The fecal microbiome of cattle plays a critical role not only in animal health and productivity but also in food safety, pathogen shedding, and the performance of fecal pollution detection methods. Unfortunately, most published molecular surveys fail to provide adequate detail about variability in the community structures of fecal bacteria within and across cattle populations. Using massively parallel pyrosequencing of a hypervariable region of the rRNA coding region, we profiled the fecal microbial communities of cattle from six different feeding operations where cattle were subjected to consistent management practices for a minimum of 90 days. We obtained a total of 633,877 high-quality sequences from the fecal samples of 30 adult beef cattle (5 individuals per operation). Sequence-based clustering and taxonomic analyses indicate less variability within a population than between populations. Overall, bacterial community composition correlated significantly with fecal starch concentrations, largely reflected in changes in the Bacteroidetes, Proteobacteria, and Firmicutes populations. In addition, network analysis demonstrated that annotated sequences clustered by management practice and fecal starch concentration, suggesting that the structures of bovine fecal bacterial communities can be dramatically different in different animal feeding operations, even at the phylum and family taxonomic levels, and that the feeding operation is a more important determinant of the cattle microbiome than is the geographic location of the feedlot.The enteric microbiota of cattle affects animal health and food safety and is used as an indicator of fecal pollution, which can affect the types and concentrations of indicator organisms in recreational surface waters. The presence of pathogenic bacteria such as Escherichia coli O157:H7 in the bovine gastrointestinal tract has been linked to disease outbreaks due to the consumption of contaminated beef, milk, and drinking water (3). The average feedlot steer produces 1.62 kg of feces (dry matter) per day (2), resulting in more than 18 million metric tons of feces (dry matter) per year in the United States alone. When bovine fecal waste is moved from feedlot operations for land application as fertilizer or is accidentally discharged into the environment due to severe storms, hazardous events, or failure of onsite waste management practices, pathogenic members of this microbial community, such as E. coli O157:H7, Campylobacter jejuni, Salmonella spp., Leptospira interrogans, and Cryptosporidium parvum (5,14,22,41,44), can pose a serious public health risk.Because of the enormous influence the fecal bacterial community of cattle has on the beef and dairy industry, the economy, and public health, a great deal of research has been conducted to characterize the effects of animal age, disease state, feeding practices, and antibiotic treatments on cattle fecal microorganisms. Many of the most comprehensive studies use DNA-based methodologies, such as sequencing of the full-length 16S rRNA gen...

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There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.The ability to discriminate between bovine and other sources of fecal contamination is necessary for the accurate evaluation of human health risks associated with agricultural runoff and focused water quality management to make waters safe for human use. Many methods have been proposed to identify bovine fecal pollution using a variety of different microbiology and molecular techniques. One of the most widely used approaches utilizes a PCR to amplify a gene target that is specifically found in a host population. Currently, there are numerous PCR-based assays for the detection and/or quantitative assessment of bovine fecal pollution available for microbial source-tracking (MST) applications (1, 5-7, 11, 14, 17, 18, 21, 23). These assays target genes ranging from mitochondrial DNA to ribosomal rRNA to other functional genes involved in microorganism-host interactions.The majority of the reported bovine-associated PCR assays target 16S rRNA genes from the order Bacteroidales. This bacterial group constitutes a large proportion of the normal gut microbiota of most animals, including bovines (28), and contains subpopulations closely associated with other animal hosts such as swine, horse, and human (1,3,6,18,24). Hostassociated PCR-based assays targeting Bacteroidales genetic markers have been used to investigate the sources and levels of fecal pollution at a number of beaches and inland watersheds, with variable levels of success (10,13,22,27). Researchers have postulated that differences in host animal age, health, diet, and geographic lo...