Microbial diversity and host-specific sequences of Canada goose feces

Abstract

Methods to assess the impact of goose fecal contamination are needed as the result of the increasing number of Canada geese (Branta canadensis) near North American inland waters. However, there is little information on goose fecal microbial communities, and such data are important for the development of host-specific source-tracking methods. To address this issue, 16S rRNA gene clone libraries for Canada goose fecal samples from Ontario, Canada, and Ohio were analyzed. Analyses of fecal clones from Ontario (447) and Ohio (302) showed that goose fecal communities are dominated by the classes "Clostridia" (represented by 33.7% of clones) and "Bacilli" (38.1% of clones) and the phylum "Bacteroidetes" (10.1% of clones). Sequences not previously found in other avian fecal communities were used to develop host-specific assays. Fecal DNA extracts from sewage plants (10 samples) and different species of birds (11 samples) and mammals (18 samples) were used to test for host specificity. Of all the assays tested, one assay showed specificity for Canada goose fecal DNA. The PCR assay was positive for Canada goose fecal DNA extracts collected from three locations in North America (Ohio, Oregon, and Ontario, Canada). Additionally, of 48 DNA extracts from Lake Ontario waters presumed to be impacted by waterfowl feces, 19 tested positive by the assay, although 10 were positive only after a nested PCR approach was used. Due to the level of host specificity and the presence of signals in environmental waters, the assay is proposed as a part of the toolbox to detect Canada goose contamination in waterfowl-contaminated waters.

FIG. 1.

Unrooted neighbor-joining tree of 16S rRNA gene sequences from low-G+C-content gram-positive bacteria, obtained from clone libraries. Sequences were aligned and a bootstrap consensus tree was created using MEGA 3.1. The values along branches indicate the percent confidence. The numbers in parentheses indicate numbers of sequences analyzed. CAN, Canada; OH, Ohio.

FIG. 2.

Unrooted neighbor-joining tree of 16S rRNA gene sequences (Ohio Canada goose clones including Prevatella-like sequences) compared to other closely related clone library sequences retrieved from GenBank. Sequences were aligned and a bootstrap consensus tree was created using MEGA 3.1.

FIG. 3.

Detection limits of the CGf5r1 assay. (A) Lanes: 2 to 5, 10-fold dilutions of Canada goose fecal DNA ranging from 20 to 0.02 ng per PCR assay mixture; 6, negative control. (B) Lanes: 10 to 15, plasmid DNA (10-fold dilutions of plasmid DNA ranging from 2 × 10⁵ to 2 copies per PCR mixture) containing a targeted insert; 16, negative control. Lanes 1, 8, and 17 contain molecular size markers.