Human and Animal Fecal Contamination of Community Water Sources, Stored Drinking Water and Hands in Rural India Measured with Validated Microbial Source Tracking Assays.

We examined pathways of exposure to fecal contamination of human and animal origin in 24 villages in Odisha, India. In a cross-sectional study during the monsoon season, fecal exposure via community water sources (N = 123) and in the home (N = 137) was assessed using human- and nonhuman-associated Bacteroidales microbial source tracking (MST) markers and fecal coliforms (FCs). Detection rates and marker concentrations were examined to pinpoint pathways of human fecal exposure in the public and domestic domains of disease transmission in study communities. Human fecal markers were detected much more frequently in the domestic domain (45% of households) than in public domain sources (8% of ponds; 4% of groundwater drinking sources). Animal fecal markers were widely detected in both domains (74% of ponds, 96% of households, 10% of groundwater drinking sources), indicating ubiquitous risks of exposure to animal feces and zoonotic pathogens. This study confirms an often suggested contamination link from hands to stored water in the home in developing countries separately for mothers' and children's hands and both human and animal fecal contamination. In contrast to MST markers, FCs provided a poor metric to assess risks of exposure to fecal contamination of human origin in this rural setting

Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India.

We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India

Human fecal and pathogen exposure pathways in rural Indian villages and the effect of increased latrine coverage.

Efforts to eradicate open defecation and improve sanitation access are unlikely to achieve health benefits unless interventions reduce microbial exposures. This study assessed human fecal contamination and pathogen exposures in rural India, and the effect of increased sanitation coverage on contamination and exposure rates. In a cross-sectional study of 60 villages of a cluster-randomized controlled sanitation trial in Odisha, India, human and domestic animal fecal contamination was measured in community tubewells and ponds (n = 301) and via exposure pathways in homes (n = 354), using Bacteroidales microbial source tracking fecal markers validated in India. Community water sources were further tested for diarrheal pathogens (rotavirus, adenovirus and Vibrio cholerae by quantitative PCR; pathogenic Escherichia coli by multiplex PCR; Cryptosporidium and Giardia by immunomagnetic separation and direct fluorescent antibody microscopy). Exposure pathways in intervention and control villages were compared and relationships with child diarrhea examined. Human fecal markers were rarely detected in tubewells (2.4%, 95%CI: 0.3-4.5%) and ponds (5.6%, 95%CI: 0.8-10.3%), compared to homes (35.4%, 95%CI: 30.4-40.4%). In tubewells, V. cholerae was the most frequently detected pathogen (19.8%, 95%CI: 14.4-25.2%), followed by Giardia (14.8%, 95%CI: 10.0-19.7%). In ponds, Giardia was most often detected (74.5%, 95%CI: 65.7-83.3%), followed by pathogenic E. coli (48.1%, 95%CI: 34.8-61.5%) and rotavirus (44.4%, 95%CI: 34.2-54.7%). At village-level, prevalence of fecal pathogen detection in community drinking water sources was associated with elevated prevalence of child diarrhea within 6 weeks of testing (RR 2.13, 95%CI: 1.25-3.63) while within homes, higher levels of human and animal fecal marker detection were associated with increased risks of subsequent child diarrhea (P = 0.044 and 0.013, respectively). There was no evidence that the intervention, which increased functional latrine coverage and use by 27 percentage points, reduced human fecal contamination in any tested pathway, nor the prevalence of pathogens in water sources. In conclusion, the study demonstrates that (1) improved sanitation alone may be insufficient and further interventions needed in the domestic domain to reduce widespread human and animal fecal contamination observed in homes, (2) pathogens detected in tubewells indicate these sources are microbiologically unsafe for drinking and were associated with child diarrhea, (3) domestic use of ponds heavily contaminated with multiple pathogens presents an under-recognized health risk, and (4) a 27 percentage point increase in improved sanitation access at village-level did not reduce detectable human fecal and pathogen contamination in this setting

Effects of salinity and transparent exopolymer particles on formation of aquatic aggregates and their association with norovirus

