Using flow cytometry and <Emphasis Type="Italic">Bacteroidales</Emphasis> 16S rRNA markers to study the hygienic quality of source water

Six source water fountains in the community of Berne, Switzerland were sampled monthly over the period of 1 year. The samples were tested for total counts by flow cytometry, and for fecal contamination by using the Bacteroidales 16S rRNA markers HF183, BacR and AllBac. The total counts varied considerably between the different fountains with a minimal value of 5115 counts/L and with a maximal count of 198,508 counts/L. The long-term patterns of total counts over 1 year were typical for each fountain. Comparison of rainfall data and data for the non-specific fecal marker AllBac was shown to be a suitable approach to highlight the vulnerability of sources to environmental influences. HF183, indicating contamination of human origin, occurred only sporadically and in insignificant amounts. Furthermore, as indicated by BacR, the studied fountains showed no evidence of contamination by ruminant feces. Further work is suggested in order to establish threshold values for molecular Bacteroidales markers, which could in future replace the currently used criteria for fecal indicator bacteria.     

Relative decay of fecal indicator bacteria and human-associated markers: a microcosm study simulating wastewater input into seawater and freshwater

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations.     

Incidence of the enterococcal surface protein (esp) gene in human and animal fecal sources

The occurrence of the enterococcal surface protein (esp) gene in the opportunistic pathogens Enterococcus faecalis and E. faecium is well-documented in clinical research. Recently, the esp gene has been proposed as a marker of human pollution in environmental waters; however, information on its relative incidence in various human and animal fecal sources is limited. We have determined the occurrence of the esp gene in enterococci from human (n=64) and animal (n=233) fecal samples by polymerase chain reaction using two primer sets: one presumably specific for E. faecium (esp(fm)) and the other for both E. faecalis and E. faecium (esp(fs/fm)). We believe that this research is the first to explore the use of esp(fs/fm) for the detection of human waste in natural environmental settings. The incidence in human sources was 93.1% esp(fm) and 100% esp(fs/fm) in raw sewage influent; 30% for both esp(fm) and esp(fs/fm) in septic waste; and 0% esp(fm) and 80% esp(fs/fm) in active pit toilets. The overall occurrence of the gene in animal feces was 7.7% (esp(fs/fm)) and 4.7% (esp(fm)); animal types with positive results included dogs (9/43, all esp(fm)), gulls (10/34, esp(fs/fm); 2/34, esp(fm)), mice (3/22, all esp(fs/fm)), and songbirds (5/55, all esp(fs/fm)). The esp gene was not detected in cat (0/34), deer (0/4), goose (0/18), or raccoon (0/23) feces. The inconsistent occurrence, especially in septic and pit toilet sewage, suggests a low statistical power of discrimination between animal and human sources, which means a large number of replicates should be collected. Both esp(fm) and esp(fs/fm) were common in raw sewage, but neither one efficiently differentiated between animal and other human sources.     

Antioxidant effect of a crude phenolic extract from sorghum bran in sunflower oil in the presence of ferric ions

Whole grain condensed tannin sorghum, its bran and a crude phenolic extract (CPE) prepared from the bran were evaluated for total phenols (TP), condensed tannins (CT) and antioxidant activity (AA). Antioxidant effect of the CPE from the sorghum bran was evaluated in sunflower oil in the presence of ferric ions by measuring peroxide values (PVs) and anisidine values (AVs) during storage at 65 °C, in comparison with tertiary butyl hydroquinone (TBHQ). Sorghum bran contained three times more TP and AA, and seven times more CT than the whole grain. The CPE had highest levels of TP, CT and AA. Sunflower oil with CPE had lower PVs and AVs compared to control samples. Oil samples with TBHQ had PVs lower than, but AVs similar to samples containing CPE. In the presence of ferric ions, the CPE was less effective in reducing PVs, but was more effective than TBHQ in reducing AVs.     

Speciation and frequency of virulence genes of Enterococcus spp. isolated from rainwater tank samples in Southeast Queensland, Australia

