacter freundii, 1 of which was carbapenem susceptible by standard testing methods. Use of a gastrostomy tube was associated with CRE carriage (P = .04). We demonstrated fecal carriage of carbapenem-resistant or carbapenemase-producing gram-negative bacteria in 4.6% of nursing home patients admitted to 2 acute care hospitals, but only 0.4% of such admissions were patients with fecal carriage of CPE. Use of gastrostomy tubes was associated with fecal carriage of gram-negative bacteria with detectable carbapenem resistance. CRE fecal carriage is uncommon in our hospital admissions from nursing homes. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Conservation Physiology of an Uncatchable Animal: The North Atlantic Right Whale (Eubalaena glacialis).

PubMed

Hunt, Kathleen E; Rolland, Rosalind M; Kraus, Scott D

2015-10-01

The North Atlantic right whale, Eubalaena glacialis (NARW), a critically endangered species that has been under intensive study for nearly four decades, provides an excellent case study for applying modern methods of conservation physiology to large whales. By combining long-term sighting histories of known individuals with physiological data from newer techniques (e.g., body condition estimated from photographs; endocrine status derived from fecal samples), physiological state and levels of stress can be estimated despite the lack of any method for nonlethal capture of large whales. Since traditional techniques for validating blood assays cannot be used in large whales, assays of fecal hormones have been validated using information on age, sex, and reproductive state derived from an extensive NARW photo-identification catalog. Using this approach, fecal glucocorticoids have been found to vary dramatically with reproductive state. It is therefore essential that glucocorticoid data be interpreted in conjunction with reproductive data. A case study correlating glucocorticoids with chronic noise is presented as an example. Keys to a successful research program for this uncatchable species have included: consistent population monitoring over decades, data-sharing across institutions, an extensive photo-identification catalog that documents individual histories, and consistent efforts at noninvasive collection of samples over years. Future research will require flexibility to adjust to changing distributions of populations. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Microbiological indicators of water quality in the Xochimilco canals, Mexico City.

PubMed

Juárez-Figueroa, Luis Alfredo; Silva-Sánchez, Jesús; Uribe-Salas, Felipe Javier; Cifuentes-García, Enrique

2003-01-01

To quantify microbiology indicators of fecal contamination in the effluents of two waste water treatment plants and in samples collected in several canals in Xochimilco. A cross sectional study was performed. Ten sites, 5 from plant effluents and 5 from canals, were selected for sampling during November and December 2001. Fecal coliforms and enterococci were quantified by membrane filtration, male specific (F+) and somatic coliphages by double agar layer technique, and Cryptosporidium oocysts and Giardia cysts by concentration with Envirocheck filter followed by immunofluorescence microscopy quantification. The average of organisms counts from effluents and canal water were compared with t Student test. Treated water discharge in canals showed a low count of Fecal Coliforms (average 40.4/100 ml), enterococci (average 58.8/100 ml) and Cryptosporidium oocysts (average 13.2/100 l), while coliphages and Giardia cyst rendered higher counts (average 1467.5/100 ml and 1199.8/100 l, respectively) suggesting the water treatment methods could fail to remove these agents. A significant lower count of Giardia cysts (average 45/100 l) and no Cryptosporidium oocysts were found in irrigation canals, which suggests a natural clearance of these pathogens. Strains of Escherichia coli isolated in one of the canals contaminated with sewage had antimicrobial multi-resistance that was transferred by conjugation suggesting that resistance is encoded in a plasmid potentially transferable to other pathogenic bacteria. Cost effective and culturally acceptable waste treatment methods will require careful planning and consultation if they are to be adopted and mantained by local populations.

Development and Evaluation of a Loop-Mediated Isothermal Amplification (Lamp) Assay for the Detection of Haemonchus contortus in Goat Fecal Samples.

PubMed

Yang, X; Qi, M W; Zhang, Z Z; Gao, C; Wang, C Q; Lei, W Q; Tan, L; Zhao, J L; Fang, R; Hu, M

2017-04-01

Haemonchus contortus is one of the most significant strongylid nematodes infecting small ruminants, and it causes great economic losses to the livestock industry worldwide. Accurate diagnosis of H. contortus is crucial to control strategies. Traditional microscopic examinations are the most common methods for the diagnosis of H. contortus , but they are time-consuming and inaccurate. Molecular methods based on PCR are more accurate, but need expensive machines usually only used in the laboratory. Loop-mediated isothermal amplification (LAMP) is a rapid, simple, specific, and sensitive method that has been widely used to detect viruses, bacteria, and parasites. In the present study, a LAMP method targeting ribosomal ITS-2 gene for detection of the H. contortus in goat fecal samples has been established. The established LAMP method was H. contortus specific, and the sensitivity of LAMP was the same as that of the H. contortus species-specific PCR, with the lowest DNA level detected as being 1 pg. Examination of the clinical samples indicated that the positive rate of LAMP was higher than that of PCR, but no statistical difference was observed between LAMP and PCR (χ 2 = 17.991, P = 0.053). In conclusion, a LAMP assay with a high specificity and a good sensitivity has been developed to detect H. contortus infection in goats. The established LAMP assay is useful for clinical diagnosis of H. contortus .

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

USGS Publications Warehouse

Stoeckel, D.M.; Stelzer, E.A.; Stogner, R.W.; Mau, D.P.

2011-01-01

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections-augmented with local observations and land-use patterns-indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination. ?? 2011 Elsevier Ltd.

Frequent detection of a human fecal indicator in the urban ocean: environmental drivers and covariation with enterococci.

PubMed

Jennings, Wiley C; Chern, Eunice C; O'Donohue, Diane; Kellogg, Michael G; Boehm, Alexandria B

2018-03-01

Fecal pollution of surface waters presents a global human health threat. New molecular indicators of fecal pollution have been developed to address shortcomings of traditional culturable fecal indicators. However, there is still little information on their fate and transport in the environment. The present study uses spatially and temporally extensive data on traditional (culturable enterococci, cENT) and molecular (qPCR-enterococci, qENT and human-associated marker, HF183/BacR287) indicator concentrations in marine water surrounding highly-urbanized San Francisco, California, USA to investigate environmental and anthropogenic processes that impact fecal pollution. We constructed multivariable regression models for fecal indicator bacteria at 14 sampling stations. The human marker was detected more frequently in our study than in many other published studies, with detection frequency at some stations as high as 97%. The odds of cENT, qENT, and HF183/BacR287 exceeding health-relevant thresholds were statistically elevated immediately following discharges of partially treated combined sewage, and cENT levels dissipated after approximately 1 day. However, combined sewer discharges were not important predictors of indicator levels typically measured in weekly monitoring samples. Instead, precipitation and solar insolation were important predictors of cENT in weekly samples, while precipitation and water temperature were important predictors of HF183/BacR287 and qENT. The importance of precipitation highlights the significance of untreated storm water as a source of fecal pollution to the urban ocean, even for a city served by a combined sewage system. Sunlight and water temperature likely control persistence of the indicators via photoinactivation and dark decay processes, respectively.

Assessment of possible sources of microbiological contamination in the water column and streambed sediment of the Jacks Fork, Ozark National Scenic Riverways, Missouri - Phase III

USGS Publications Warehouse

Davis, Jerri V.; Barr, Miya N.

2006-01-01

In 1998, a 5 river-mile reach of the Jacks Fork was included on Missouri's list of impaired waters as required by Section 303(d) of the Federal Clean Water Act. The identified pollutant on the Jacks Fork was fecal coliform bacteria. The length of the impaired reach was changed to 7 miles on the Missouri 2002 303(d) list because of data indicating the fecal coliform bacteria problem existed over a broader area. The U.S. Geological Survey, in cooperation with the National Park Service, conducted a study to better understand the extent and sources of microbiological contamination within the Jacks Fork from Alley Spring to the mouth, which includes the 7-mile 303(d) reach. Ten sites were sampled from June 2003 through October 2003 and from June 2004 through October 2004. Water-column and streambed sediment samples were collected from main-stem and tributary sites mostly during base-flow conditions during a variety of recreational season river uses and analyzed for fecal coliform and Escherichia coli bacteria. Isolates of Escherichia coli obtained from water samples collected at five sites were submitted for rep-PCR analysis to identify presumptive sources of fecal indicator bacteria in the Jacks Fork. Results indicate that recreational users (including boaters and swimmers) are not the primary source of fecal coliform bacteria in the Jacks Fork; rather, the presence of fecal coliform bacteria is associated with other animals, of which horses are the primary source. Increases in fecal coliform bacteria densities in the Jacks Fork are associated with cross-country horseback trail-riding events.

Diagnostic strategies to reveal covert infections with intestinal helminths in dogs.

PubMed

Adolph, Chris; Barnett, Sharon; Beall, Melissa; Drake, Jason; Elsemore, David; Thomas, Jennifer; Little, Susan

2017-11-30

Intestinal helminths are common in dogs in the United States, particularly non-treated dogs in animal shelters, but surveys by fecal flotation may underestimate their prevalence. To determine the prevalence of intestinal helminths and evaluate the ability of fecal flotation and detection of nematode antigen to identify those infections, contents of the entire gastrointestinal tract of 97 adult (>1year) dogs previously identified for humane euthanasia at two animal control shelters in northeastern Oklahoma, USA, were screened. All helminths recovered were washed in saline and fixed prior to enumeration and identification to genus and species. Fecal samples from each dog were examined by passive sodium nitrate (SG 1.33) and centrifugal sugar solution (SG 1.25) flotation. Fecal antigen detection assays were used to confirm the presence of nematode antigen in frozen fecal samples from 92 dogs. Necropsy examination revealed Ancylostoma caninum in 45/97 (46.4%), Toxocara canis in 11/97 (11.3%), Trichuris vulpis in 38/97 (39.2%), Dipylidium caninum in 48/97 (49.5%), and Taenia sp. in 7/97 (7.2%) dogs. Passive fecal flotation identified 38/45 (84.4%) A. caninum, 6/11 (54.5%) T. canis, 26/38 (68.4%) T. vulpis, 2/48 (4.2%) D. caninum, and 1/7 (14.3%) Taenia sp. infections, while centrifugal flotation combined with antigen detection assays identified A. caninum in 97.7% (43/44), T. canis in 77.8% (7/9), and T. vulpis in 83.3% (30/36) of infected dogs based on necropsy recovery of nematodes. Taken together, these data indicate that detection of nematode antigen is a useful adjunct to microscopic examination of fecal samples for parasite eggs, and that this approach can improve diagnostic sensitivity for intestinal nematode infections in dogs. Copyright © 2017 Elsevier B.V. All rights reserved.

