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Appl Environ Microbiol. 2010 July; 76(14): 4886–4889.
Published online 2010 May 28. doi:  10.1128/AEM.00110-10
PMCID: PMC2901712

Quantitative Real-Time PCR Assays for Sensitive Detection of Canada Goose-Specific Fecal Pollution in Water Sources [down-pointing small open triangle]

Abstract

Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

The Canada goose (Branta canadensis) is a prevalent waterfowl species in North America. The population density of Canada geese has doubled during the past 15 years, and the population was estimated to be close to 3 million in 2007 (4). Canada geese often congregate within urban settings, likely due to available water sources, predator-free grasslands, and readily available food supplied by humans (6). They are suspected to contribute to pollution of aquatic environments due to the large amounts of fecal matter that can be transported into the water. This can create a public health threat if the fecal droppings contain pathogenic microorganisms (6, 7, 9, 10, 12, 13, 19). Therefore, tracking transient fecal pollution of water due to fecal inputs from waterfowl, such as Canada geese, is of importance for protecting public health.

PCR detection of host-specific 16S rRNA gene sequences from Bacteroidales of fecal origin has been described as a promising microbial source-tracking (MST) approach due to its rapidity and high specificity (2, 3). Recently, Lu et al. (15) characterized the fecal microbial community from Canada geese by constructing a 16S rRNA gene sequence database using primers designed to amplify all bacterial 16S rRNA gene sequences. The authors reported that the majority of the 16S rRNA gene sequences obtained were related to Clostridia or Bacilli and to a lesser degree Bacteroidetes, which represent possible targets for host-specific source-tracking assays.

The main objective of this study was to identify novel Bacteroidales 16S rRNA gene sequences that are specific to Canada goose feces and design primers and TaqMan fluorescent probes for sensitive and specific quantification of Canada goose fecal contamination in water sources.

Primers 32F and 708R from Bernhard and Field (2) were used to construct a Bacteroidales-specific 16S rRNA gene clone library from Canada goose fecal samples (n = 15) collected from grass lawns surrounding Wascana Lake (Regina, SK, Canada) in May 2009 (for a detailed protocol, see File S1 in the supplemental material). Two hundred eighty-eight clones were randomly selected and subjected to DNA sequencing (at the Plant Biotechnology Institute DNA Technologies Unit, Saskatoon, SK, Canada). Representative sequences of each operational taxonomic unit (OTU) were recovered using an approach similar to that described by Mieszkin et al. (16). Sequences that were less than 93% similar to 16S rRNA gene sequences from nontarget host species in GenBank were used in multiple alignments to identify regions of DNA sequence that were putatively goose specific. Subsequently, two TaqMan fluorescent probe sets (targeting markers designated CGOF1-Bac and CGOF2-Bac) were designed using the RealTimeDesign software provided by Biosearch Technologies (http://www.biosearchtech.com/). The newly designed primer and probe set for the CGOF1-Bac assay included CG1F (5′-GTAGGCCGTGTTTTAAGTCAGC-3′) and CG1R (5′-AGTTCCGCCTGCCTTGTCTA-3′) and a TaqMan probe (5′-6-carboxyfluorescein [FAM]-CCGTGCCGTTATACTGAGACACTTGAG-Black Hole Quencher 1 [BHQ-1]-3′), and the CGOF2-Bac assay had primers CG2F (5′-ACTCAGGGATAGCCTTTCGA-3′) and CG2R (5′-ACCGATGAATCTTTCTTTGTCTCC-3′) and a TaqMan probe (5′-FAM-AATACCTGATGCCTTTGTTTCCCTGCA-BHQ-1-3′). Oligonucleotide specificities for the Canada goose-associated Bacteroides 16S rRNA primers were verified through in silico analysis using BLASTN (1) and the probe match program of the Ribosomal Database Project (release 10) (5). Host specificity was further confirmed using DNA extracts from 6 raw human sewage samples from various geographical locations in Saskatchewan and 386 fecal samples originating from 17 different animal species in Saskatchewan, including samples from Canada geese (n = 101) (Table (Table1).1). An existing nested PCR assay for detecting Canada goose feces (15) (targeting genetic marker CG-Prev f5) (see Table S1 in the supplemental material) was also tested for specificity using the individual fecal and raw sewage samples (Table (Table1).1). All fecal DNA extracts were obtained from 0.25 g of fecal material by using the PowerSoil DNA extraction kit (Mo Bio Inc., Carlsbad, CA) (File S1 in the supplemental material provides details on the sample collection).

