All livestock operations generate fecal waste and manure management is an essential aspect of livestock production. Each livestock enterprise, residential communities and wildlife near a stream, river or lake are a potential source of fecal waste in surface waters. Fecal waste in surface waters is generally referred to as non-point source contamination but in reality, all fecal waste has a vertebrate animal origin, and the species of origin varies with adjacent land-use practices. 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. We have developed a second stage assay that detects the presence of host species mitochondrial DNA (mtDNA) in surface waters. During these studies we expanded the host range of our assays by improving detection of poultry waste and developing new assays for white-tailed deer and Canada goose. We retested samples from a prior National Pork Board study using the poultry assay and also examined surface water samples from sites previously monitored by the NC Department of Environmental Quality (DEM) in the Stockinghead Creek Watershed, Duplin, County, NC. Additional samples were obtained from NC Rivernet program sites and previously monitored sites in Stockinghead Creek. Surface water grab samples and samples collected with automated samplers were collected and tested with a non-specific Enterococcus spp. assay that identified the presence of fecal waste in the samples. Positive samples were then tested using the second stage mitochondrial DNA assay to attribute the waste to be of human, cattle, swine, poultry. Additional testing has been conducted to screen samples collected with Isco automated samplers during 2021-2022 for white-tailed deer, and Canada Goose mtDNA.  

Elevated levels ofnitrate were most prominent at Sikes Mill Run and Stockinghead Creek. Ammonia levels (mg/L) were consistently high at Maple Branch at Wards Bridge Road in the Goshen watershed and in Stockinghead Creek at Stocking Head Road and sporadically at Sikes Mill Run at Beasly Road. Phosphate concentrations were most prominent in Stewart’s Creek. Enterococcus spp. as an indicator of the presence of fecal waste was detected in 46% of the samples collected at five sites in Stockinghead Creek and five Rivernet sites in the Neuse and Cape Fear River basins. Enterococcus spp. were present in 55% of the samples collected at 18 prior Division of Environmental Quality monitoring sites. Second stage testing with the mtDNA Droplet Digital PCR assay documented that the sources of fecal waste in the studies creeks and rivers originated from multiple sources and the prominence of origin from each species varied markedly.

These studies documented that fecal waste in the multi-use Stockinghead Creek watershed is derived from multiple species. Fecal waste in individual samples was attributed to humans, cattle, swine, and poultry. We also compared and contrasted the mtDNA assay with the bacterial based assays, Pig2Bac for swine, and HF 183 for humans. Although the sensitivity of the assays was different, both the mtDNA assays and bacterial-based assays proved useful as second stage assays for attributing the source of fecal waste in surface waters.

Aerosols in samples collected in close proximity to livestock operations are often monitored for small particulates (particulate matter 2.5, PM2.5), odors, and at times microbes. As with water sample monitoring, these assays are often non-specific. We adapted our mtDNA testing to identify the potential presence of host species (mtDNA) in air samples. A sampling system was devised using a vacuum pump and glass filters. Samples were collected within swine barns, poultry houses, dairy barns and at a sewage treatment plant as well as at 50 ft, and 100 ft. from the barns. Positive tests for swine, cattle and poultry were detected within the barn. However, with one exception a positive sample collected at a distance of 50 ft. from a swine barn, all other samples taken at 50 ft and 100 ft. were negative. Although the mtDNA assay can detect the species of origin in air samples taken within livestock facilities, it does not appear useful for monitoring aerosol deposition at distances outside the perimeter of farms.