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

PubMed

Erlandsen, S L; Sherlock, L A; Bemrick, W J

1990-04-01

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Fecal Contamination on Produce from Wholesale and Retail Food Markets in Dhaka, Bangladesh.

PubMed

Harris, Angela R; Islam, Mohammad Aminul; Unicomb, Leanne; Boehm, Alexandria B; Luby, Stephen; Davis, Jennifer; Pickering, Amy J

2018-01-01

Fresh produce items can become contaminated with enteric pathogens along the supply chain at the preharvest (e.g., irrigation water, soil, fertilizer) or postharvest (e.g., vendor handling or consumer handling) stages. This study assesses the concentrations of fecal indicator bacteria Escherichia coli , enterococci (ENT), and Bacteriodales on surfaces of carrots, eggplants, red amaranth leaves, and tomatoes obtained from both a wholesale market (recently harvested) and neighborhood retail markets in Dhaka, Bangladesh. We detected E. coli in 100% of carrot and red amaranth rinses, 92% of eggplant rinses, and 46% of tomato rinses. Using a molecular microbial source tracking assay, we found that 32% of produce samples were positive for ruminant fecal contamination. Fecal indicator bacteria were more likely to be detected on produce collected in retail markets compared with that in the wholesale market; retail market produce were 1.25 times more likely to have E. coli detected ( P = 0.03) and 1.24 times more likely to have ENT detected ( P = 0.03) as compared with wholesale market produce. Bacteriodales was detected in higher concentrations in retail market produce samples compared with wholesale market produce samples (0.40 log 10 gene copies per 100 cm 2 higher, P = 0.03). Our results suggest that ruminant and general fecal contamination of produce in markets in Dhaka is common, and suggest that unsanitary conditions in markets are an important source of produce fecal contamination postharvest.

Probabilistic analysis showing that a combination of bacteroides and methanobrevibacter source tracking markers is effective for identifying waters contaminated by human fecal pollution

USGS Publications Warehouse

Johnston, Christopher; Byappanahalli, Muruleedhara N.; Gibson, Jacqueline MacDonald; Ufnar, Jennifer A.; Whitman, Richard L.; Stewart, Jill R.

2013-01-01

Microbial source tracking assays to identify sources of waterborne contamination typically target genetic markers of host-specific microorganisms. However, no bacterial marker has been shown to be 100% host-specific, and cross-reactivity has been noted in studies evaluating known source samples. Using 485 challenge samples from 20 different human and animal fecal sources, this study evaluated microbial source tracking markers including the Bacteroides HF183 16S rRNA, M. smithii nifH, and Enterococcus esp gene targets that have been proposed as potential indicators of human fecal contamination. Bayes' Theorem was used to calculate the conditional probability that these markers or a combination of markers can correctly identify human sources of fecal pollution. All three human-associated markers were detected in 100% of the sewage samples analyzed. Bacteroides HF183 was the most effective marker for determining whether contamination was specifically from a human source, and greater than 98% certainty that contamination was from a human source was shown when both Bacteroides HF183 and M. smithii nifH markers were present. A high degree of certainty was attained even in cases where the prior probability of human fecal contamination was as low as 8.5%. The combination of Bacteroides HF183 and M. smithii nifH source tracking markers can help identify surface waters impacted by human fecal contamination, information useful for prioritizing restoration activities or assessing health risks from exposure to contaminated waters.

Screening and rapid identification of Campylobacter spp. DNA by FlaA PCR based method on chicken and human fecal samples in Egypt

USDA-ARS?s Scientific Manuscript database

Campylobacter is a foodborne pathogen which has a potential public health concern worldwide. Due to discriminatory problems encountered by conventional isolation of Campylobacter spp. and its genetic similarities, rapid molecular techniques for its genetic characterization are useful. In this study,...

EVALUATE THE UTILITY OF ENTEROCOCCI AS INDICATORS OF THE SOURCES OF FECAL CONTAMINATION IN IMPAIRED SUBWATERSHEDS THROUGH DNA-BASED MOLECULAR TECHNIQUES

EPA Science Inventory

Microbial source tracking (MST) is based on the assumption that specific strains of bacteria are associated with specific host species. MST methods are attractive because their application on environmental samples could help define the nature of water quality problems in impaire...

Multiplex PCR for Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

PubMed Central

Vidal, Roberto; Vidal, Maricel; Lagos, Rossana; Levine, Myron; Prado, Valeria

2004-01-01

A multiplex PCR for detection of three categories of diarrheagenic Escherichia coli was developed. With this method, enterohemorrhagic E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were identified in fecal samples from patients with hemorrhagic colitis, watery diarrhea, or hemolytic-uremic syndrome and from food-borne outbreaks. PMID:15071051

DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY THE MERGING PATHOGEN HEPATITIS E IN WATER SAMPLES

EPA Science Inventory

Hepatitis E virus (HEV) is an emerging pathogen that causes significant illness in the developing world. Like the hepatitis A virus, it is transmitted via the fecal-oral route and can cause short-term, acute hepatitis. In addition, hepatitis E has been found to cause a signific...

DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY THE EMERGING PATHOGEN HEPATITIS E IN WATER SAMPLES

EPA Science Inventory

Hepatitis E virus (HEV) is an emerging pathogen that causes significant illness in the developing world. Like the hepatitis A virus, it is transmitted via the fecal-oral route and can cause short-term, acute hepatitis. In addition, hepatitis E has been found to cause a signific...

Is "dried stool spots on filter paper method (DSSFP)" more sensitive and effective for detecting Blastocystis spp. and their subtypes by PCR and sequencing?

PubMed

Seyer, Ayse; Karasartova, Djursun; Ruh, Emrah; Güreser, Ayse Semra; Imir, Turgut; Taylan-Ozkan, Aysegul

2016-12-01

PCR and DNA sequencing are currently the diagnostic methods of choice for detection of Blastocystis spp. and their suptypes. Fresh or frozen stool samples have disadvantages in terms of several aspects such as transportation, storage, and existence of PCR inhibitors. Filter paper technology may provide a solution to these issues. The aim of the present study was to detect Blastocystis spp. and their subtypes by employing two different preservation methods: conventional frozen stool (FS) and dried stool spots on filter paper (DSSFP). Concentration and purity of DNA, sensitivity of PCR, and DNA sequencing results obtained from the two methods were also compared. A total of 230 fecal samples were included and separated into two parts: one part of the fecal samples were directly frozen and stored at -20 °C. The remaining portion of the specimens were homogenized with saline and spread onto the filter papers as thin layer with a diameter of approximately 3 cm. After air-dried, the filter papers were stored at room temperature. DSSFP samples were collected by scraping from the filter papers. DNA were extracted by EURx Stool DNA Extraction Kit from both samples. Concentration and purity were measured with Nano-Drop, then PCR and sequencing were conducted for detection of Blastocystis spp. and its genotypes. Pure DNA was obtained with a A260/A280 ratio of 1.7-2.2 in both methods. DNA yield from FS was 25-405 ng/μl and average DNA concentration was 151 ng/μl, while these were 7-339 and 122 ng/μl for DSSFP, respectively. No PCR inhibition was observed in two methods. DNA from DSSFP were found to be stable and PCR were reproducible for at least 1 year. FS-PCR- and DSSFP-PCR-positive samples were 49 (21.3 %) and 58 (25.3 %), respectively (p = 0.078). The 43 specimens were concordantly positive by both FS-PCR and DSSFP-PCR. When the microscopy was taken as the gold standard, sensitivity of DSSFP-PCR and FS-PCR was 95.5 and 86.4 %, while specificity of both tests was 99.4 and 98.3 %, respectively. DNA sequencing results of 19 microscopically confirmed cases were strictly identical (concordance 100 %) in both methods, and ST2:6, ST3:8, ST4:3, and ST6:2 were the detected subtypes. Among the 230 fecal samples, the most predominant subtypes were ST3, ST2, ST4, and ST1 by both FS and DSSFP methods. Concordance of DNA sequencing results obtained from the two methods was noted to be 90.7 %. To our knowledge, this is the first study that demonstrates DNA extraction from DSSFP is more sensitive and effective than the FS method for diagnosis of Blastocystis spp. and their subtypes by PCR and DNA sequencing.

Fecal indicator bacteria persistence under natural conditions in an ice-covered river.

PubMed Central

Davenport, C V; Sparrow, E B; Gordon, R C

1976-01-01

Total coliform (TC), fecal coliform (FC), and fecal streptococcus (FS) survival characteristics, under natural conditions at 0 degrees C in an ice-covered river, were examined during February and March 1975. The membrane filter (MF) technique was used throughout the study, and the multiple-tube (MPN) method was used in parallel on three preselected days for comparative recovery of these bacteria. Survival was studied at seven sample stations downstream from all domestic pollution sources in a 317-km reach of the river having 7.1 days mean flow time (range of 6.0 to 9.1 days). The mean indicator bacteria densities decreased continuously at successive stations in this reach and, after adjustment for dilution, the most rapid die-off was found to occur during the first 1.9 days, followed by a slower decrease. After 7.1 days, the relative survival was TC less than FC less than FS, with 8.4%, 15.7%, and 32.8% of the initial populations remaining viable, respectively. These rates are higher than previously reported and suggest that the highest survival rates for these bacteria in receiving streams can be expected at 0 degree C under ice cover. Additionally, the FC-FS ratio was greater than 5 at all stations, indicating that this ratio may be useable for determining the source of fecal pollution in receiving streams for greater than 7 days flow time at low water temperatures. The MPN and MF methods gave comparable results for the TC and FS at all seven sample stations, with both the direct and verified MF counts within the 95% confidence limits of the respective MPNs in most samples, but generally lower than the MPN index. Although FC recovery on membrane filters was comparable results at stations near the pollution source. However, the results became more comparable with increasing flow time. The results of this study indicate that heat shock is a major factor in suppression of the FC counts on the membrane filters at 44.5 degree C. Heat shock may be minimized by extended incubation at 35 degrees C before exposure to the higher temperature. PMID:825042

Comparison of fecal culture and Danish Mix-ELISA for determination of Salmonella enterica subsp. enterica prevalence in growing swine.