TABLE 1.
Specificities of the CGOF1-Bac, CGOF2-Bac, and CG-Prev f5 PCR assays for different species present in Saskatchewan, Canada

The majority of the Canada goose feces analyzed in this study (94%; 95 of 101) carried the Bacteroidales order-specific genetic marker designated All-Bac, with a relatively high median concentration of 8.2 log10 copies g1 wet feces (Table (Table11 and Fig. Fig.1).1). The high prevalence and abundance of Bacteroidales in Canada goose feces suggested that detecting members of this order could be useful in identifying fecal contamination associated with Canada goose populations.

FIG. 1.
Concentrations of the Bacteroidales (All-Bac, CGOF1-Bac, and CGOF2-Bac) genetic markers in feces from various individual Canada geese.

The composition of the Bacteroidales community in Canada goose feces (n = 15) was found to be relatively diverse since 52 OTUs (with a cutoff of 98% similarity) were identified among 211 nonchimeric 16S rRNA gene sequences. Phylogenetic analysis of the 52 OTUs (labeled CGOF1 to CGOF52) revealed that 43 (representing 84% of the 16S rRNA gene sequences) were Bacteroides like and that 9 (representing 16% of the 16S rRNA gene sequences) were likely to be members of the Prevotella-specific cluster (see Fig. S2 in the supplemental material). Similarly, Jeter et al. (11) reported that 75.7% of the Bacteroidales 16S rRNA clone library sequences generated from goose fecal samples were Bacteroides like. The majority of the Bacteroides- and Prevotella-like OTUs were dispersed among a wide range of previously characterized sequences from various hosts and did not occur in distinct clusters suitable for the design of Canada goose-associated real-time quantitative PCR (qPCR) assays (see Fig. S2 in the supplemental material). However, two single Bacteroides-like OTU sequences (CGOF1 and CGOF2) contained putative goose-specific DNA regions that were identified by in silico analysis (using BLASTN, the probe match program of the Ribosomal Database Project, and multiple alignment). The primers and probe for the CGOF1-Bac and CGOF2-Bac assays were designed with no mismatches to the clones CGOF1 and CGOF2, respectively.

The CGOF2-Bac assay demonstrated no cross-amplification with fecal DNA from other host groups, while cross-amplification for the CGOF1-Bac assay was limited to one pigeon fecal sample (1 of 25, i.e., 4% of the samples) (Table (Table1).1). Since the abundance in the pigeon sample was low (3.3 log10 marker copies g1 feces) and detection occurred late in the qPCR (with a threshold cycle [CT] value of 37.1), it is unlikely that this false amplification would negatively impact the use of the assay as a tool for detection of Canada goose-specific fecal pollution in environmental samples. In comparison, the nested PCR CG-Prev f5 assay described by Lu and colleagues (15) demonstrated non-host-specific DNA amplification with fecal DNA samples from several animals, including samples from humans, pigeons, gulls, and agriculturally relevant pigs and chickens (Table (Table11).

Both CGOF1-Bac and CGOF2-Bac assays showed limits of quantification (less than 10 copies of target DNA per reaction) similar to those of other host-specific Bacteroidales real-time qPCR assays (14, 16, 18). The sensitivities of the CGOF1-Bac and CGOF2-Bac assays were 57% (with 58 of 101 samples testing positive) and 50% (with 51 of 101 samples testing positive) for Canada goose feces, respectively (Table (Table1).1). A similar sensitivity of 58% (with 59 of 101 samples testing positive) was obtained using the CG-Prev f5 PCR assay. The combined use of the three assays increased the detection level to 72% (73 of 101) (Fig. (Fig.2).2). Importantly, all markers were detected within groups of Canada goose feces collected each month from May to September, indicating relative temporal stability of the markers. The CG-Prev f5 PCR assay is an end point assay, and therefore the abundance of the gene marker in Canada goose fecal samples could not be determined. However, development of the CGOF1-Bac and CGOF2-Bac qPCR approach allowed for the quantification of the host-specific CGOF1-Bac and CGOF2-Bac markers. In the feces of some individual Canada geese, the concentrations of CGOF1-Bac and CGOF2-Bac were high, reaching levels up to 8.8 and 7.9 log10 copies g1, respectively (Fig. (Fig.11).