In this study, 212 Enterococcus isolates from 23 rainwater tank samples in Southeast Queensland (SEQ), Australia were identified to the species level. The isolates were also tested for the presence of 6 virulence genes associated with Enterococcus related infections. Among the 23 rainwater tank samples, 20 (90%), 10 (44%), 7 (30%), 5 (22%), 4 (17%), 2 (9%), and 1 (4%) samples yielded E. faecalis, E. mundtii, E. casseliflavus, E. faecium, E. hirae, E. avium, and E. durans, respectively. Among the 6 virulence genes tested, gelE and efaA were most prevalent, detected in 19 (83%) and 18 (78%) of 23 rainwater tank samples, respectively. Virulence gene ace was also detected in 14 (61%) rainwater tank samples followed by AS, esp (E. faecalis variant), and cylA genes which were detected in 3 (13%), 2 (9%), and 1 (4%) samples, respectively. In all, 120 (57%) Enterococcus isolates from 20 rainwater tank samples harbored virulence genes. Among these tank water samples, Enterococcus spp. from 5 (25%) samples harbored a single virulence gene and 15 (75%) samples were harboring two or more virulence genes. The significance of these strains in terms of health implications remains to be assessed. The potential sources of these strains need to be identified for the improved management of captured rainwater quality. Finally, it is recommended that Enterococcus spp. should be used as an additional fecal indicator bacterium in conjunction with E. coli for the microbiological assessment of rainwater tanks.     

ESCHERICHIA COLI O157 IN IRISH FEEDLOT CATTLE: A LONGITUDINAL STUDY INVOLVING PREHARVEST AND HARVEST PHASES OF THE FOOD CHAIN

The aim of this study was to investigate fecal shedding and transmission of E. coli O157 in cohorts of cattle within a feedlot, to assess subsequent contamination of carcasses with this pathogen and to identify risk factors associated with fecal shedding of E. coli O157. A cohort of 133 heifers housed infour adjacent pens was examined over a five month period, from entering the feedlot to slaughter. Individual rectal fecal samples and pen environmental samples were taken at monthly intervals. The entire outer and inner surfaces of a carcass side of each animal were swabbed immediately following slaughter.
E. coli O157 was isolated from 136 (23%) of the 600 rectal fecal samples; 96% of which contained virulent markers. One hundred and sixty environmental samples were examined and E. coli O157 was isolated from 46 (29%), all of which contained virulent markers. E. coli O157 was not isolated from any of the dressed carcasses. The prevalence of E. coli O157 fecal shedding may be related to the pen and E. coli O157 contamination of the pen floor feces, water trough and feed.
E. coli O157 should be considered as a pathogen shed in the feces of a substantial proportion of feedlot cattle. However, with good hygienic practice at harvest, a very low level of this pathogen can be achieved on dressed carcasses.     

Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution

Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.     

Sewage exfiltration as a source of storm drain contamination during dry weather in urban watersheds

Separating storm drains and sanitary sewers is expected to control sewage pollution, for example, from combined sewer overflows, and to reduce excessive stormwater flow to wastewater treatment plants. However, sewage contamination has been found in such separated storm drain systems in urban areas during dry-weather flow. To determine whether transmission of sewage is occurring from leaking sanitary sewers directly to leaking separated storm drains, field experiments were performed in three watersheds in Santa Barbara, CA. Areas with high and low risks for sewage exfiltration into storm drains were identified, and rhodamine WT (RWT) dye pulses were added to the sanitary sewers. RWT was monitored in nearby storm drain manholes using optical probes set up for unattended continuous monitoring. Above-background RWT peaks were detected in storm drains in high-risk areas, and multiple locations of sewage contamination were found. Sewage contamination during the field studies was confirmed using the human-specific Bacteroidales HF183 and Methanobrevibacter smithii nifH DNA markers. This study is the first to provide direct evidence that leaking sanitary sewers can directly contaminate nearby leaking storm drains with untreated sewage during dry weather and suggests that chronic sanitary sewer leakage contributes to downstream fecal contamination of coastal beaches.     

Application of host‐specific source‐tracking tools for rapid identification of fecal contamination in fresh produce by humans and livestock

BACKGROUND: Fecal contamination in fresh produce is a public health concern because it may contain human pathogens. We introduced host‐specific quantitative real‐time polymerase chain reaction (qPCR) assays for the rapid detection and identification of fecal contamination sources from humans and farm animals (cow, pig, chicken) in fresh produce. Each composite fecal sample was spiked on lettuce at two contamination levels (0.2 mg or 2 mg feces g^?1), followed by qPCR assays for detecting each host‐specific genetic marker: BoBac (cow); PF163 (pig); CP3‐49 (chicken); and HF183 and gyrB (human). Two commercial DNA extraction kits were compared to evaluate DNA recovery yields and removal of PCR inhibition. Sketa2 assay was conducted to assess the presence of PCR inhibition in the contaminated lettuce. RESULTS: All the qPCR assays yielded reliable detection from contaminated lettuce (2 mg feces g^?1), where their target gene numbers were 1.5–5.0 × 10³ (HF183), 0.8–2.2 × 10³ (gyrB), 0.6–1.6 × 10³ (BoBac), 1.6–3.0 × 10³ (CP3‐49) and 1.1–2.2 × 10³ (PF163) copies g^?1 of lettuce. Among the two extraction kits, QIAamp DNA Stool Kit resulted in 2–3 times higher sensitivity and 20% less PCR inhibition than the PowerFood^? kit. CONCLUSION: This study provides information on the optimized host‐specific qPCR assay in identifying sources of fecal contamination in fresh produce and is useful for tracking the contamination source and improving agricultural practice. © 2012 Society of Chemical Industry     