PubMed

Funk, J A; Harris, I T; Davies, P R

2005-04-25

In the USA, control of food-borne salmonellosis associated with meat consumption has been predominantly focused at slaughter and processing. It is expected that standards at slaughter and processing will become more stringent, creating pressure to reduce prevalence of Salmonella-positive food animals through on-farm interventions. The aim of this study was to compare traditional fecal culture and the Danish Mix-ELISA (DME) for determination of Salmonella prevalence pre-harvest in swine. In Trial 1, five cohorts of individually identified pigs were longitudinally sampled during the growing period to compare the kinetics of prevalence as estimated by fecal culture and the DME. In Trial 2, the correlation between fecal prevalence and seroprevalence was estimated pre-marketing in 49 groups of pigs. In Trial 1, fecal prevalence and seroprevalence showed similar kinetics, with a tendency of a higher OD% cut-off to more closely approximate fecal prevalence. In Trial 2, correlations between fecal culture and the DME were 0.40, 0.36, 0.43, and 0.43 (p<0.001) for OD% cut-offs > or =10, 20, 30, and 40, respectively. Based on these results, a higher OD% cut-off would be recommended if more approximate estimation of fecal prevalence is desired and longitudinal sampling would be suggested for evaluating the impact of on-farm interventions for Salmonella reduction whether utilizing fecal culture or the DME. Further evaluation of the impact of Salmonella serovar present on farms on seroprevalence and the relationship of on-farm seroprevalence with food safety risk are needed prior to utilizing the DME for pre-harvest Salmonella diagnostics in the US swine herd.

Validation of a fecal scoring scale in puppies during the weaning period.

PubMed

Grellet, Aurélien; Feugier, Alexandre; Chastant-Maillard, Sylvie; Carrez, Bruno; Boucraut-Baralon, Corine; Casseleux, Gregory; Grandjean, Dominique

2012-10-01

In puppies weaning is a high risk period. Fecal changes are frequent and can be signs of infection by digestive pathogens (bacteria, viruses, parasites) and indicators of nutritional and environmental stress. The aim of this study was to define a pathological fecal score for weaning puppies, and to study the impact on that score of two intestinal viruses (canine parvovirus type 2 and canine coronavirus). For this, the quality of stools was evaluated on 154 puppies between 4 and 8 weeks of age (100 from small breeds and 54 from large breeds). The scoring was performed immediately after a spontaneous defecation based on a 13-point scale (from 1; liquid to 13; dry and hard feces). Fecal samples were frozen for further viral analysis. Each puppy was weighed once a week during the study period. The fecal score regarded as pathological was the highest score associated with a significant reduction in average daily gain (ADG). Fecal samples were checked by semi-quantitative PCR or RT-PCR for canine parvovirus type 2 and canine coronavirus identification, respectively. The quality of feces was affected by both age and breed size. In small breeds, the ADG was significantly reduced under a fecal score of 6 and 7 for puppies at 4-5 and 6-8 weeks of age, respectively. In large breeds, the ADG was significantly reduced under a fecal score of 5 whatever the age of the puppy. Whereas a high viral load of canine parvovirus type 2 significantly impacted feces quality, no effect was recorded for canine coronavirus. This study provides an objective threshold for evaluation of fecal quality in weaning puppies. It also emphasizes the importance to be given to age and breed size in that evaluation. Copyright © 2012 Elsevier B.V. All rights reserved.

Classification of Antibiotic Resistance Patterns of Indicator Bacteria by Discriminant Analysis: Use in Predicting the Source of Fecal Contamination in Subtropical Waters

PubMed Central

Harwood, Valerie J.; Whitlock, John; Withington, Victoria

2000-01-01

The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida. PMID:10966379

Fecal and Mucosa-Associated Intestinal Microbiota in Patients with Diarrhea-Predominant Irritable Bowel Syndrome.

PubMed

Maharshak, Nitsan; Ringel, Yehuda; Katibian, David; Lundqvist, Ashley; Sartor, R Balfour; Carroll, Ian M; Ringel-Kulka, Tamar

2018-05-17

Irritable bowel syndrome (IBS) has been associated with changes in the intestinal microbiota. Only a few studies have explored differences in the mucosa-associated microbiota between IBS patients and healthy controls (HC). To characterize and compare the microbiota in mucosal and fecal samples from carefully selected patients with IBS-D and HC. The cohort was composed of 23 diarrhea-predominant IBS (IBS-D) patients and 24 HC. Fresh stool samples were collected from participants prior to the collection of colonic mucosal samples from an unprepped bowel. After DNA extraction, 16S rRNA genes were sequenced by 454 pyrosequencing and analyzed using the QIIME pipeline. The fecal microbiota (luminal niche) of IBS-D patients was found to have reduced enteric richness compared to HC (P < 0.05), whereas no differences were observed between the two groups within the mucosal microbiota. Within the luminal niche, the relative proportions of Faecalibacterium genus were found to be lower in IBS-D than in HC and the Dorea genus was higher in IBS-D. None of the taxa proportions were significantly different in IBS-D patients versus HC using an FDR of ≤ 0.1 when analyzing samples that appeared in > 25% samples of either niche. Fecal and mucosal microbiota of IBS-D patients and HC are very similar and are not sufficient to explain the reported altered physiology and symptomatology of IBS-D. Future studies should investigate intestinal microbiome-dependent functional activity in addition to the fecal and mucosal-associated microbial composition.

Quantitative PCR measurements of Escherichia coli including shiga toxin-producing E. coli (STEC) in animal feces and environmental waters.

PubMed

Ahmed, W; Gyawali, P; Toze, S

2015-03-03

Quantitative PCR (qPCR) assays were used to determine the concentrations of E. coli including shiga toxin-producing E. coli (STEC) associated virulence genes (eaeA, stx1, stx2, and hlyA) in ten animal species (fecal sources) and environmental water samples in Southeast Queensland, Australia. The mean Log10 concentrations and standard deviations of E. coli 23S rRNA across fecal sources ranged from 1.3 ± 0.1 (horse) to 6.3 ± 0.4 (cattle wastewater) gene copies at a test concentration of 10 ng of DNA. The differences in mean concentrations of E. coli 23S rRNA gene copies among fecal source samples were significantly different from each other (P < 0.0001). Among the virulence genes, stx2 (25%, 95% CI, 17-33%) was most prevalent among fecal sources, followed by eaeA (19%, 95% CI, 12-27%), stx1 (11%, 95% CI, 5%-17%) and hlyA (8%, 95% CI, 3-13%). The Log10 concentrations of STEC virulence genes in cattle wastewater samples ranged from 3.8 to 5.0 gene copies at a test concentration of 10 ng of DNA. Of the 18 environmental water samples tested, three (17%) were positive for eaeA and two (11%) samples were also positive for the stx2 virulence genes. The data presented in this study will aid in the estimation of quantitative microbial risk assessment (QMRA) from fecal pollution of domestic and wild animals in drinking/recreational water catchments.

Survival and persistence of fecal host-specific Bacteroidales cells and their DNA assessed by PMA-qPCR

NASA Astrophysics Data System (ADS)

Bae, S.; Bombardelli, F.; Wuertz, S.

2008-12-01

Understanding and managing microbial pollutions in water is one of the foremost challenges of establishing effective managements and remediation strategies to impaired water bodies polluted by uncharacterized fecal sources. Quantitative microbial source tracking (MST) approaches using fecal Bacteroidales and quantitative PCR (qPCR) assays to measure gene copies of host-specific 16S rRNA genetic markers are promising because they can allow for identifying and quantifying fecal loadings from a particular animal host and understanding the fate and transport of host-specific Bacteroidales over a range of conditions in water bodies. Similar to the case of traditional fecal indicator bacteria, a relatively long persistence of target DNA may hamper applied MST studies, if genetic markers cannot be linked to recent fecal pollution in water. We report a successful approach to removing the qPCR signal derived from free DNA and dead host-specific Bacteroidales cells by selectively binding the DNA and consequently inhibiting PCR amplification using light- activated propidium monoazide (PMA). Optimal PMA-qPCR conditions were determined as 100 µM of PMA concentration and a 10-min light exposure time at different solids concentrations in order to mimic a range of water samples. Under these conditions, PMA-qPCR resulted in the selective exclusion of DNA from heat- treated cells of non-culturable Bacteroidales in human feces and wastewater influent and effluent samples. Also, the persistence of feces-derived host-specific Bacteroidales DNA and their cells (determined by universal, human-, cow- and dog-specific Bacteroidales qPCR assays) in seawater was investigated in microcosms at environmental conditions. The average T99 (two log reduction) value for host-specific viable Bacteroidales cells was 28 h, whereas that for total host-specific Bacteroidales DNA was 177 h. Natural sunlight did not have a strong influence on the fate of fecal Bacteroidales cells and their DNA, presumably because the presence of oxygen significantly affected the viability and persistence of these obligate anaerobes. In conclusion, measuring Bacteroidales DNA in viable cells is recommended in applied MST studies because extracellular Bacteroidales DNA persists longer in the environment. The methods and results presented are helpful to improve the accuracy of MST applications, to develop a model of fate and transport of host-specific Bacteroidales, and to implement management practices to protect water quality.

FECAL COLIFORM INCREASE AFTER CENTRIFUGATION

EPA Science Inventory

The Water Environment Research Foundation (WERF) recently published a report titled Examination of Reactivation and Regrowth of Fecal Coliforms in Anaerobically Digested Sludges. Seven full-scale publicly owned treatment facilities were sampled several times to determine if bacte...

