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Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern. [ABSTRACT FROM AUTHOR]

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment. [ABSTRACT FROM AUTHOR]

Abstract Introduction In the United States and elsewhere, recreational water quality is monitored for fecal indicator bacteria to help prevent swimming-associated illnesses. Standard methods to measure these bacteria take at least 24 hours to obtain results. Molecular approaches such as quantitative polymerase chain reaction (qPCR) can estimate these bacteria faster, in under 3 hours. Previously, we demonstrated that measurements of the fecal indicator bacteria Enterococcus using qPCR were associated with gastrointestinal (GI) illness among swimmers at freshwater beaches. In this paper, we report on results from three marine beach sites. Methods We interviewed beach-goers and collected water samples at marine beaches affected by treated sewage discharges in Mississippi in 2005, and Rhode Island and Alabama in 2007. Ten to twelve days later, we obtained information about gastrointestinal, respiratory, eye, ear and skin symptoms by telephone. We tested water samples for fecal indicator organisms using qPCR and other methods. Results We enrolled 6,350 beach-goers. The occurrence of GI illness among swimmers was associated with a log10-increase in exposure to qPCR-determined estimates of fecal indicator organisms in the genus Enterococcus (AOR = 2.6, 95% CI 1.3-5.1) and order Bacteroidales (AOR = 1.9, 95% CI 1.3-2.9). Estimates of organisms related to Clostridium perfringens and a subgroup of organisms in the genus Bacteroides were also determined by qPCR in 2007, as was F+ coliphage, but relationships between these indicators and illness were not statistically significant. Conclusions This study provides the first evidence of a relationship between gastrointestinal illness and estimates of fecal indicator organisms determined by qPCR at marine beaches.

Abstract Background In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. Results Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. Conclusion The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.

A performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays which are associated with either ruminant or bovine feces are reported. The large discrepancies in the performance of bovine-associated assays across different bovine populations and variability in assay performance between host populations indicated that the use of bovine microbial source-tracking applications would need a prior characterization at each water-shed of interest.

The quantitative PCR arrays are developed for the quantification of two human-specific genetic markers targeting Bacteriodales-like cell surface-associated genes. Assay performance combined with the prevalence of DNA targets in sewage samples have provided evidence that has supported the application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

Quantitative PCR assays are developed for the detection of bovine feces-specific genetic markers and a method is designed for the enumeration of these markers by using a Markov chain Monte Carlo approach. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations have shown that these quantitative assays are useful in monitoring bovine fecal pollution in ambient waters.