Analysis of adenoviruses and polyomaviruses quantified by qPCR as indicators of water quality in source and drinking-water treatment plants

Three drinking-water treatment plants were analyzed for the presence of human adenoviruses (HAdV) and JC polyomavirus (JCPyV), previously suggested as viral contamination indicators, in order to define their water quality in relation to the presence of viral pathogens and the efficiency of the treatments applied.The 90% of the river water samples had positive results of HAdV (10¹-10⁴ genome copies (GC)/L); and 48%, of JCPyV (10⁰-10³ GC/L). Lower concentrations of HAdV and JCPyV were found in different treatment steps of the plants in absence of bacterial standards. Virus removal efficiencies were higher than 5 logs in plants 1 and 3 and could be quantified as >2 logs in plant 2. However, three post-chlorinated samples from plants 2 and 3 (11%) were found to be positive for HAdV by qPCR, but did not show infectivity in the cell cultures assayed. Simple methods based on the adsorption-elution of viruses from glass wool give low-cost and efficient virus recovery from source water and large-volume water samples. Quantification of JCPyV and HAdV using qPCR is useful for evaluating virus removal efficiency in water treatment plants, identification of Hazard Analysis and Critical Control Points (HACCP) and as a molecular index of the virological quality of water. Though infectivity is not guaranteed when using qPCR techniques in water treated with disinfection processes, the quality of source water, where viruses have proved to have infective capabilities, and the removal efficiency of viral particles may be efficiently quantified.     

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10¹ to 1.07 × 10⁶ genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.     

One year environmental surveillance of rotavirus specie A (RVA) genotypes in circulation after the introduction of the Rotarix® vaccine in Rio de Janeiro, Brazil

Rotavirus specie A (RVA) infection is the leading cause of severe acute diarrhea among young children worldwide. To reduce this major RVA health impact, the Rotarix^® vaccine (GlaxoSmithKline, Rixensart, Belgium) was introduced in the Brazilian Expanded Immunization Program in March 2006 and became available to the entire birth cohort. The aim of this study was to evaluate the spread of RVA in the environment after the introduction of Rotarix^® in Brazil. For this purpose, a Wastewater Treatment Plant (WTP) in Rio de Janeiro was monitored for one year to detect, characterize and discriminate RVA genotypes and identify possible circulation of vaccine strains. Using TaqMan^® quantitative PCR (qPCR), RVA was detected in 100% (mean viral loads from 2.40 × 10⁵ to 1.16 × 10⁷ genome copies (GC)/L) of sewage influent samples and 71% (mean viral loads from 1.35 × 10³ to 1.64 × 10⁵ GC/L) of sewage effluent samples. The most prevalent RVA genotypes were P[4], P[6] and G2, based on VP4 and VP7 classification. Direct nucleotide sequencing (NSP4 fragment) and restriction enzyme digestion (NSP3) analysis did not detect RVA vaccine-like strains from the sewage samples. These data on RVA detection, quantification and molecular characterization highlight the importance of environmental monitoring as a tool to study RVA epidemiology in the surrounding human population and may be useful on ongoing vaccine monitoring programs, since sewage may be a good screening option for a rapid and economical overview of the circulating genotypes.     

Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments

There is a widespread need for cultivation-free methods to quantify viability of natural microbial communities in aquatic environments. Adenosine tri-phosphate (ATP) is the energy currency of all living cells, and therefore a useful indicator of viability. A luminescence-based ATP kit/protocol was optimised in order to detect ATP concentrations as low as 0.0001 nM with a standard deviation of <5%. Using this method, more than 100 water samples from a variety of aquatic environments (drinking water, groundwater, bottled water, river water, lake water and wastewater effluent) were analysed for extracellular ATP and microbial ATP in comparison with flow-cytometric (FCM) parameters. Microbial ATP concentrations ranged between 3% and 97% of total ATP concentrations, and correlated well (R² = 0.8) with the concentrations of intact microbial cells (after staining with propidium iodide). From this correlation, we calculated an average ATP-per-cell value of 1.75 × 10⁻¹⁰ nmol/cell. An even better correlation (R² = 0.88) was observed between intact biovolume (derived from FCM scatter data) and microbial ATP concentrations, and an average ATP-per-biovolume value of 2.95 × 10⁻⁹ nmol/μm³ was calculated. These results support the use of ATP analysis for both routine monitoring and research purposes, and contribute towards a better interpretation of ATP data.     