Variability of Hormonal Stress Markers Collected from a Managed Dolphin Population

DTIC Science & Technology

2014-09-30

physiological indicators of stress in wild marine mammals and the interrelationships between different stress markers can be used to estimate the impact...and thyroid hormones via radioimmunoassay (RIA). The methods have been validated for cortisol and aldosterone in this species (Houser et al., 2011...measurement methods. Metabolites of cortisol, aldosterone and thyroid hormone will be extracted from fecal samples and measured via RIA using established

40 CFR 141.202 - Tier 1 Public Notice-Form, manner, and frequency of notice.

Code of Federal Regulations, 2010 CFR

2010-07-01

... fecal coliform or E. coli are present in the water distribution system (as specified in § 141.63(b)), or when the water system fails to test for fecal coliforms or E. coli when any repeat sample tests...) Detection of E. coli, enterococci, or coliphage in source water samples as specified in § 141.402(a) and...

40 CFR 141.202 - Tier 1 Public Notice-Form, manner, and frequency of notice.

Code of Federal Regulations, 2011 CFR

2011-07-01

... fecal coliform or E. coli are present in the water distribution system (as specified in § 141.63(b)), or when the water system fails to test for fecal coliforms or E. coli when any repeat sample tests...) Detection of E. coli, enterococci, or coliphage in source water samples as specified in § 141.402(a) and...

Associations between fecal indicator bacteria prevalence and demographic data in private water supplies in Virginia.

PubMed

Smith, Tamara; Krometis, Leigh-Anne H; Hagedorn, Charles; Lawrence, Annie H; Benham, Brian; Ling, Erin; Ziegler, Peter; Marmagas, Susan West

2014-12-01

Over 1.7 million Virginians rely on private water sources to provide household water. The heaviest reliance on these systems occurs in rural areas, which are often underserved with respect to available financial resources and access to environmental health education. This study aimed to identify potential associations between concentrations of fecal indicator bacteria (FIB) (coliforms, Escherichia coli) in over 800 samples collected at the point-of-use from homes with private water supply systems and homeowner-provided demographic data (household income and education). Of the 828 samples tested, 349 (42%) of samples tested positive for total coliform and 55 (6.6%) tested positive for E. coli. Source tracking efforts targeting optical brightener concentrations via fluorometry and the presence of a human-specific Bacteroides marker via quantitative real-time polymerase chain reaction (qPCR) suggest possible contamination from human septage in over 20 samples. Statistical methods implied that household income has an association with the proportion of samples positive for total coliform, though the relationship between education level and FIB is less clear. Further exploration of links between demographic data and private water quality will be helpful in building effective strategies to improve rural drinking water quality.

Sphingolipid metabolism potential in fecal microbiome and bronchiolitis in infants: a case-control study.

PubMed

Hasegawa, Kohei; Stewart, Christopher J; Mansbach, Jonathan M; Linnemann, Rachel W; Ajami, Nadim J; Petrosino, Joseph F; Camargo, Carlos A

2017-07-26

Emerging evidence demonstrated that the structure of fecal microbiome is associated with the likelihood of bronchiolitis in infants. However, no study has examined functional profiles of fecal microbiome in infants with bronchiolitis. In this context, we conducted a case-control study. As a part of multicenter prospective study, we collected stool samples from 40 infants hospitalized with bronchiolitis (cases). We concurrently enrolled 115 age-matched healthy controls. First, by applying 16S rRNA gene sequencing to these 155 fecal samples, we identified the taxonomic profiles of fecal microbiome. Next, based on the taxonomy data, we inferred the functional capabilities of fecal microbiome and tested for differences in the functional capabilities between cases and controls. Overall, the median age was 3 months and 45% were female. Among 274 metabolic pathways surveyed, there were significant differences between bronchiolitis cases and healthy controls for 37 pathways, including lipid metabolic pathways (false discovery rate [FDR] <0.05). Particularly, the fecal microbiome of bronchiolitis cases had consistently higher abundances of gene function related to the sphingolipid metabolic pathways compared to that of controls (FDR <0.05). These pathways were more abundant in infants with Bacteroides-dominant microbiome profile compared to the others (FDR <0.001). On the basis of the predicted metagenome in this case-control study, we found significant differences in the functional potential of fecal microbiome between infants with bronchiolitis and healthy controls. Although causal inferences remain premature, our data suggest a potential link between the bacteria-derived metabolites, modulations of host immune response, and development of bronchiolitis.

An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids.

PubMed Central

Peltonen, R; Ling, W H; Hänninen, O; Eerola, E

1992-01-01

The effect of an uncooked extreme vegan diet on fecal microflora was studied by direct stool sample gas-liquid chromatography (GLC) of bacterial cellular fatty acids and by quantitative bacterial culture by using classical microbiological techniques of isolation, identification, and enumeration of different bacterial species. Eighteen volunteers were divided randomly into two groups. The test group received an uncooked vegan diet for 1 month and a conventional diet of mixed Western type for the other month of the study. The control group consumed a conventional diet throughout the study period. Stool samples were collected. Bacterial cellular fatty acids were extracted directly from the stool samples and measured by GLC. Computerized analysis of the resulting fatty acid profiles was performed. Such a profile represents all bacterial cellular fatty acids in a sample and thus reflects its microflora and can be used to detect changes, differences, or similarities of bacterial flora between individual samples or sample groups. GLC profiles changed significantly in the test group after the induction and discontinuation of the vegan diet but not in the control group at any time, whereas quantitative bacterial culture did not detect any significant change in fecal bacteriology in either of the groups. The results suggest that an uncooked extreme vegan diet alters the fecal bacterial flora significantly when it is measured by direct stool sample GLC of bacterial fatty acids. PMID:1482187

Use of fecal volatile organic compound analysis to discriminate between non-vaccinated and BCG—Vaccinated cattle prior to and after Mycobacterium bovis challenge

PubMed Central

Stahl, Randal; Waters, W. Ray; Palmer, Mitchell V.; Nol, Pauline; Rhyan, Jack C.; VerCauteren, Kurt C.; Koziel, Jacek A.

2017-01-01

Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge. PMID:28686691

Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.

PubMed

Belshaw, Nigel J; Elliott, Giles O; Williams, Elizabeth A; Bradburn, David M; Mills, Sarah J; Mathers, John C; Johnson, Ian T

2004-09-01

Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

Distribution, Detection of Enterotoxigenic Strains and Antimicrobial Drug Susceptibility Patterns of Bacteroides Fragilis Group in Diarrheic and Non-Diarrheic Feces from Brazilian Infants

PubMed Central

Ferreira, Débora Paula; Silva, Vânia Lúcia; Guimarães, Danielle Aparecida; Coelho, Cíntia Marques; Zauli, Danielle Alves Gomes; Farias, Luiz Macêdo; Carvalho, Maria Auxiliadora Roque; Diniz, Claudio Galuppo

2010-01-01

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0–5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children. PMID:24031535

Exploration of Fecal Microbiota Transplantation in the Treatment of Refractory Diarrhea After Renal Transplantation.

PubMed

Gu, B; Bo, G Z; Ke, C

2018-06-01

Exploration of fecal microbiota transplantation in the treatment of refractory diarrhea after renal transplantation. Summarize the etiology of 120 cases with diarrhea after renal transplantation from 2014 to 2017 in our hospital. There were 4 recipients of refractory diarrhea who accepted fecal microbiota transplantation with informed consent, and we collected clinical data of stool and bacterial culture, gut microbiota analysis, graft function, electrolytes, immunosuppressant concentrations of prognostic evaluation of patients with fecal transplantation. The absorption of electrolyte is slightly higher and concentration of tacrolimus and creatinine were not significantly changed compared with before. Fecal microbiota transplantation provides a new choice to refractory diarrhea after renal transplantation as an innovative treatment, but the effectiveness of fecal microbiota transplantation needs long-term observation and further evaluation through large sample data. Copyright © 2018. Published by Elsevier Inc.

Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

PubMed

Laurin, Emilie L; Sanchez, Javier; Chaffer, Marcelo; McKenna, Shawn L B; Keefe, Greg P

2017-01-01

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Metabolome and fecal microbiota in monozygotic twin pairs discordant for weight: a Big Mac challenge.

PubMed

Bondia-Pons, Isabel; Maukonen, Johanna; Mattila, Ismo; Rissanen, Aila; Saarela, Maria; Kaprio, Jaakko; Hakkarainen, Antti; Lundbom, Jesper; Lundbom, Nina; Hyötyläinen, Tuulia; Pietiläinen, Kirsi H; Orešič, Matej

2014-09-01

Postprandial responses to food are complex, involving both genetic and environmental factors. We studied postprandial responses to a Big Mac meal challenge in monozygotic co-twins highly discordant for body weight. This unique design allows assessment of the contribution of obesity, independent of genetic liability. Comprehensive metabolic profiling using 3 analytical platforms was applied to fasting and postprandial serum samples from 16 healthy monozygotic twin pairs discordant for weight (body mass index difference >3 kg/m(2)). Nine concordant monozygotic pairs were examined as control pairs. Fecal samples were analyzed to assess diversity of the major bacterial groups by using 5 different validated bacterial group specific denaturing gradient gel electrophoresis methods. No differences in fecal bacterial diversity were detected when comparing co-twins discordant for weight (ANOVA, P<0.05). We found that within-pair similarity is a dominant factor in the metabolic postprandial response, independent of acquired obesity. Branched chain amino acids were increased in heavier as compared with leaner co-twins in the fasting state, but their levels converged postprandially (paired t tests, FDR q<0.05). We also found that specific bacterial groups were associated with postprandial changes of specific metabolites. Our findings underline important roles of genetic and early life factors in the regulation of postprandial metabolite levels. © FASEB.

Optimization of GC/TOF MS analysis conditions for assessing host-gut microbiota metabolic interactions: Chinese rhubarb alters fecal aromatic amino acids and phenol metabolism.