FIG. 2.
Venn diagram for Canada goose fecal samples testing positive with the CGOF1-Bac, CGOF2-Bac, and/or CG-Prev f5 PCR assay. The number outside the circles indicates the number of Canada goose fecal samples for which none of the markers were detected.

The potential of the Canada goose-specific Bacteroides qPCR assays to detect Canada goose fecal pollution in an environmental context was tested using water samples collected weekly during September to November 2009 from 8 shoreline sampling sites at Wascana Lake (see File S1 and Fig. S1 in the supplemental material). Wascana Lake is an urban lake, located in the center of Regina, that is routinely frequented by Canada geese. In brief, a single water sample of approximately 1 liter was taken from the surface water at each sampling site. Each water sample was analyzed for Escherichia coli enumeration using the Colilert-18/Quanti-Tray detection system (IDEXX Laboratories, Westbrook, ME) (8) and subjected to DNA extraction (with a PowerSoil DNA extraction kit [Mo Bio Inc., Carlsbad, CA]) for the detection of Bacteroidales 16S rRNA genetic markers using the Bacteroidales order-specific (All-Bac) qPCR assay (14), the two Canada goose-specific (CGOF1-Bac and CGOF2-Bac) qPCR assays developed in this study, and the human-specific (BacH) qPCR assay (17). All real-time and conventional PCR procedures as well as subsequent data analysis are described in the supplemental material and methods. The E. coli and All-Bac quantification data demonstrated that Wascana Lake was regularly subjected to some form of fecal pollution (Table (Table2).2). The All-Bac genetic marker was consistently detected in high concentrations (6 to 7 log10 copies 100 ml1) in all the water samples, while E. coli concentrations fluctuated according to the sampling dates and sites, ranging from 0 to a most probable number (MPN) of more than 2,000 100 ml1. High concentrations of E. coli were consistently observed when near-shore water experienced strong wave action under windy conditions or when dense communities of birds were present at a given site and time point.

TABLE 2.
Levels of E. coli and incidences of the Canada goose-specific (CGOF1-Bac and CGOF2-Bac), human-specific (BacH), and generic (All-Bac) Bacteroidales 16S rRNA markers at the different Wascana Lake sites sampled weeklya

The frequent detection of the genetic markers CGOF1-Bac (in 65 of 75 water samples [87%]), CGOF2-Bac (in 55 of 75 samples [73%]), and CG-Prev f5 (in 60 of 75 samples [79%]) and the infrequent detection of the human-specific Bacteroidales 16S rRNA gene marker BacH (17) (in 5 of 75 water samples [7%[) confirmed that Canada geese significantly contributed to the fecal pollution in Wascana Lake during the sampling period. Highest mean concentrations of both CGOF1-Bac and CGOF2-Bac markers were obtained at the sampling sites W3 (3.8 and 3.9 log10 copies 100 ml1) and W4 (3.4 log10 copies 100 ml1 for both), which are heavily frequented by Canada geese (Table (Table2),2), further confirming their significant contribution to fecal pollution at these particular sites. It is worth noting that concentrations of the CGOF1-Bac and CGOF2-Bac markers in water samples displayed a significant positive relationship with each other (correlation coefficient = 0.87; P < 0.0001), supporting the accuracy of both assays for identifying Canada goose-associated fecal pollution in freshwater.

In conclusion, the CGOF1-Bac and CGOF2-Bac qPCR assays developed in this study are efficient tools for estimating freshwater fecal inputs from Canada goose populations. Preliminary results obtained during the course of the present study also confirmed that Canada geese can serve as reservoirs of Salmonella and Campylobacter species (see Fig. S3 in the supplemental material). Therefore, future work will investigate the cooccurence of these enteric pathogens with the Canada goose fecal markers in the environment.

Nucleotide sequence accession numbers.

The Bacteroidales 16S rRNA gene sequences have been submitted to GenBank under accession numbers from GU222166 (clone CGOF1) to GU222217 (clone CGOF52).

Supplementary Material

[Supplemental material]

Acknowledgments

We acknowledge the generous support of Saskatchewan Agriculture through the Agriculture Development Fund. This work was also supported by funding through the Canada Research Chairs program to C. K. Yost.

We also acknowledge Chris Somers and Arjunn Sachdev, who have assisted in the collection of fecal samples, and thank Jared Clarke of the Wascana Centre Authority for supplying the aerial photo displayed in the supplemental material.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 May 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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