Phosphorus concentration and solubility in dairy feces: variability and affecting factors

Recent data from phosphorus (P) feeding trials have demonstrated that P concentration in dairy feces is directly affected by P levels in diets and that farm P surpluses as well as potential environmental losses can be reduced through dietary manipulation. The current study was conducted to examine the variability of fecal P under farm conditions and to elucidate factors affecting the concentration and solubility of fecal P. Feed and fecal samples from >30 commercial dairies in the Northeast and Mid-Atlantic regions were analyzed. Dietary P concentrations ranged from 3.45 to 5.78 g/kg of feed DM (DM), and P determined in acid digests (TP) of feces from 5.84 to 12.84 g/kg of fecal DM. On average, 50% of fecal TP was water soluble; of the latter, 83% was inorganic (Pi). Across-farm variability (n=33) had CV averaging 18.9% for fecal TP and >20% for Pi and total P (Pt) in water extracts. Within-farm variability based on multiple samples per herd had the same magnitude as across-farm and was independent of sample numbers from individual farms (n=7 to 30). Of all fecal parameters determined, pH and DM had the lowest variability (CV <10%), water-soluble Pi, Pt, and Ca the highest (CV of 20 to 30%), and total P, Ca, and Mg determined by acid digests were intermediate (CV 10 to 20%). Water-soluble Pi concentrations determined in dried-ground fecal samples were lower than in wet samples. The drying-grinding process changes Pi solubility and the change is not linear. This study confirms that dietary P concentration is the dominating factor affecting fecal P excretion; however, Ca concentration, DIM, and fecal pH also made small, but statistically significant contributions, although some of the mechanisms remain to be thoroughly investigated.     

Detection of short tandem repeat (STR) polymorphisms by microchip electrophoresis for individual identification of cattle

A simple and rapid detection of short tandem repeat (STR) markers was studied as a screening test for individual identification of cattle. DNAs were extracted from eight commercial beef samples by a proteinase K-boil method followed by purification with 2-propanol precipitation. Five STR markers, known to be highly polymorphic, were amplified by PCR and analyzed both by a conventional sequencing analysis (SEQ) and by a proposed microchip electrophoresis (MEP). Every marker revealed high polymorphism, such as 5-9 alleles in SEQ analysis, and 4-6 alleles in MEP analysis. This simple and rapid MEP analysis is expected to be an effective screening tool with use of confirmatory SEQ analysis.     

Long-term trends and opportunities for managing regional water supply and wastewater greenhouse gas emissions

Greenhouse gas emissions are likely to rise faster than growth in population and more than double for water supply and wastewater services over the next 50 years in South East Queensland (SEQ), Australia. New sources of water supply such as rainwater tanks, recycled water, and desalination currently have greater energy intensity than traditional sources. In addition, direct greenhouse gas emissions from reservoirs and wastewater treatment and handling have potentially the same magnitude as emissions from the use of energy. Centralized and decentralized water supply and wastewater systems are considered for a scenario based upon a government water supply strategy for the next 50 years. Many sources of data have large uncertainties which are estimated following the IPCC Good Practice Guidelines. Important sources of emissions with large uncertainties such as rainwater tanks and direct emissions were identified for further research and potential mitigation of greenhouse gas emissions.     

Validating intrinsic markers and optimizing spot sampling frequency to estimate fecal outputs