PubMed

Yin, Shan; Guo, Pan; Hai, Dafu; Xu, Li; Shu, Jiale; Zhang, Wenjin; Khan, Muhammad Idrees; Kurland, Irwin J; Qiu, Yunping; Liu, Yumin

2017-12-01

In this paper, an optimized method based on gas chromatography/time-of-flight mass spectrometry (GC-TOFMS) platform has been developed for the analysis of gut microbial-host related co-metabolites in fecal samples. The optimization was performed with proportion of chloroform (C), methanol (M) and water (W) for the extraction of specific metabolic pathways of interest. Loading Bi-plots from the PLS regression model revealed that high concentration of chloroform emphasized the extraction of short chain fatty acids and TCA intermediates, while the higher concentration of methanol emphasized indole and phenyl derivatives. Low level of organic solution emphasized some TCA intermediates but not for indole and phenyl species. The highest sum of the peak area and the distribution of metabolites corresponded to the extraction of methanol/chloroform/water of 225:75:300 (v/v/v), which was then selected for method validation and utilized in our application. Excellent linearity was obtained with 62 reference standards representing different classes of gut microbial-host related co-metabolites, with correlation coefficients (r 2 ) higher than 0.99. Limit of detections (LODs) and limit of qualifications (LOQs) for these standards were below 0.9 nmol and 1.6 nmol, respectively. The reproducibility and repeatability of the majority of tested metabolites in fecal samples were observed with RSDs lower than 15%. Chinese rhubarb-treated rats had elevated indole and phenyl species, and decreased levels of polyamine such as putrescine, and several amino acids. Our optimized method has revealed host-microbe relationships of potential importance for intestinal microbial metabolite receptors such as pregnane X receptor (PXR) and aryl hydrocarbon receptor (AHR) activity, and for enzymes such as ornithine decarboxylase (ODC). Copyright © 2017 Elsevier B.V. All rights reserved.

First record of entodiniomorph ciliates in a carnivore, the maned wolf (Chrysocyon brachyurus), from Brazil.

PubMed

Vynne, Carly; Kinsella, John M

2009-06-01

The entodiniomorph ciliates (Ciliophora: Entodiniomorphida) are endosymbiotes widely found in the intestines of herbivorous mammals. These commensals commonly occur in the Artiodactyla and Perissodactyla and have also been described in the Proboscidea, Primates, Rodentia, and Diprotodontia. This study reports the first finding of a ciliate in a member of order Carnivora, the maned wolf (Chrysocyon brachyurus). Fecal samples from wild and captive maned wolves were screened using ethyl acetate sedimentation. Prevalence in fecal samples collected from free-ranging maned wolves in Brazil was 40% (6 of 15). Fecal samples from two of four captive individuals from the St. Louis Zoo also had the same species of ciliate. The largely frugivorous diet of the maned wolf likely explains the occurrence of these normally herbivore-associated endosymbiotes in a carnivore.

Analysis of the Microbial Diversity in the Fecal Material of Giraffes.

PubMed

Schmidt, Jessica M; Henken, Susan; Dowd, Scot E; McLaughlin, Richard William

2018-03-01

Using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing, the microbiota of the fecal material of seven giraffes living in captivity at the Jacksonville Zoo and Gardens, Jacksonville, FL was investigated. In all samples, the most predominant bacterial phylum was the Firmicutes followed by Bacteroidetes. The most predominant fungi were members of the phylum Ascomycota followed by Neocallimastigomycota in five of seven samples. The reverse was true in the other two samples.

The Endemicity of Human Fascioliasis in Guilan Province, Northern Iran: the Baseline for Implementation of Control Strategies

PubMed Central

ASHRAFI, Keyhan; SAADAT, Farshid; O’NEILL, Sandra; RAHMATI, Behnaz; AMIN TAHMASBI, Hadid; PIUS DALTON, John; NADIM, Abolhassan; ASADINEZHAD, Mohsen; REZVANI, Sayed Mahmood

2015-01-01

Background: The largest global outbreaks of liver fluke disease (Fascioliasis) in humans, caused by species of the genus Fasciola, have occurred in Guilan Province of Iran, affecting more than 15000 people. Although, different aspects of fascioliasis have been the subject of various researches during last two decades, nevertheless no community-based study has been performed in endemic regions of Guilan. The aim of present study was to obtain the basic information needed to develop future control strategies. Methods: Fecal and blood samples were collected from 1,984 volunteers in the Bandar-Anzali district, the region where previous epidemics occurred. Fecal samples were examined by Kato-Katz and formalin-ether methods for the presence of Fasciola eggs. Sera samples were analyzed by ELISA to detect anti-cathepsin L antibodies. Results: Twenty-seven (1.36%) individuals were seropositive, 9 (0.45%) individuals were egg positive (mean egg count 50.7 (±30.36) eggs per gram of faeces) and 30 individuals (1.51%) were positive using both methods. No statistical association was observed between infection and age, gender, location, occupation, educational status and dietary habits. The prevalence of intestinal parasites is also included. Conclusion: Human fascioliasis is hypoendemic in this region and recommends a passive case-finding approach, effective primary prevention measures, health education through mass media and effective veterinary public health measures for control of human disease. PMID:26056669

Parasite prevalence in fecal samples from shelter dogs and cats across the Canadian provinces.

PubMed

Villeneuve, Alain; Polley, Lydden; Jenkins, Emily; Schurer, Janna; Gilleard, John; Kutz, Susan; Conboy, Gary; Benoit, Donald; Seewald, Wolfgang; Gagné, France

2015-05-21

In Canada, surveys of enteric parasites in dogs and cats have been reported sporadically over the past 40 years, mostly focusing on a specific region. The present work was performed to determine the current prevalence of various parasites in fecal samples from shelter dogs and cats across the Canadian provinces. A total of 1086 dog and 636 cat fecal samples from 26 shelters were analysed using a sugar solution double centrifugal flotation technique. Prevalences (national, regional, provincial, age and parasite-specific), were calculated and compared using the Fisher-Exact test. A multiplex PCR was performed to distinguish Taenia spp, Echinococcus granulosus and E. multilocularis on samples positive for taeniid eggs. Overall, 33.9% of dogs and 31.8% of cats were positive for at least one parasite. Toxocara canis and T. cati were the most prevalent parasite present in fecal samples followed by Cystoisospora spp. Prevalence in dogs was similar across the Atlantic, East, West and Pacific regions, while prevalence in cats varied regionally. Eggs of E. granulosus/E. canadensis were detected in samples from dogs from BC, AB, and ON. Data from this study will help in the development of strategies, based on the level of risk per geographic location for the prevention and response to these parasites in pets and free-roaming and shelter animals in Canada.

The impact of fecal sample processing on prevalence estimates for antibiotic-resistant Escherichia coli.

PubMed

Omulo, Sylvia; Lofgren, Eric T; Mugoh, Maina; Alando, Moshe; Obiya, Joshua; Kipyegon, Korir; Kikwai, Gilbert; Gumbi, Wilson; Kariuki, Samuel; Call, Douglas R

2017-05-01

Investigators often rely on studies of Escherichia coli to characterize the burden of antibiotic resistance in a clinical or community setting. To determine if prevalence estimates for antibiotic resistance are sensitive to sample handling and interpretive criteria, we collected presumptive E. coli isolates (24 or 95 per stool sample) from a community in an urban informal settlement in Kenya. Isolates were tested for susceptibility to nine antibiotics using agar breakpoint assays and results were analyzed using generalized linear mixed models. We observed a <3-fold difference between prevalence estimates based on freshly isolated bacteria when compared to isolates collected from unprocessed fecal samples or fecal slurries that had been stored at 4°C for up to 7days. No time-dependence was evident (P>0.1). Prevalence estimates did not differ for five distinct E. coli colony morphologies on MacConkey agar plates (P>0.2). Successive re-plating of samples for up to five consecutive days had little to no impact on prevalence estimates. Finally, culturing E. coli under different conditions (with 5% CO 2 or micro-aerobic) did not affect estimates of prevalence. For the conditions tested in these experiments, minor modifications in sample processing protocols are unlikely to bias estimates of the prevalence of antibiotic-resistance for fecal E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

Canine scent detection and microbial source tracking of human waste contamination in storm drains.

PubMed

Van De Werfhorst, Laurie C; Murray, Jill L S; Reynolds, Scott; Reynolds, Karen; Holden, Patricia A

2014-06-01

Human fecal contamination of surface waters and drains is difficult to diagnose. DNA-based and chemical analyses of water samples can be used to specifically quantify human waste contamination, but their expense precludes routine use. We evaluated canine scent tracking, using two dogs trained to respond to the scent of municipal wastewater, as a field approach for surveying human fecal contamination. Fecal indicator bacteria, as well as DNA-based and chemical markers of human waste, were analyzed in waters sampled from canine scent-evaluated sites (urban storm drains and creeks). In the field, the dogs responded positively (70% and 100%) at sites for which sampled waters were then confirmed as contaminated with human waste. When both dogs indicated a negative response, human waste markers were absent. Overall, canine scent tracking appears useful for prioritizing sampling sites for which DNA-based and similarly expensive assays can confirm and quantify human waste contamination.

A study of the level and dynamics of Eimeria populations in naturally infected, grazing beef cattle at various stages of production in the Mid-Atlantic USA.

PubMed

Lucas, Aaron S; Swecker, William S; Lindsay, David S; Scaglia, Guillermo; Neel, James P S; Elvinger, Francois C; Zajac, Anne M

2014-05-28

There is little information available on the species dynamics of eimerian parasites in grazing cattle in the central Appalachian region of the United States. Therefore, the objective of this study was to describe the level of infection and species dynamics of Eimeria spp. in grazing beef cattle of various age groups over the course of a year in the central Appalachian region. Rectal fecal samples were collected from male and female calves (n=72) monthly from May through October 2005, heifers only (n=36) monthly from November 2005 to April 2006, and cows (n=72) in May, July, and September, 2005. Eimeria spp. oocysts were seen in 399 of 414 (96%) fecal samples collected from the calves from May through October. Fecal oocysts counts (FOC) in the calves were lower (P<0.05) in May than all other months and no significant differences were detected from June through September. Eimeria spp. oocysts were detected in 198 of 213 (92%) of fecal samples collected from the 36 replacement heifers monthly from November to April and monthly mean FOC did not differ during this time period. The prevalence of oocyst shedding increased to 100% in calves in September and remained near 100% in the replacement heifers during the sampling period. Eimeria spp. oocysts were also detected in 150 of 200 (75%) samples collected in May, July, and September from the cows and mean FOC did not differ significantly over the sampling period. Eimeria spp. composition was dominated by Eimeria bovis in fecal samples collected from calves, replacement heifers and cows. Mixed Eimeria spp. infections were, however, common in all groups and 13 Eimeria spp. oocysts were identified throughout the sampling period. Copyright © 2014 Elsevier B.V. All rights reserved.

