Date Full Report Received

Date Abstract Report Received

All livestock operations generate fecal waste and manure management is an essential aspect of livestock production. Each livestock enterprise and residential community near a stream, river or lake is a potential source of fecal waste in surface waters. Monitoring programs established to protect public health have traditionally relied on detection of fecal coliforms or other enteric bacteria as indicators of fecal contamination. These enteric organisms are non-specific indicators of the presence of fecal waste and do not attribute contamination to specific animal hosts. A second stage assay is often used to detect the presence of host-specific enteric bacteria, and target identification of waste from specific types of livestock.

All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA). The detection of mitochondrial DNA is a routine aspect of forensic investigation and can be applied to identify the species of origin associated with fecal waste. Detection of mtDNA is highly specific; if mitochondrial DNA is detected in a water sample, the vertebrate animal associated with that mtDNA can be determined. Initial assays, however, at times did not detect the presence of mtDNA when fecal waste was detected. These studies supported by the National Pork Board, Pork Checkoff were conducted to improve the mtDNA detection assay and integrate the use of the assay with other promising technologies to enhance our ability to correctly classify the species of origin of fecal waste in surface waters. We adapted new assay technology, Droplet digital PCR, to refine mtDNA detection. The sensitivity of the assay for identifying the presence of host mtDNA in surface waters was markedly improved. Assay protocols were developed for detecting the presence of fecal waste from cattle, poultry, humans and pigs.

Field studies were conducted in Stockinghead Creek, in Duplin County, NC to assess the validity of the assay. Stockinghead Creek is a mixed agricultural use watershed, which includes cattle on pasture, poultry facilities, swine facilities and small residential communities that rely on septic systems to process household waste. Prior studies in the watershed had identified the presence of fecal waste in Stockinghead Creek, and efforts to identify the species contributing to the waste had particularly focused on swine farms. Automated water samplers where placed at sites in the watershed. Water samples were collected from July 2019-October 2020. Enterococcus positive samples and randomly selected negative samples were tested using the mtDNA assay. Our studies confirmed the presence of fecal contamination in Stockinghead Creek in Duplin County, NC and documented that the fecal contamination in the creek originates from at least four species, cattle, humans, poultry and swine. These studies have documented the importance of taking a broader approach when fecal waste is detected in a watershed. Testing should reflect the variety of livestock operations and other potential sources of fecal waste in the watershed. Wildlife also serve as a potential source of fecal contamination in surface waters and additional studies are in progress to develop assays for the detection of fecal waste from white-tailed deer and Canada geese. Correct attribution of the species of origin is an essential first step in efforts to mitigate the contamination of surface waters with fecal waste.

Jay Levine, Department of Marine, Earth and Atmospheric Sciences, North Carolina State University. Email: jflevine@ncsu.edu

Key Findings:

• Mitochondrial DNA detection using Droplet Digital PCR provides a sensitive and specific way to identify the vertebrate source of fecal waste in surface waters.
• Fecal waste from four species, cattle, poultry, swine and humans was detected in Stockinghead Creek, in Duplin County in eastern NC.
• Testing single grab water samples may not accurately reflect the presence of fecal waste in surface waters
• Assays used to identify the species contributing to fecal waste in surface waters should screen for multiple species and reflect the varied mixed uses of watersheds.
• Correct attribution of the source of fecal waste is a key first step in mitigating the contamination of fecal waste in surface waters.