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Antibiotic resistance analysis (ARA), frequency of sampling, and seasonality were evaluated in a rural Virginia watershed dominated by cattle. The selected watershed (Mill Creek) was 3767 ha in size, included two small communities (one sewered and one unsewered), and several farms that when combined contained over 3800 beef and dairy cattle. Monthly monitoring of fecal coliforms at two sampling sites in Mill Creek from January to December, 2001, revealed that the recreational standard (1000 colony forming units, CFUs/100 ml) was exceeded a total of eight times for a 33% violation rate at each site. In addition, stream samples were collected weekly for 4 consecutive weeks during seasonal high flows (March) and seasonal low flows (September-October), plus daily for 7 consecutive days within the weekly schedules for a combined total of 60 stream samples (30 at each of two sites). The recreational standard was exceeded once during seasonal high flow and nine times during seasonal low flow. Microbial source tracking (MST) was performed by ARA to assess the impact of cattle on water quality within the different sampling routines. The resistance patterns of 2880 water isolates and 1158 known source (host-origin) isolates were determined with seven antibiotics at 28 different concentrations. The 1158 isolate database was reduced to 562 unique isolates when clonal ARA patterns were removed. This database of 562 unique isolates had an average rate of correct classification (ARCC) of 95.4%, and several statistical procedures confirmed the library as accurate and representative. Sixty-five percent of 50 challenge-set isolates from sources, but not samples, used in the library were correctly identified. The 562 unique pattern database was used to classify Escherichia coli isolates from water samples into six host source categories. The ARA results showed that cattle were the major source of pollution in the stream and cattle were the dominant source in over 60% of the water samples. Sampling frequency and seasonality had no effect on the MST results, as cattle dominated both seasons and samplings. Deer were a minor contributor in the summer (high water demand), and geese were a minor contributor in the winter when migratory flocks were observed moving through the watershed. An unexpected human allocation was found, especially under seasonal high flow conditions. The exact origin of this human allocation is not known. This project demonstrated that a host-origin library, based on a phenotypic method, could be developed for a well-defined watershed and was both representative of the sources in the watershed and performed reasonably well against a challenge set.