Viral pollution determination of superficial waters (river water and sea-water) from the urban area of Barcelona (Spain)

Various surface water systems in the metropolitan area of Barcelona (Spain), including two rivers and three beaches, were analysed for human viruses using glass powder as adsorbent to concentrate them. The method used allows concentration of large volumes (up to 2001), of water in a single step. Values obtained varied from 18.4 to values higher than 55 MPNCU 1⁻¹in the Besôs river and 0.44-44 MPNCU 1⁻¹in the Llobregat river. Viruses were isolated from approx. 70% of sea-water samples, the values ranging from 0 to 1.60 MPNCU 1⁻¹. Findings are discussed in terms of correlation between virus pollution and bacterial indicators of fecal contamination. Also, the suitability of glass powder adsorption to concentrate viruses in sea-water is discussed.     

Determination of aromatic amines in water samples by capillary electrophoresis with amperometric detection

Aromatic amines such as aniline and its derivatives are an important class of environmental water pollutants. A method based on capillary zone electrophoresis with amperometric detection (CZE-AD) at carbon disk electrode was developed for the determination of aromatic amines in water samples. The effects of working potential, pH and concentration of running buffer, separation voltage and injection time were investigated. Under the optimum conditions, 2,3-diaminonaphthalene, aniline, o-phenylenediamine and p-chloroaniline could be separated in 0.16 mol/L Na₂HPO₄-citric acid buffer (pH 4.6) within 23 min. The detection limits of them were 1.0 × 10⁻⁷, 3.3 × 10⁻⁸, 5.0 × 10⁻⁸, and 1.3 × 10⁻⁷ mol/L (S/N = 3), respectively. The method can be applied directly for the determination of aromatic amines in real water samples with satisfactory results.     

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10−⁷ to 10² g⁻¹ (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.     

Level and source of 129I of environmental samples in Xi'an region, China

Iodine-129 is widely used as a tracer in various environmental practices such as monitoring of nuclear environmental safety, seawater exchange and transport, geochemical cycle of stable iodine and dating of geological events. The spatial distribution of ¹²⁹I concentration varies significantly on global scale because of anthropogenic input from nuclear activities coupled with scarcity of data on environmental ¹²⁹I variability in many parts of the world including Asia. Here we report new data on ¹²⁹I and ¹²⁷I concentrations in soil, vegetation, river water and precipitation collected from Xi'an area, China. The results indicate values for environmental ¹²⁹I/¹²⁷I ratios in the investigated area range from 1.1 × 10^− 10 to 43.5 × 10^− 10 with a mean of 20.6 × 10^− 10, which is 1-3 orders of magnitude lower than the ratios observed in Europe, but comparable with those observed in the locations far from direct effect of point release sources and at similar latitude. The main source of ¹²⁹I in the investigated area is attributed to the global fallout of both atmospheric nuclear weapons testing and long distance dispersion of fuel reprocessing releases.     

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P < 0.0001, N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P < 0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated (R = 0.565, P < 0.0001, N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P < 0.0001, N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R² = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R² = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.     

Characterisation of virus transport and attenuation in epikarst using short pulse and prolonged injection multi-tracer testing

Attenuation processes controlling virus fate and transport in the vadose zone of karstified systems can strongly influence groundwater quality. This research compares the breakthrough of two bacteriophage tracers (H40/1 and T7), with contrasting properties, at subsurface monitoring points following application onto an overlying composite sequence of thin organic soil and weathered limestone (epikarst). Short pulse multi-tracer test results revealed that T7 (Source concentration, Co = 1.8 × 10⁶ pfu/mL) and H40/1 (Co = 5.9 × 10⁶ pfu/mL) could reach sampling points 10 m below ground less than 30 min after tracer application. Contrasting deposition rates, determined from simulated tracer responses, reflected the potential of the ground to differentially attenuate viruses. Prolonged application of both T7 (Co = 2.3 × 10⁴ pfu/mL) and H40/1 (Co = 1.3 × 10⁵ pfu/mL) over a five hour period during a subsequent test, in which ionic strength levels observed at monitoring points rose consistently, corresponded to a rapid rise in T7 levels, followed by a gradual decline before the end of tracer injection; this reflected reaction-limited deposition in the system. T7's response contrasted with that of H40/1, whose concentration remained constant over a three hour period before declining dramatically prior to the end of tracer injection. Subsequent application of lower ionic strength tracer-free flush water generated a rapid rise in H40/1 levels and a more gradual release of T7. Results highlight the benefits of employing prolonged injection multi-tracer tests for identifying processes not apparent from conventional short pulse tests. Study findings demonstrate that despite rapid transport rates, the epikarst is capable of physicochemical filtration of viruses and their remobilization, depending on virus type and hydrochemical conditions.     

