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

139

Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10 ؊6 in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10 ؊4 . Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context. Fecal indicator bacteria (FIB), including fecal coliforms, Escherichia coli, and enterococci, have been approved indicators of sewage contamination in recreational waters for decades (49, 51). Swimmers exposed to waters contaminated with sewage are at a greater risk of infection by human pathogens, and subsequent gastrointestinal or other illnesses, than those exposed to unimpacted waters (57). The use of FIB to detect sewage contamination, however, relies on the assumptions that elevated FIB concentrations are representative of pathogen presence and that the fate of these bacteria mimics that of pathogens. Unfortunately, previous studies have shown that concentrations of FIB do not necessarily correlate well with bacterial, protozoan, and viral pathogens (2,20).To address the limitations of FIB, microbial source tracking (MST) methods have been developed to identify human fecal contamination (e.g., sewage) in recreational waters (7, 32). These methods are culture independent and are therefore more rapid than FIB methodologies that require a culture step. The potential for same-day results provides the possibility of advisories that reflect current conditions, rather than day-old ones, which allows more-immediate action to protect against public health risks posed by recreational water use (12, 56). The selecti...

Section: Discussionmentioning

confidence: 99%

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Staley

Gordon

Schoen

et al. 2012

139

“…All amplifications were performed in a 7900 HT Fast real-time sequence detector (Applied Biosystems). Reaction conditions and thermal cycling parameters for CowM2, CowM3, GenBac3, EC23S857, and Entero1 are reported elsewhere (14,17,18). For CampF2 Experiment controls.…”

Section: Methodsmentioning

confidence: 99%

“…A total of 9 PCRbased assays were used in this study (Table 1), including four endpoint PCR assays (CF128 and CF193) and seven qPCR assays (CowM2, CowM3, Entero1, EC23S857, GenBac3, CampF2, and ttr-6) (14-21). The plasmid DNA construct used as an absolute DNA standard for the CowM2 and CowM3 qPCR assays is described elsewhere (18). Two additional plasmid constructs (Integrated DNA Technologies) were generated for the remaining qPCR assays (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…For CampF2 Experiment controls. An internal amplification control (IAC) designed to evaluate the suitability of isolated DNA for PCR-based amplifications was performed on each DNA extract using the CowM2 IAC multiplex assay as described previously (18). The criterion for concluding the presence of amplification inhibition in fecal DNA preparations was established as a C q value of 35.9 ϩ 1.5, based on repeated experiments used to calculate a mean C q value from control reaction mixtures containing 50 copies of the CowM2 IAC in buffer alone.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces

Shanks

Kelty

Peed

et al. 2014

Self Cite

cCalves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR-and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under-or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.

“…Salmonella enterica serovar Typhimurium and E. coli O157:H7 are two examples of bacterial pathogens that may enter VBNC states (8, 62). In addition, host-associated qPCR genetic markers are available and are reported to discriminate between human, cattle, swine, and other animal sources (4,23,25,32,35,(43)(44)(45), providing fecal pollution source information that may lead to better-informed remedial actions to improve water quality.Several of the emerging FIB and host-associated qPCR assays target gDNA from obligate anaerobic bacteria, primarily Bacteroidales. These organisms offer improved host specificity for development of host-associated qPCR assays and comprise a large fraction of the bacterial community in feces.…”

mentioning

confidence: 99%

Decay of Bacterial Pathogens, Fecal Indicators, and Real-Time Quantitative PCR Genetic Markers in Manure-Amended Soils

Rogers

Donnelly

Peed

et al. 2011

Self Cite

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manureamended agricultural soils. Known concentrations of transformed green fluorescent protein-expressing Escherichia coli O157:H7/pZs and red fluorescent protein-expressing Salmonella enterica serovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of ؊20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (Enterococcus spp., E. coli, and Bacteroidales) exhibited decay rate coefficients similar to that of E. coli O157:H7/pZs but not of S. enterica serovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (stx1 and ttrRSBCA, respectively), and FIB genetic markers were also correlated (r ‫؍‬ 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Hostassociated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB, Salmonella enterica serovar Typhimurium/pDs, and E. coli O157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.Cultivation-based methods for fecal indicator bacteria (FIB) such as Escherichia coli and Enterococcus spp. have long been used to indicate potential public health risks associated with water impacted by human and other animal feces (53). FIB cultivation methods are simple to perform and inexpensive. However, these methods require 18 to 24 h following sampling to generate test results; this allows potential exposure of the public to fecal pathogens in the interim. Regulatory agencies, business owners, and other stakeholders have expressed interest in more rapid and specific methods to identify water quality impairment.Emerging real-time quantitative PCR (qPCR) methods designed to estimate the concentration of fecal pollution by targeting genomic DNA (gDNA) from FIB such as Bacteroidales, Enterococcus spp., and E. coli are now available and can generate test results in just a few hours after sampling (10,16,48). Some of these genetic markers can be correlated to public health risk and may soon be incorporated by the U.S. Environmental Protection Agency into water quality standards in the United States (16,59). These genetic markers may also detect viable but nonculturable (VBNC) cells tha...