Indirect methods of spot sampling with intrinsic markers to estimate fecal output and nutrient digestibility often have been used in dairy nutrition research as alternatives to total collection of feces (TC) because of labor and expense. However, fecal output and nutrient digestibility estimated from the indirect method must be accurate regardless of altering dietary conditions. This experiment was designed to validate the accuracy of using indigestible neutral detergent fiber (iNDF) or acid-insoluble ash (AIA) as intrinsic markers to estimate fecal outputs and nutrient digestibility compared with TC and to determine the optimal number of spot sampling events to accurately determine fecal output and then nutrient excretion. The experiment used 12 multiparous lactating Holstein cows in a randomized complete block design. Cows were blocked by days in milk and milk yield and randomly assigned to 1 of 2 diets: a diet containing about 49% corn silage on a dry matter basis and a diet containing about 48% alfalfa silage with high by-product (soyhulls) and supplemental K. During the final 3 d of 21-d periods, TC was performed, and 12 spot samples were collected for the same 3 d to represent every 2 h in a 24-h cycle. Fecal outputs and nutrient digestibility of dry matter, organic matter, or nitrogen estimated with iNDF or AIA as an intrinsic marker were compared with TC. Overall, fecal outputs and digestibility estimated with iNDF were similar to that estimated with TC, whereas AIA overestimated fecal output by 44 to 61% and underestimated nutrient digestibilities by 16 to 32%. However, potential differences in statistical inference of dietary effects between iNDF and TC were found. Data from individual spot samples were aggregated to represent spot sampling frequencies of 12 (SP12), 6 (SP6), 4 (SP4), or 2 (SP2) evenly spaced events starting at feeding time. Compared with TC, SP12 produced similar fecal content of iNDF, organic matter, and nitrogen, but fecal AIA content was greater. Furthermore, compared with SP12, SP6 produced similar fecal content of all nutrients, whereas marker and nutrient concentrations in SP4 and SP2 were different. In this experiment, iNDF was a better fecal marker than AIA, and a spot sampling frequency of at least 6 events was necessary. However, interpretation of dietary effects could be confounded when iNDF was used to estimate fecal outputs.     

Quantitative application of fecal sterols using gas chromatography-mass spectrometry to investigate fecal pollution in tropical waters: western Malaysia and Mekong Delta,Vietnam

This is the first report on fecal pollution using molecular markers in Southeast Asia where serious sewage pollution has occurred. A simple and sensitive analytical method using gas chromatography-mass spectrometry for 10 sterols in various environmental samples was developed to monitor extensive areas of tropical Asia. First, the method was applied to wastewater to confirm that >95% of sterols existed in the particulate phase. Then the approach was applied to a tropical Asian region, Malaysia and Vietnam, with a selection of 59 sampling stations in total. River water and sediment samples were collected and analyzed for chemical markers (coprostanol and other sterols) and microbiological markers (fecal coliforms and fecal streptococci). Particulate coprostanol concentrations ranged from <0.0001 to 13.47 microg/L in tropical river and estuarine waters, indicating severe fecal pollution in populous areas. Coprostanol concentrations in the sediments ranged from 0.005 to 15.5 microg/g-dry. The sedimentary coprostanol concentrations were lower than those reported in some urban areas of industrialized countries. This is probably because frequent heavy rain induces intensive input of eroded soil, which dilutes fecal material in river sediments. The relationship between the concentrations of fecal sterols and bacterial indicators was examined in an attempt to develop public health criteria for coprostanol levels applicable to the tropical region. Coprostanol concentrations of 30-100 ng/L or percent coprostanol levels of 2% corresponded to approximately 1000 fecal coliforms per 100 mL, which is set for secondary contact limit in many countries. These coprostanol concentrations were lower than those proposed as criteria in temperate countries, probably owing to greater survival of bacteria in warmer tropical waters. On the basis of these criteria, extensive monitoring of sediments suggests that poor sanitary conditions exist in most of the urbanized area of Malaysia and in several urban and rural sites in Vietnam.     

桑叶水提物对高脂饮食小鼠粪便中胆固醇代谢产物的影响

本文研究了桑叶水提物对高脂饮食小鼠粪便中胆固醇代谢产物的影响,为桑叶饮料降血脂功能确定提供数据支持。将桑叶水提物按其DNJ含量设置成高(8.0 mg/kg·bw)、中(4.0 mg/kg·bw)、低(2.0 mg/kg·bw)3个剂量组,分别灌胃高脂饮食小鼠4、8、12、16周,测定各组小鼠粪便pH、粪固醇、胆汁酸、短链脂肪酸(Short Chain Fatty Acids,SCFAs)含量,并对SCFAs和血清胆固醇(Total Cholesterol,TC)水平、动脉粥样硬化指数(Atherosclerosis Index,AI)进行相关性分析。与高脂对照组相比,每天灌胃8mg/kg·bwDNJ的桑叶水提物16周,小鼠粪固醇升高22.31%,胆汁酸升高22.31%,SCFAs升高36.76%,乙酸升高39.25%,丙酸升高96.52%,丁酸升高60.43%,异丁酸升高68.05%,戊酸升高74.21%,异戊酸升高23.78%,粪便pH降低2.05%,上述指标的变化均具有显著性(p0.05)。血清TC、AI分别与乙酸、丙酸、丁酸、异丁酸、戊酸和异戊酸呈极显著负相关(p0.01)。桑叶水提物可改善高脂饮食小鼠肠道环境,促进肠道微生物发酵产生SCFAs,促进粪固醇、胆汁酸排出,从而有助于降血脂作用。     

Detection of Bacteroidales fecal indicators and the zoonotic pathogens E. coli 0157:H7, salmonella, and campylobacter in river water

Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.     