Chemical weathering inferred from riverine water chemistry in the lower Xijiang basin, South China

Seasonal sampling was conducted on 13 sites involving the lower stem of the Xijiang river and its three tributaries to determine the spatial patterns of the riverine water chemistry and to quantify the chemical weathering rates of carbonate and silicate of the bedrock. Results indicate that the major ions in the Xijiang river system are dominated by Ca²⁺ and HCO₃⁻ with a higher concentration of total dissolved solids, characteristic of the drainages developed on typical carbonate regions. Obvious spatial variations of major ion concentrations are found at various spatial scales, which are dominantly controlled by the lithology particularly carbonate distribution in the region. The four selected rivers show similar seasonal variations in major ions, with lower concentrations during the rainy season. Runoff is the first important factor for controlling the weathering rate in the basin, although increasing temperature and duration of water-rock interaction could make positive contributions to the enhancement of chemical weathering. The chemical weathering rates range from 52.6 to 73.7 t/km²/yr within the lower Xijiang basin and carbonate weathering is over one order of magnitude higher than that of silicates. CO₂ consumption rate by rock weathering is 2.0 × 10¹¹ mol/yr, of which more than 60% is contributed by carbonate weathering. The flux of CO₂ released to the atmosphere-ocean system by sulfuric acid-induced carbonate weathering is 1.1 × 10⁵ mol/km²/yr, comparable with the CO₂ flux consumed by silicate weathering.     

Identification of airborne bacterial and fungal community structures in an urban area by T-RFLP analysis and quantitative real-time PCR

This study explores the characteristics of bacterial and fungal communities of total suspended particles (TSP) in the atmosphere by using various molecular methods. TSP samples were collected on a glass fiber filter at an urban location in the middle of the Korean Peninsula (Seoul) between middle autumn and early winter in 2007. From the aerosol samples, DNA could be extracted and DNA sequences were determined for bacteria and fungi. Terminal restriction length polymorphism (T-RFLP) analysis was applied to analyze the community structure of them. To estimate the concentration of DNA originating from bacterial and fungal communities, we used the quantitative real-time polymerase chain reaction (Q-PCR). Sequence analyses were also used to determine the identity of biological organisms. The number of bacteria and fungi in the air were between 5.19 × 10¹ and 4.31 × 10³ cells m^− 3 and from 9.56 × 10¹ to 4.22 × 10⁴ cells m^− 3, respectively and bacterium/fungus ratios ranged from 0.09 to 0.76 across the seven sampling dates. Most of the bacterial sequences found in our TSP samples were from Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The fungal sequences were characteristic for Ascomycota, Basidiomycota, and Glomeromycota which are known to actively discharge spores into the atmosphere. The plant sequences could be also detected. We found that large shifts in the community structure of bacteria and fungi were present in our TSP samples collected on different dates. The results demonstrated that in our TSP samples collected at the urban site; (1) there were very diverse bacterial and fungal groups including potential pathogens and allergens and (2) there were temporal shifts in both bacterial and fungal communities in terms of both diversity and abundances across an inter-seasonal period.     

Toxic metals in the atmosphere in Lahore, Pakistan

Aerosol mass (PM₁₀ and PM_2.5) and detailed elemental composition were measured in monthly composites during the calendar year of 2007 at a site in Lahore, Pakistan. Elemental analysis revealed extremely high concentrations of Pb (4.4 μg m^− 3), Zn (12 μg m^− 3), Cd (0.077 μg m^− 3), and several other toxic metals. A significant fraction of the concentration of Pb (84%), Zn (98%), and Cd (90%) was contained in the fine particulate fraction (PM_2.5 and smaller); in addition, Zn and Cd were largely (≥ 60%) water soluble. The 2007 annual average PM₁₀ mass concentration was 340 μg m^− 3, which is well above the WHO guideline of 20 μg m^− 3. Dust sources were found to contribute on average (maximum) 41% (70%) of PM₁₀ mass and 14% (29%) of PM_2.5 mass on a monthly basis. Seasonally, concentrations were found to be lowest during the monsoon season (July-September). Principle component analysis identified seven factors, which combined explained 91% of the variance of the measured components of PM₁₀. These factors included three industrial sources, re-suspended soil, mobile sources, and two regional secondary aerosol sources likely from coal and/or biomass burning. The majority of the Pb was found to be associated with one industrial source, along with a number of other toxic metals including As and Cr. Cadmium, another toxic metal, was found at concentrations 16 times higher than the maximum exposure level recommended by the World Health Organization, and was concentrated in one industrial source that was also associated with Zn. These results highlight the importance of focusing control strategies not only on reducing PM mass concentration, but also on the reduction of toxic components of the PM as well, to most effectively protect human health and the environment.     

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10² and 4.3 × 10² genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.     

Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants

Hospital wastewater has been described as an important source of spreading pathogenic microorganisms in the environment. However, there are few studies reporting the presence and concentrations of gastroenteric viruses and hepatitis A viruses in these environmental matrices. The aim of this study was to assess the contamination by viruses responsible for acute gastroenteritis and hepatitis derived from hospital wastewater treatment plants (WWTPs). Rotavirus A (RV-A), human adenoviruses (HAdV), norovirus genogroup I and II (NoV GI/GII) and hepatitis A viruses (HAV) were detected and quantified in sewage samples from two WWTPs located in Rio de Janeiro (Brazil) that operates different sewage treatments. WWTP-1 uses an Upflow Anaerobic Sludge Blanket (UASB reactor) and three serial anaerobic filters while WWTP-2 uses aerobic processes, activated sludge with extended aeration and final chlorination of the effluents. Viruses’ detection was investigated by using conventional PCR/RT-PCR, quantitative real-time PCR (qPCR) and partial sequencing of the genome of the viruses detected. Rate of viruses detection ranged from 7% (NoV GI in WWTP-1) to 95% (RV-A in WWTP-2) and genome from all viruses were detected. The most prevalent genotypes were RV-A SG I, HAdV species D and F, NoV GII/4 and HAV subgenotype IA. Mean values of viral loads (genome copies (GC)/ml) obtained in filtered effluents from anaerobic process was 1.9 × 10³ (RV-A), 2.8 × 10³ (HAdV) and 2.4 × 10³ (NoV GII). For chlorinated effluents from activated sludge process, the mean values of viral loads (GC/ml) was 1.2 × 10⁵ (RV-A), 1.4 × 10³ (HAdV), 8.1 × 10² (NoV GII) and 2.8 × 10⁴ (HAV). Data on viral detection in treated effluents of hospital WWTPs confirmed the potential for environmental contamination by viruses and could be useful to establish standards for policies on wastewater management.     

One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan

To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is utilized for the main production of drinking water for the Tokyo Metropolitan Area from October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L. The number of acute infectious gastroenteritis patients in the upper river basin was highly correlated with all the viral concentrations, while general microbial indicator data such as turbidity and heterotrophic plate count were independent of viral concentration as suggested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin.     

Occurrence of enteric viruses and faecal indicators in submarine groundwater discharges in the coastal environment of the Mexican Caribbean

The occurrence of faecal microorganisms such as total coliforms (TCs), Escherichia coli (E. coli), coliphages (somatic and F+ specific) and the genomes of faecal viruses: human adenovirus (HAdV), norovirus (NoV) genogroup I (GI) and II (GII), enterovirus (EV), aichi virus (AiV) and Pepper mild mottle virus (PMMoV), was explored in submarine groundwater discharges (SGDs) in the Mexican Caribbean. Ultra-filtrated water samples were subjected to nucleic acid extraction and real-time quantitative polymerase chain reaction (PCR). Results showed the presence of TC bacteria at all sites, whereas the detection of E. coli occurred only during the dry season. Coliphages occurred during the rainy and dry seasons, with concentrations ranging from 10 to 2560 plaque forming units (PFU/100 ml). HAdV and AiV were detected in 50% and 41% of the samples, at concentrations ranging from 10² to 10⁴ genome copies per litre (GC/L) and from 10² to 10⁶ GC/L, respectively. NoV GI and II were detected in 25% and 16% of the samples, at concentrations of 10¹ GC/L and 10²–10³ GC/L, respectively. PMMoV was detected in 50% of the samples at concentrations ranging from 10¹ to 10³ GC/L. The EV genomes were not detected. These findings demonstrate that faecal microorganisms can be transported through SGDs in the Mexican Caribbean, potentially contributing to human health risks for recreation.     

Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces

We measured the concentrations of four host-specific (human, dog, cow, and horse Bacteroidales), four generic fecal (16S total Bacteroidales and Escherichia coli, 23S Enterococcus and uidA E. coli,) and two universal bacterial (16S universal and rpoB universal) DNA targets by qPCR in raw sewage and pooled fecal samples from dogs, cows, horses, and Canada Geese. A spiking protocol using the non-fecal bacterium Pseudomonas syringae pph6 was developed to estimate the recovery of DNA from fecal and environmental samples. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host type. The host-specific markers were found in high concentrations (8-9 log₁₀ copies/g dry wt.) in their respective hosts' samples, which were equal to or greater than the concentrations of generic E. coli and Enterococcus markers, lending support to the use of host-specific and generic Bacteroidales as sensitive indicators of fecal pollution. The host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4% and horse-specific 2%. Based on this limited data set, the measurement of host-specific indicators by qPCR has several promising applications. These applications include determining the percentage of total Bacteroidales contributed by a specific host type, using the ratios of host-specific markers to E. coli or Enterococcus to estimate the contribution of each source to these regulated fecal indicator bacteria, and estimating the mass of feces from each host type in environmental samples.