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Title: Performance assessment PCR-based assays targeting Bacteroidales genetic markers of bovine fecal pollution

item SHANKS, O - Environmental Protection Agency (EPA)
item WHITE, K - Environmental Protection Agency (EPA)
item KELTY, C - Environmental Protection Agency (EPA)
item HAYES, S - Environmental Protection Agency (EPA)
item SIVAGANESAN, M - Environmental Protection Agency (EPA)
item Jenkins, Michael
item VARMA, M - Environmental Protection Agency (EPA)
item HAUGLAND, R - Environmental Protection Agency (EPA)

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2009
Publication Date: 3/1/2010
Citation: Shanks, O.C., White, K., Kelty, C.A., Hayes, S., Sivaganesan, M., Jenkins, M., Varma, M., Haugland, R.A. 2010. Performance assessment PCR-based assays targeting Bacteroidales genetic markers of bovine fecal pollution. Applied and Environmental Microbiology. 76:1359-1366.

Interpretive Summary: To protect public health it is necessary to identify sources of fecal pollution in waters used for recreation or domestic consumption. This protection has become a global issue and focus of research. Several methodologies of fecal source tracking have been developed and tested for identifying human fecal pollution from agricultural animals (cattle, pigs, chickens, sheep, etc.), and wildlife (deer, geese, etc.) sources. The idea behind fecal source tracking is that if a source of fecal pollution is identified then a solution can be devised. This study compared assays that target different genes that are presumed to be specific for cattle. The problem being addressed is (1) to determine which method is most specific or does not cross react with non-cattle fecal samples, and (2) to determine which method is the most sensitive, when the contamination is at a low concentration. In addition, we wanted to determine if differences in the diets of cattle (grass-fed or grain-fed) could influence the presence of a method’s target gene. This is because different diets are known to lead to differences in population of bacteria in the feces. In cooperation with scientists at USEPA, scientists at USDA-ARS J. Phil Campbell, Sr., Natural Resource Conservation Center organized the procurement of cattle feces from ARS facilities across the country (and under different diets), and fecal samples from horses, and chickens. DNA was extracted from these various fecal samples which were tested against the different source tracking methods. Results of this study indicated variation in assay performance and also indicated that animal diet influences whether the target DNA marker is present or not. This is important knowledge for further developing and modifying the fecal source tracking methods for improved effectiveness and general use by regulatory agencies such as USEPA.

Technical Abstract: There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. Each assay targets a different gene and microorganism leading to differences in method performance, making it difficult to determine which approach is most suitable for field applications. We describe a single laboratory evaluation of seven PCR and qPCR assays reported to be specific for either ruminant or bovine fecal pollution. Each assay was tested against a reference collection of 247 individual bovine fecal samples representing 11 different populations and 184 fecal DNA extracts from 30 different animal species. Host-specific assays demonstrated a broad distribution among individual bovine samples ranging from 38.5-93.2% and exhibited specificity levels spanning 54.1% to 100%. PCR sensitivity testing indicated variable detection levels between assays and among different bovine populations. For quantitative real-time PCR assays, the abundance of each host-specific genetic marker was measured within each bovine population and compared to quantities of traditional general fecal indicator bacteria determined by real-time 16S rRNA PCR assays specific for total Bacteroidales and Clostridia spp. fecal microorganisms. Experiments indicated large discrepancies in assay performance across bovine populations suggesting that different diet management practices may influence the distribution of host-specific genetic markers.