Modeling the transport and inactivation of E. coli and enterococci in the near-shore region of Lake Michigan

To investigate the transport and fate of fecal pollution at Great Lakes beaches and the health risks associated with swimming, the near-shore waters of Lake Michigan and two tributaries discharging into it were examined for bacterial indicators of human fecal pollution. 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%) in the tributaries draining into Lake Michigan and in 6/30 samples (20%) in Lake Michigan beaches. This was indicative of human fecal pollution being transported in the tributaries and occurrence at Lake Michigan beaches. To understand the relative importance of different processes influencing pollution transport and inactivation, a finite-element model of surf-zone hydrodynamics (coupled with models for temperature, E. coli and enterococci) was used. Enterococci appear to survive longer than E. coli, which was described using an overall first-order inactivation coefficient in the range 0.5-2.0 per day. Our analysis suggests that the majority of fecal indicator bacteria variation can be explained based on loadings from the tributaries. Sunlight is a major contributor to inactivation in the surf-zone and the formulation based on sunlight, temperature and sedimentation is preferred over the first-order inactivation formulation.     

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

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 10(1) to 10(8) copies/microl. 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 x 10(7) copies/g) to thatfound in human feces (1.1 x 10(7) 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 x 10(4) 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.     

Polyethylene glycol as an indigestible marker to estimate fecal output in dairy cows

The objective of this study was to evaluate the accuracy of fecal output measurements using polyethylene glycol (PEG) as an external marker determined by near-infrared reflectance spectroscopy. In addition, the accuracy of dry matter intake predictions based on fecal output and digestibility estimated using an internal marker [indigestible neutral detergent fiber (iNDF)] was assessed. The experiment was conducted using 6 lactating dairy cows fed 2 different diets. Polyethylene glycol was administered twice daily into the rumen and the diurnal pattern of fecal concentrations and recovery in feces were determined. To evaluate the effects of alternative marker administration and sampling schemes on fecal output estimates, the passage kinetics of PEG in the digestive tract of dairy cows was determined and used for simulation models. The results indicate that PEG was completely recovered in feces and, thus, fecal output was accurately estimated using PEG. Good agreement between measured and predicted dry matter intake (standard error of prediction = 0.86 kg/d, R² = 0.81) indicates good potential to determine feed intake using PEG in combination with iNDF. The precision of cow-specific digestibility estimates based on iNDF was unsatisfactory, but for a group of cows iNDF provided an accurate estimate of dry matter digestibility. The current study indicated that, to overcome inherent day-to-day variation in feed intake and fecal output, the minimum of 4 fecal spot samples should be collected over 4 d. Preferably, these samples should be distributed evenly over the 12-h marker administration interval to compensate for the circadian variation in fecal PEG concentrations.     

Use of fecal steroids to infer the sources of fecal indicator bacteria in the Lower Santa Ana River Watershed, California: sewage is unlikely a significant source

The Santa Ana River (SAR), CA and adjacent wetlands have been identified as potential sources of fecal indicator bacteria (FIB) to the surf zone at Huntington Beach, CA. A suite of fecal steroids, including coprostanol (COP), epicoprostanol (eCOP), cholesterol (CHOE), cholestanol (CHOA), alpha-cholestanone (aONE), beta-cholestanone (bONE), beta-sitosterol (bSIT), stigmasterol (STIG), stigmastanol (STAN), and campesterol (CAM), were used as chemical markers to examine whether sewage was a significant source of FIB within the lower Santa Ana River watershed. A total of 54 water samples were collected from three locations in the intertidal zone near the mouth of the Santa Ana River at different tidal stages. Steroid ratios in SAR samples were different from those found in raw and treated sewage from a local wastewater treatment plant or in nearby effluent plume and did not appear to be influenced by the sampling location, daily tides, and spring/neap tidal cycle. The characteristics of steroid ratios suggested a diagenetic ratherthan a biogenic source forthe COP content of the samples. The log-based concentrations of COP and FIB in the SAR samples were not significantly correlated, inconsistent with sewage being the source of FIB in the study area. In addition, multivariate statistical analysis showed that the concentrations of FIB were better correlated with bird fecal steroids than with the typical sewage sterols. The results implied that sewage was not a significant source of fecal steroids, and therefore perhaps FIB to the study area. Instead, birds may be one possible source of the intermittently high levels of FIB observed in the lower Santa Ana River watershed and the nearby surf zone.