Dynamic changes of the respiratory microbiota and its relationship to fecal and blood microbiota in healthy young cats

PubMed Central

Rindt, Hansjorg; Grobman, Megan E.; Graham, Amber; Bishop, Kaitlin; Cohn, Leah A.; Reinero, Carol R.

2017-01-01

Advances in the field of metagenomics using culture-independent methods of microbial identification have allowed characterization of rich and diverse communities of bacteria in the lungs of healthy humans, mice, dogs, sheep and pigs. These data challenge the long held belief that the lungs are sterile and microbial colonization is synonymous with pathology. Studies in humans and animals demonstrate differences in the composition of airway microbiota in health versus disease suggesting respiratory dysbiosis occurs. Using 16S rRNA amplicon sequencing of DNA extracted from rectal and oropharyngeal (OP) swabs, bronchoalveolar lavage fluid (BALF), and blood, our objective was to characterize the fecal, OP, blood, and lower airway microbiota over time in healthy cats. This work in healthy cats, a species in which a respiratory microbiota has not yet been characterized, sets the stage for future studies in feline asthma in which cats serve as a comparative and translational model for humans. Fecal, OP and BALF samples were collected from six healthy research cats at day 0, week 2, and week 10; blood was collected at week 10. DNA was extracted, amplified via PCR, and sequenced using the Illumina MiSeq platform. Representative operational taxonomic units (OTUs) were identified and microbial richness and diversity were assessed. Principal component analysis (PCA) was used to visualize relatedness of samples and PERMANOVA was used to test for significant differences in microbial community composition. Fecal and OP swabs provided abundant DNA yielding a mean±SEM of 65,653±6,145 and 20,6323±4,360 sequences per sample, respectively while BALF and blood samples had lower coverage (1,489±430 and 269±18 sequences per sample, respectively). Oropharyngeal and fecal swabs were significantly richer than BALF (mean number OTUs 93, 88 and 36, respectively; p < 0.001) with no significant difference (p = 0.180) in richness between time points. PCA revealed site-specific microbial communities in the feces, and upper and lower airways. In comparison, blood had an apparent compositional similarity with BALF with regard to a few dominant taxa, but shared more OTUs with feces. Samples clustered more by time than by individual, with OP swabs having subjectively greater variation than other samples. In summary, healthy cats have a rich and distinct lower airway microbiome with dynamic bacterial populations. The microbiome is likely to be altered by factors such as age, environmental influences, and disease states. Further data are necessary to determine how the distinct feline microbiomes from the upper and lower airways, feces and blood are established and evolve. These data are relevant for comparisons between healthy cats and cats with respiratory disease. PMID:28278278

Dynamic changes of the respiratory microbiota and its relationship to fecal and blood microbiota in healthy young cats.

PubMed

Vientós-Plotts, Aida I; Ericsson, Aaron C; Rindt, Hansjorg; Grobman, Megan E; Graham, Amber; Bishop, Kaitlin; Cohn, Leah A; Reinero, Carol R

2017-01-01

Advances in the field of metagenomics using culture-independent methods of microbial identification have allowed characterization of rich and diverse communities of bacteria in the lungs of healthy humans, mice, dogs, sheep and pigs. These data challenge the long held belief that the lungs are sterile and microbial colonization is synonymous with pathology. Studies in humans and animals demonstrate differences in the composition of airway microbiota in health versus disease suggesting respiratory dysbiosis occurs. Using 16S rRNA amplicon sequencing of DNA extracted from rectal and oropharyngeal (OP) swabs, bronchoalveolar lavage fluid (BALF), and blood, our objective was to characterize the fecal, OP, blood, and lower airway microbiota over time in healthy cats. This work in healthy cats, a species in which a respiratory microbiota has not yet been characterized, sets the stage for future studies in feline asthma in which cats serve as a comparative and translational model for humans. Fecal, OP and BALF samples were collected from six healthy research cats at day 0, week 2, and week 10; blood was collected at week 10. DNA was extracted, amplified via PCR, and sequenced using the Illumina MiSeq platform. Representative operational taxonomic units (OTUs) were identified and microbial richness and diversity were assessed. Principal component analysis (PCA) was used to visualize relatedness of samples and PERMANOVA was used to test for significant differences in microbial community composition. Fecal and OP swabs provided abundant DNA yielding a mean±SEM of 65,653±6,145 and 20,6323±4,360 sequences per sample, respectively while BALF and blood samples had lower coverage (1,489±430 and 269±18 sequences per sample, respectively). Oropharyngeal and fecal swabs were significantly richer than BALF (mean number OTUs 93, 88 and 36, respectively; p < 0.001) with no significant difference (p = 0.180) in richness between time points. PCA revealed site-specific microbial communities in the feces, and upper and lower airways. In comparison, blood had an apparent compositional similarity with BALF with regard to a few dominant taxa, but shared more OTUs with feces. Samples clustered more by time than by individual, with OP swabs having subjectively greater variation than other samples. In summary, healthy cats have a rich and distinct lower airway microbiome with dynamic bacterial populations. The microbiome is likely to be altered by factors such as age, environmental influences, and disease states. Further data are necessary to determine how the distinct feline microbiomes from the upper and lower airways, feces and blood are established and evolve. These data are relevant for comparisons between healthy cats and cats with respiratory disease.

FECAL COLIFORM INCREASE AFTER CENTRIFUGATION: EPA PERSPECTIVE

EPA Science Inventory

The Water Environment Research Foundation (WERF) recently published a report titled Examination of Reactivation and Regrowth of Fecal Coliforms in Anaerobically Digested Sludges. Seven full-scale publicly owned treatment facilities were sampled several times to determine if bacte...

Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal

PubMed Central

Hamad, Ibrahim; Sokhna, Cheikh; Raoult, Didier; Bittar, Fadi

2012-01-01

Background Microbial eukaryotes represent an important component of the human gut microbiome, with different beneficial or harmful roles; some species are commensal or mutualistic, whereas others are opportunistic or parasitic. The diversity of eukaryotes inhabiting humans remains relatively unexplored because of either the low abundance of these organisms in human gut or because they have received limited attention from a whole-community perspective. Methodology/Principal Finding In this study, a single fecal sample from a healthy African male was studied using both culture-dependent methods and extended molecular methods targeting the 18S rRNA and ITS sequences. Our results revealed that very few fungi, including Candida spp., Galactomyces spp., and Trichosporon asahii, could be isolated using culture-based methods. In contrast, a relatively a high number of eukaryotic species could be identified in this fecal sample when culture-independent methods based on various primer sets were used. A total of 27 species from one sample were found among the 977 analyzed clones. The clone libraries were dominated by fungi (716 clones/977, 73.3%), corresponding to 16 different species. In addition, 187 sequences out of 977 (19.2%) corresponded to 9 different species of plants; 59 sequences (6%) belonged to other micro-eukaryotes in the gut, including Entamoeba hartmanni and Blastocystis sp; and only 15 clones/977 (1.5%) were related to human 18S rRNA sequences. Conclusion Our results revealed a complex eukaryotic community in the volunteer’s gut, with fungi being the most abundant species in the stool sample. Larger investigations are needed to assess the generality of these results and to understand their roles in human health and disease. PMID:22808282

Prevalence and distribution of Baylisascaris procyonis in urban raccoons (Procyon lotor) in Winnipeg, Manitoba.

PubMed

Sexsmith, Jennifer L; Whiting, Terry L; Green, Chris; Orvis, Sheldon; Berezanski, Dean J; Thompson, Amy B

2009-08-01

The prevalence of Baylisascaris procyonis was estimated in the urban raccoon population of Winnipeg through the fecal flotation of raccoon feces collected at active latrines and through gross postmortem and fecal flotation of samples collected from nuisance raccoons. Fecal flotation of latrine-collected feces was positive in 33 of 89 samples and, of 52 latrines identified, 26 were positive on 1 or more occasions. Trapped individual raccoons subjected to postmortem examination were positive in 57 of 114 animals captured. Comparing a single fecal flotation to the gold standard of finding adult worms in the small intestine had a sensitivity of 78.9% and specificity of 92.9%. This study suggests that carriage of Baylisascaris procyonis is widespread in raccoons in the Winnipeg urban ecosystem. Raccoon latrines in Winnipeg should be treated as infectious sites and efforts should be made to limit access of pets and people at risk to those sites.

Levels of fecal corticosterone in sandhill cranes during a human-led migration.

PubMed

Hartup, Barry K; Olsen, Glenn H; Czekala, Nancy M; Paul-Murphy, Joanne; Langenberg, Julia A

2004-04-01

Fourteen captive-reared greater sandhill cranes (Grus canadensis tabida) were conditioned to follow ultralight aircraft to promote migration between Wisconsin and Florida (USA) after release. Fecal samples were collected throughout the training period in Wisconsin and during a l977-km human-led migration to Florida to determine fecal corticosterone (FC) concentrations by radioimmunnoassay. The mean (+/-SE) FC concentration during the training period was 109.5 +/- 7.5 ng/g and was representative of baseline levels recorded previously from sandhill cranes. Fecal corticosterone concentrations increased in early migration compared to concentrations I mo prior to departure (P < 0.01) but were not different from baseline concentrations at tile end of the 6-wk migration period. The variability of FC concentrations in individual samples was greater throughout the migration than the training period. Increases in FC during migration were modest and generally consistent with normal corticosterone elevations observed in migrating birds.