Human noroviruses (NoVs) are responsible for 50% of food-related disease outbreaks and are notably associated with shellfish consumption. Despite the detrimental health impacts of human NoV-contaminated seafood to public health, there is a lack of knowledge on the physicochemical conditions that govern NoV transmission in aquatic ecosystems. In the present study, we investigated the propensity for NoVs to associate with aquatic aggregates, which have been shown to efficiently deliver nano-sized particles to shellfish. Specific physicochemical conditions characteristic of shellfish cultivation waters, specifically salinity and transparent exopolymer particles (TEP), were targeted in this study for investigating aggregate formation and NoV association dynamics. Murine norovirus (MNV) was used in aggregation experiments as a model surrogate for NoVs. Results demonstrate increased aggregate formation as a function of increasing salinity and TEP concentrations, as well as greater numbers of MNV genomes incorporated into aggregates under conditions that favor aggregation. As aggregate formation was enhanced in waters representing optimal conditions for shellfish production, specifically saline and high TEP waters, the implications to virus transport and shellfish food safety are profound: more aggregates implies increased scavenging of virus particles from surrounding waters and therefor greater risk for bivalve contamination with nano-sized pathogens. These novel data provide insight into where and when NoVs are most likely to be ingested by shellfish via contaminated aggregates, thereby informing best management and water quality monitoring practices aimed at providing safe seafood to consumers

Consideration of Natural Sources in a Bacteria TMDLLines of Evidence, Including Beach Microbial Source Tracking

Total Maximum Daily Load (TMDL) stipulations remained unmet at a southern California beach despite a suite of management actions carried out since 2001, prompting exploration of a Natural Sources Exclusion (NSE) provision within the TMDL. Quantitative Microbial Source Tracking (MST) was employed from 2012 to 2015 to inventory sources of natural and anthropogenic fecal indicator bacteria (FIB). Data suggested FIB exceedances could be traced to gulls based on gull marker prevalence and correlations with FIB concentrations in seawater, sand, and eelgrass. In contrast, human marker concentrations and a tracer dye test did not indicate prevalent human sources. Exponential decay of gull marker in sand amended with live <i>Catellicoccus marimammalium</i> suggested that measured marker reflected fecal inputs versus growth outside the host. Improved water quality was coincident with a 2013 bird exclusion structure, consistent with NSE. However, load allocation needed for TMDL reconsideration was hampered by variable ratios of FIB, MST markers, and pathogens measured in seawater and in gull, cat, and raccoon feces. Quantitative Microbial Risk Assessment is a suggested path forward because such models can incorporate distributions from a combination of FIB sources and communicate criteria in terms of human health risk

Spatial and hydrologic variation of Bacteroidales, adenovirus and enterovirus in a semi-arid, wastewater effluent-impacted watershed

Bacteroidales and viruses were contemporaneously measured during dry and wet weather conditions at a watershed-scale in a semi-arid watershed impacted by a mixture of agricultural runoff, municipal wastewater effluent and municipal runoff. The results highlight the presence of municipal wastewater effluent as a confounding factor for microbial source tracking (MST) studies, and thus data were segregated into groups based on whether they were impacted by wastewater effluent. In semi-arid environments such as the Calleguas Creek watershed, located in southern California, the relative contribution of municipal wastewater effluent is dependent on hydrology as storm events lead to conditions where agricultural and municipal stormwater dominate receiving waters (rather than municipal wastewater, which is the case during dry weather). As such, the approach to data segregation was dependent on hydrology/storm conditions. Storm events led to significant increases in ruminant- and dog-associated Bacteroidales concentrations, indicating that overland transport connects strong non-human fecal sources with surface waters. Because the dataset had a large number of non-detect samples, data handling included the Kaplan–Meir estimator and data were presented graphically in a manner that reflects the potential effect of detection limits. In surface water samples with virus detections, Escherichia coli concentrations were often below (in compliance with) the recreational water quality criteria. In fact, sites downstream of direct inputs of municipal wastewater effluent exhibited the lowest concentrations of E. coli, but the highest concentrations of human-associated Bacteroidales and highest detection rates of human viruses. The toolkit, comprised of the four Bacteroidales assays and human virus assays used, can be successfully applied to inform watershed managers seeking to comply with recreational water quality criteria. However, care should be taken when analyzing data to account for the effect of non-detect samples, sources with differing microbial viability, and diverging hydrologic conditions.Fil: Bambic, Dustin G.. University of California at Davis; Estados UnidosFil: Kildare Hann, Beverly J.. University of California at Davis; Estados UnidosFil: Rajal, Verónica Beatriz. University of California at Davis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sturm, Belinda S. M.. University of California at Davis; Estados UnidosFil: Minton, Chris B.. Larry Walker Associates; Estados UnidosFil: Schriewer, Alexander. University of California at Davis; Estados UnidosFil: Wuertz, Stefan. University of California at Davis; Estados Unidos. Nanyang Technological University; Singapu