Increased prevalence of antibiotic-resistant E. coli in gulls sampled in southcentral Alaska is associated with urban environments

USGS Publications Warehouse

Atterby, Clara; Ramey, Andrew M.; Gustafsson Hall, Gabriel; Jarhult, Josef; Borjesson, Stefan; Bonnedahl, Jonas

2016-01-01

BackgroundAntibiotic-resistant bacteria pose challenges to healthcare delivery systems globally; however, limited information is available regarding the prevalence and spread of such bacteria in the environment. The aim of this study was to compare the prevalence of antibiotic-resistant bacteria in large-bodied gulls (Larus spp.) at urban and remote locations in Southcentral Alaska to gain inference into the association between antibiotic resistance in wildlife and anthropogenically influenced habitats.MethodsEscherichia coli was cultured (n=115 isolates) from fecal samples of gulls (n=160) collected from a remote location, Middleton Island, and a more urban setting on the Kenai Peninsula.ResultsScreening of E. coli from fecal samples collected from glaucous-winged gulls (Larus glaucescens) at Middleton Island revealed 8% of isolates were resistant to one or more antibiotics and 2% of the isolates were resistant to three or more antibiotics. In contrast, 55% of E. coli isolates derived from fecal samples collected from large-bodied gulls (i.e. glaucous, herring [Larus argentatus], and potentially hybrid gulls) on the Kenai Peninsula were resistant to one or more antibiotics and 22% were resistant to three or more antibiotics. In addition, total of 16% of the gull samples from locations on the Kenai Peninsula harbored extended-spectrum cephalosporin-resistant E. coli isolates (extended-spectrum beta-lactamases [ESBL] and plasmid-encoded AmpC [pAmpC]), in contrast to Middleton Island where no ESBL- or pAmpC-producing isolates were detected.ConclusionOur findings indicate that increased prevalence of antibiotic resistance is associated with urban environments in Southcentral Alaska and presumably influenced by anthropogenic impacts. Further investigation is warranted to assess how migratory birds may maintain and spread antimicrobial-resistant bacteria of relevance to human and animal health.

Novel Insights in the Fecal Egg Count Reduction Test for Monitoring Drug Efficacy against Soil-Transmitted Helminths in Large-Scale Treatment Programs

PubMed Central

Levecke, Bruno; Speybroeck, Niko; Dobson, Robert J.; Vercruysse, Jozef; Charlier, Johannes

2011-01-01

Background The fecal egg count reduction test (FECRT) is recommended to monitor drug efficacy against soil-transmitted helminths (STHs) in public health. However, the impact of factors inherent to study design (sample size and detection limit of the fecal egg count (FEC) method) and host-parasite interactions (mean baseline FEC and aggregation of FEC across host population) on the reliability of FECRT is poorly understood. Methodology/Principal Findings A simulation study was performed in which FECRT was assessed under varying conditions of the aforementioned factors. Classification trees were built to explore critical values for these factors required to obtain conclusive FECRT results. The outcome of this analysis was subsequently validated on five efficacy trials across Africa, Asia, and Latin America. Unsatisfactory (<85.0%) sensitivity and specificity results to detect reduced efficacy were found if sample sizes were small (<10) or if sample sizes were moderate (10–49) combined with highly aggregated FEC (k<0.25). FECRT remained inconclusive under any evaluated condition for drug efficacies ranging from 87.5% to 92.5% for a reduced-efficacy-threshold of 90% and from 92.5% to 97.5% for a threshold of 95%. The most discriminatory study design required 200 subjects independent of STH status (including subjects who are not excreting eggs). For this sample size, the detection limit of the FEC method and the level of aggregation of the FEC did not affect the interpretation of the FECRT. Only for a threshold of 90%, mean baseline FEC <150 eggs per gram of stool led to a reduced discriminatory power. Conclusions/Significance This study confirms that the interpretation of FECRT is affected by a complex interplay of factors inherent to both study design and host-parasite interactions. The results also highlight that revision of the current World Health Organization guidelines to monitor drug efficacy is indicated. We, therefore, propose novel guidelines to support future monitoring programs. PMID:22180801

Fecal /sup 13/C analysis for the detection and quantitation of intestinal malabsorption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, P.D.; MacLean, W.C. Jr.; Watkins, J.B.

Use of /sup 14/CO/sub 2/ breath tests and fecal analyses for the detection and quantitation of intestinal malabsorption has been extensively documented in adult subjects. The use of radioisotopes has extended the range of breath test applications to include pediatric and geriatric subjects. Here we report a fecal /sup 13/C analysis that can be used in conjunction with /sup 14/CO/sub 2/ breath tests. Twenty-four-hour fecal samples were collected before and after the administration of a labeled substrate. Simultaneous cholyglycine /sup 13/CO/sub 2/ breath tests and fecal assays were performed in five children. One child with bacterial overgrowth had an abnormalmore » breath test and a normal fecal test. Of three children with ileal dysfunction, only one had an abnormal breath test, whereas the fecal test was abnormal in all three. Both the breath test and fecal test were abnormal for a child who had undergone an ileal resection. Both tests were normal for a child with ulcerative colitis.« less

The networks of human gut microbe–metabolite associations are different between health and irritable bowel syndrome

PubMed Central

Shankar, Vijay; Homer, Daniel; Rigsbee, Laura; Khamis, Harry J; Michail, Sonia; Raymer, Michael; Reo, Nicholas V; Paliy, Oleg

2015-01-01

The goal of this study was to determine if fecal metabolite and microbiota profiles can serve as biomarkers of human intestinal diseases, and to uncover possible gut microbe–metabolite associations. We employed proton nuclear magnetic resonance to measure fecal metabolites of healthy children and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D). Metabolite levels were associated with fecal microbial abundances. Using several ordination techniques, healthy and irritable bowel syndrome (IBS) samples could be distinguished based on the metabolite profiles of fecal samples, and such partitioning was congruent with the microbiota-based sample separation. Measurements of individual metabolites indicated that the intestinal environment in IBS-D was characterized by increased proteolysis, incomplete anaerobic fermentation and possible change in methane production. By correlating metabolite levels with abundances of microbial genera, a number of statistically significant metabolite–genus associations were detected in stools of healthy children. No such associations were evident for IBS children. This finding complemented the previously observed reduction in the number of microbe–microbe associations in the distal gut of the same cohort of IBS-D children. PMID:25635640

The networks of human gut microbe-metabolite associations are different between health and irritable bowel syndrome.

PubMed

Shankar, Vijay; Homer, Daniel; Rigsbee, Laura; Khamis, Harry J; Michail, Sonia; Raymer, Michael; Reo, Nicholas V; Paliy, Oleg

2015-08-01

The goal of this study was to determine if fecal metabolite and microbiota profiles can serve as biomarkers of human intestinal diseases, and to uncover possible gut microbe-metabolite associations. We employed proton nuclear magnetic resonance to measure fecal metabolites of healthy children and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D). Metabolite levels were associated with fecal microbial abundances. Using several ordination techniques, healthy and irritable bowel syndrome (IBS) samples could be distinguished based on the metabolite profiles of fecal samples, and such partitioning was congruent with the microbiota-based sample separation. Measurements of individual metabolites indicated that the intestinal environment in IBS-D was characterized by increased proteolysis, incomplete anaerobic fermentation and possible change in methane production. By correlating metabolite levels with abundances of microbial genera, a number of statistically significant metabolite-genus associations were detected in stools of healthy children. No such associations were evident for IBS children. This finding complemented the previously observed reduction in the number of microbe-microbe associations in the distal gut of the same cohort of IBS-D children.

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags

PubMed Central

2010-01-01

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80°C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method. PMID:20673359

Fecal coliforms on environmental surfaces in two day care centers.

PubMed Central

Weniger, B G; Ruttenber, A J; Goodman, R A; Juranek, D D; Wahlquist, S P; Smith, J D

1983-01-01

A survey of environmental surfaces in two Atlanta area day care centers was conducted to determine the prevalence of fecal coliform bacteria, considered a marker for the presence of fecal contamination which might contain pathogenic parasites, bacteria, or viruses. Fecal coliforms were found in 17 (4.3%) of 398 representative samples of building surfaces, furniture, and other objects. These surfaces may be involved in the chain of transmission of enteric diseases among children. Therefore, disinfection of inanimate objects, in addition to good handwashing, may be important in controlling the spread of enteric diseases in day care centers. PMID:6830225

Fecal parasite identification by microscopy and PCR in scimitar-horned oryx, Oryx dammah, managed at two sites.

PubMed

Pauling, Cassandra Dawn; Oller, Anna R; Jackson, Victoria

2016-12-01

The scimitar-horned oryx, Oryx dammah , an endangered species extinct in the wild, is managed in various captive management programs and is the focus of reintroduction efforts. Management variability can contribute to substantial parasite load differences, which can affect deworming programs and potentially transfer parasites to different regions with translocations. Parasite studies in O. dammah are lacking. In this study, we determined fecal egg/oocyst counts of O. dammah in two captive herds, Fossil Rim Wildlife Center (FRWC) and Kansas City Zoo (KCZ). Fecal egg counts (FEC) were performed on O. dammah feces collected seasonally using the modified McMaster method, and microscopy provided additional identification of parasite genera ova and oocysts. To identify parasites to species level, homogenized fecals provided DNA subjected to the polymerase chain reaction (PCR) using genus specific primers. Microscopy and sequencing results indicated the presence of Strongylus (Strongylus vulgaris, Angiostrongylus cantonensis) , Trichostrongylus (Haemonchus contortus, Camelostrongylus mentulatus) , Trichuris (T. leporis, T. ovis, and T. discolor) , Isospora (Isospora gryphoni) and Eimeria (E. zuernii and E. bovis) , with Strongylus being the most common. Nematodirus was identified through microscopy at FRWC. Fecal egg counts were significantly higher in (FRWC) than in (KCZ) in all samplings (P = <0.001). No significant difference was seen between parasite load and seasons (P = 0.103), nor site and season (P = 0.51). Both study sites maintained most animals within commonly accepted FEC levels found in domestic livestock. Individuals with high numbers of EPG or OPG were subordinate males, pregnant females, or neonates. Several significant interactions were found between genera of parasites, age, sex, season, and pregnancy status in the FRWC herd. Sampling limitations prevented further analysis of the KCZ herd. Understanding interactions between parasite loads and physiological, environmental, and regional differences can help determine inter-specific transfer of parasites, and establish appropriate anthelmintic programs for O. dammah herds.

The role of land use and environmental factors on microbial pollution of mountainous limestone aquifers

NASA Astrophysics Data System (ADS)

Allocca, V.; Celico, F.; Petrella, E.; Marzullo, G.; Naclerio, G.