Human fecal and pathogen exposure pathways in rural Indian villages and the effect of increased latrine coverage.

Efforts to eradicate open defecation and improve sanitation access are unlikely to achieve health benefits unless interventions reduce microbial exposures. This study assessed human fecal contamination and pathogen exposures in rural India, and the effect of increased sanitation coverage on contamination and exposure rates. In a cross-sectional study of 60 villages of a cluster-randomized controlled sanitation trial in Odisha, India, human and domestic animal fecal contamination was measured in community tubewells and ponds (n&nbsp;=&nbsp;301) and via exposure pathways in homes (n&nbsp;=&nbsp;354), using Bacteroidales microbial source tracking fecal markers validated in India. Community water sources were further tested for diarrheal pathogens (rotavirus, adenovirus and Vibrio cholerae by quantitative PCR; pathogenic Escherichia coli by multiplex PCR; Cryptosporidium and Giardia by immunomagnetic separation and direct fluorescent antibody microscopy). Exposure pathways in intervention and control villages were compared and relationships with child diarrhea examined. Human fecal markers were rarely detected in tubewells (2.4%, 95%CI: 0.3-4.5%) and ponds (5.6%, 95%CI: 0.8-10.3%), compared to homes (35.4%, 95%CI: 30.4-40.4%). In tubewells, V.&nbsp;cholerae was the most frequently detected pathogen (19.8%, 95%CI: 14.4-25.2%), followed by Giardia (14.8%, 95%CI: 10.0-19.7%). In ponds, Giardia was most often detected (74.5%, 95%CI: 65.7-83.3%), followed by pathogenic E.&nbsp;coli (48.1%, 95%CI: 34.8-61.5%) and rotavirus (44.4%, 95%CI: 34.2-54.7%). At village-level, prevalence of fecal pathogen detection in community drinking water sources was associated with elevated prevalence of child diarrhea within 6 weeks of testing (RR 2.13, 95%CI: 1.25-3.63) while within homes, higher levels of human and animal fecal marker detection were associated with increased risks of subsequent child diarrhea (P&nbsp;=&nbsp;0.044 and 0.013, respectively). There was no evidence that the intervention, which increased functional latrine coverage and use by 27 percentage points, reduced human fecal contamination in any tested pathway, nor the prevalence of pathogens in water sources. In conclusion, the study demonstrates that (1) improved sanitation alone may be insufficient and further interventions needed in the domestic domain to reduce widespread human and animal fecal contamination observed in homes, (2) pathogens detected in tubewells indicate these sources are microbiologically unsafe for drinking and were associated with child diarrhea, (3) domestic use of ponds heavily contaminated with multiple pathogens presents an under-recognized health risk, and (4) a 27 percentage point increase in improved sanitation access at village-level did not reduce detectable human fecal and pathogen contamination in this setting

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

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,andCowM3)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-associatedCowM2qPCR method combined with either the BacR orRum2Bacruminantassociated methods are most suitable for implementation