2008-07-01

Limestone aquifers in Southern Italy are often affected by bacterial contamination produced by pasture and agriculture. The main goals of this study were (1) to analyze the role of land use and environmental factors on microbial contamination and, (2) to identify, at field scale, the most suitable indicator of fecal pollution, by comparing fecal coliforms and fecal enterococci. Analyzing surface and spring water, it was noted that both fecal indicators showed a significant decrease during the period characterized by freezing and/or freeze-thaw intervals. The data analysis shows that fecal coliforms are characterized by a significant decrease in population (3 orders of magnitude, at least) during the freezing period, while fecal enterococci are temporarily inhibited. A taxonomic classification of fecal enterococci detected in spring water samples was performed by the API 20 Strep system and by sequencing of the ribosomal 16S DNA genes. The results showed that freezing conditions did not cause any significant change on the set of enterococcal species.

Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods.

PubMed

Soria, M C; Soria, M A; Bueno, D J; Godano, E I; Gómez, S C; ViaButron, I A; Padin, V M; Rogé, A D

2017-08-01

The performance of detection methods (culture methods and polymerase chain reaction assay) and plating media used in the same type of samples were determined as well as the specificity of PCR primers to detected Salmonella spp. contamination in layer hen farms. Also, the association of farm characteristics with Salmonella presence was evaluated. Environmental samples (feces, feed, drinking water, air, boot-swabs) and eggs were taken from 40 layer hen houses. Salmonella spp. was most detected in boot-swabs taken around the houses (30% and 35% by isolation and PCR, respectively) follow by fecal samples (15.2% and 13.6% by isolation and PCR, respectively). Eggs, drinking water, and air samples were negative for Salmonella detection. Salmonella Schwarzengrund and S. Enteritidis were the most isolated serotypes. For plating media, relative specificity was 1, and the relative sensitivity was greater for EF-18 agar than XLDT agar in feed and fecal samples. However, relative sensitivity was greater in XLDT agar than EF-18 agar for boot-swab samples. Agreement was between fair to good depending on the sample, and it was good between isolation and PCR (feces and boot-swabs), without agreement for feed samples. Salmonella spp. PCR was positive for all strains, while S. Typhimurium PCR was negative. Salmonella Enteritidis PCR used was not specific. Based in the multiple logistic regression analyses, categorization by counties was significant for Salmonella spp. presence (P-value = 0.010). This study shows the importance of considering different types of samples, plating media and detection methods during a Salmonella spp. monitoring study. In addition, it is important to incorporate the sampling of floors around the layer hen houses to learn if biosecurity measures should be strengthened to minimize the entry and spread of Salmonella in the houses. Also, the performance of some PCR methods and S. Enteritidis PCR should be improved, and biosecurity measures in hen farms must be reinforced in the region of more concentrated layer hen houses to reduce the probability of Salmonella spp. presence. © 2017 Poultry Science Association Inc.

Comparison of four-hour and twenty-four-hour refrigerated storage of nonpotable water for fecal coliform analysis.

PubMed Central

Standridge, J H; Lesar, D J

1977-01-01

The problem of extending the storage time of water samples for fecal coliform analysis was addressed. Included in this report is a literature review of the storage problem. Twenty-eight samples were analyzed in replicate to determine the effect of 24-h storage of water samples at 4 degrees C. A new statistical approach to data analysis, coupled with the concept of practical acceptability, is presented. According to our results, many samples can successfully be stored at 4 degrees C for 24 h. PMID:335972

Quantitative PCR for Genetic Markers of Human Fecal Pollution

EPA Science Inventory

Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-...

[Molecular Detection of Giardia lamblia and Cryptosporidium Species in Pet Dogs].

PubMed

GU, You-fang; WANG, Kai; LIU, De-yi; MEI, Nan; CHEN, Cheng; CHEN, Tao; HAN, Min-min; ZHOU, Li; CAO, Jia-tong; ZHANG, Heng; ZHANG, Xue-liang; FAN, Zi-lai; LI, Wen-chao

2015-10-01

To determine the prevalence of Giardia lamblia and Cryptosporidium species infection in pet dogs, and identify the G. lamblia assemblages and Cryptosporidium species. A total of 315 fresh fecal samples were collected from pet clinics in five counties of Anhui Province and in Hangzhou of Zhejiang Province. Hemi-nested-PCR targeting the GDH gene of G. lamblia and nested-PCR targeting the SSU rRNA gene of Cryptosporidium were performed in all the fecal samples. The PCR products were sequenced and analyzed using bioinformatics methods to identify the G. lamblia assemblages and Cryptosporidium species. The positive rates of G. lamblia and Cryptosporidium spp. infections in the 315 fecal samples were 3.2% (10/315) and 1.6% (5/315), respectively. Specifically, the two indicators were both significantly higher in dogs ≤12 months (17.8% and 11.1%, respectively) than in adult dogs (0.7% and 0.0%)(P<0.05). However, there was no significant difference in the two indicators between male and female dogs. In addition, two G. lamblia assemblages were identified, assemblages B (n=6) and D (n=4). Sequence analysis of PCR products of the SSU rRNA gene showed that the five Cryptosporidium isolates were C. canis (n =5). The prevalences of G. lamblia and Cryptosporidium infection in pet dogs in Anhui and Zhejiang Provinces were 3.2 % and 1.6 %, respectively. The assemblages of G. lamblia in this study are of types B and D.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress.

PubMed

Morato, R G; Bueno, M G; Malmheister, P; Verreschi, I T N; Barnabe, R C

2004-12-01

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20 degrees C until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37 degrees C for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 +/- 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (+/- SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 +/- 26.7, after: 213.7 +/- 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

EVALUATE THE UTILITY OF ENTEROCOCCI AND BACTEROIDES AS INDICATORS OF THE SOURCES OF FECAL CONTAMINATION IN IMPAIRED SUBWATERSHEDS THROUGH DNA-BASED MOLECULAR TECHNIQUES.

EPA Science Inventory

Microbial source tracking (MST) is based on the assumption that specific strains of bacteria are associated with specific host species. MST methods are attractive because their application on environmental samples could help define the nature of water quality problems in impaire...

40 CFR 141.402 - Ground water source microbial monitoring and analytical methods.

Code of Federal Regulations, 2011 CFR

2011-07-01

... approves the use of E. coli as a fecal indicator for source water monitoring under this paragraph (a). If the repeat sample collected from the ground water source is E.coli positive, the system must comply... listed in the in paragraph (c)(2) of this section for the presence of E. coli, enterococci, or coliphage...

COMPARISON OF ENTEROCOCCUS MEASUREMENTS IN FRESHWATER AT TWO RECREATIONAL BEACHES BY QUANTITATIVE POLYMERASE CHAIN REACTION AND MEMBRANE FILER CULTURE ANALYSIS

EPA Science Inventory

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (2-3 hr) quantitative PCR (QPCR) analysis based method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Enumeration...

FECAL INDICATOR BACTERIA MEASUREMENTS BY QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS IN FRESH ARCHIVED DNA EXTRACT OF WATER SAMPLE FILTRATES

EPA Science Inventory

The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this stu...

Hepatitis E Virus in Pork Production Chain in Czech Republic, Italy, and Spain, 2010

PubMed Central

Di Bartolo, Ilaria; Diez-Valcarce, Marta; Vasickova, Petra; Kralik, Petr; Hernandez, Marta; Angeloni, Giorgia; Ostanello, Fabio; Bouwknegt, Martijn; Rodríguez-Lázaro, David; Pavlik, Ivo

2012-01-01

We evaluated the prevalence of hepatitis E virus (HEV) in the pork production chain in Czech Republic, Italy, and Spain during 2010. A total of 337 fecal, liver, and meat samples from animals at slaughterhouses were tested for HEV by real-time quantitative PCR. Overall, HEV was higher in Italy (53%) and Spain (39%) than in Czech Republic (7.5%). HEV was detected most frequently in feces in Italy (41%) and Spain (39%) and in liver (5%) and meat (2.5%) in Czech Republic. Of 313 sausages sampled at processing and point of sale, HEV was detected only in Spain (6%). HEV sequencing confirmed only g3 HEV strains. Indicator virus (porcine adenovirus) was ubiquitous in fecal samples and absent in liver samples and was detected in 1 slaughterhouse meat sample. At point of sale, we found porcine adenovirus in sausages (1%–2%). The possible dissemination of HEV and other fecal viruses through pork production demands containment measures. PMID:22840221

33 CFR 159.309 - Limitations on discharge of treated sewage or graywater.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30-day period does not exceed 20 fecal coliform/100 milliliters (ml) and not more than 10 percent of the samples exceed 40 fecal coliform/100 ml; (4) Concentrations of total residual chlorine do not...

33 CFR 159.309 - Limitations on discharge of treated sewage or graywater.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30-day period does not exceed 20 fecal coliform/100 milliliters (ml) and not more than 10 percent of the samples exceed 40 fecal coliform/100 ml; (4) Concentrations of total residual chlorine do not...

Removal of Fecal Indicators, Pathogenic Bacteria, Adenovirus, Cryptosporidium and Giardia (oo)cysts in Waste Stabilization Ponds in Northern and Eastern Australia

PubMed Central

Sheludchenko, Maxim; Padovan, Anna; Katouli, Mohammad; Stratton, Helen

2016-01-01

Maturation ponds are used in rural and regional areas in Australia to remove the microbial loads of sewage wastewater, however, they have not been studied intensively until present. Using a combination of culture-based methods and quantitative real-time PCR, we assessed microbial removal rates in maturation ponds at four waste stabilization ponds (WSP) with (n = 1) and without (n = 3) baffles in rural and remote communities in Australia. Concentrations of total coliforms, E. coli, enterococci, Campylobacter spp., Salmonella spp., F+ RNA coliphage, adenovirus, Cryptosporidium spp. and Giardia (oo) cysts in maturation ponds were measured at the inlet and outlet. Only the baffled pond demonstrated a significant removal of most of the pathogens tested and therefore was subjected to further study by analyzing E. coli and enterococci concentrations at six points along the baffles over five sampling rounds. Using culture-based methods, we found a decrease in the number of E. coli and enterococci from the initial values of 100,000 CFU per 100 mL in the inlet samples to approximately 1000 CFU per 100 mL in the outlet samples for both bacterial groups. Giardia cysts removal was relatively higher than fecal indicators reduction possibly due to sedimentation. PMID:26729150

Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR

PubMed Central

Desneux, Jérémy; Pourcher, Anne-Marie

2014-01-01

Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631