Materials and Methods
Animals:
Animals in this study were used under the guidelines and approval of the Pipestone Research IACUC, protocol ID# 2020-003. Five hundred sixty-six (566) feeder pigs were challenged with PRRSV and PEDV in a Biosecurity level 2 (BSL2) research facility by an oral transmission route in the feed. The concentration of challenge material for PRRSV and PEDV was 1x10^5 Median Tissue Culture Infectious Dose (TCID50) for both pathogens. All animals were humanely euthanized by penetrating captive bolt method, according to the American Veterinary Medical Association and the American Association of Swine Veterinarians guidelines. Pigs were confirmed exposed to the pathogens via oral fluid PCR testing and confirmed infected by serum and fecal swab PCR testing for PRRSV and PEDV, respectively. Forty (40) market hogs that were dead on arrival from a processing plant were also utilized for the grinding part of this study. The bioassay contained 30 pigs housed in a BSL2 research facility challenged with their respected samples, as described in the bioassay section of the material and methods.
Diagnostics:
All PRRSV and PEDV testing were completed by reverse transcription real-time polymerase chain reaction (PCR) testing at the Animal Disease Research and Diagnostic Laboratory at South Dakota State University according to standard diagnostic procedures. Compost grab samples were processed for testing, as described in a previous study where 10g of the compost is combined with 25ml of minimal essential media (Vitosh-Sillman et al., 2017). The sample was processed by stomacher blending for 2 minutes at 230 rpm. Supernatant media is separated for testing by PCR. Laboratory standard diagnostic procedures were used to test all compost, water, and air collection elution samples by oral fluid PCR diagnostic testing. Blood collection for serum occurred via jugular venipuncture with a single-use collection system. Samples were centrifuged at 1800 xg for at least 10 minutes, after which serum was separated for testing. Fecal swabs were collected with individual sterile swabs (FisherFinest® Transport Swab, Fisher Scientific, Pittsburgh, PA) by introduction into the pig’s rectum.
Pre-processing (grinding):
All animal carcasses were pre-processed for compost by grinding using a 750hp horizontal grinder (Rotochopper® FP-66 B-series, Rotochopper, INC, St. Martin, MN). Carcasses were placed with equal volumes of biomass during the grinding. The pig and biomass blended ground material was used for the windrow compost pile formation.
Compost biomass:
Three different carbon sources were used and compared: woodchips, cornstalk bales, and a 1:1 combination of the two. A separate windrow was formed for each of the three biomass types. Pig carcasses in each specific windrow biomass type were pre-processed with their respective carbon source.
Aerosol sampling:
Aerosol collection occurred with the use of six air collectors with 200 liters per minute flow rate at 60 minute run times (Innovaprep, Drexel, MO). Collectors were positioned downwind from the grinder and were operated during the entirety of the grinding procedure. Two air collectors were placed at each distance of 46m (50 yards), 91m (100 yards), and 137m (150 yards) from the grinder. Samples for testing were collected at the end of the one-hour runtime by filter 0.075% Tween 20/PBS wet-foam elution kits (Innovaprep, Drexel, MO).
Windrow formation:
Three windrows were formed, each representing one of the biomass compost material (woodchips, cornstalks, and a 1:1 combination of the two). A base layer of each biomass type was laid down before the application of the ground carcass material. The ground carcass material was then covered with the carbon material of each windrow’s specific biomass type. Each windrow contained an end section composed of carcasses knowingly infected with PRRSV and PEDV (approximately 544kg of carcass weight per windrow). The rest of each windrow included a combination of remaining feeder pigs and the market weight hogs collected from the packing plant (approximately 3800kg of feeder pigs and 1800kg market hog carcass weight per windrow). Final windrow dimensions were three piles at 3.6m wide, 10m long, and 2.1m high.
Water well and water collection:
In the locations shown in Figure 1, within each compost biomass type under the end section of knowingly infected surrogate pigs, three water wells were made at the depths of 0.15m, 0.46m, and 0.91m below ground surface. Three wells at the same depths were also placed 7.6m downhill of the compost piles. Wells were drilled to their respective depths, and 0.15m of slotted 0.102m in width polyvinyl chloride (PVC) was placed at the bottom of each well. Sodium bentonite was placed around the top of the well where it extended from the ground. The slotted PVC was connected enough solid pipe to sit above the ground high enough to remain accessible after windrow formation was complete. All wells were capped to prevent water introduction from sources other than the ground in-between water collections. Water samples were collected by the use of a 3-way catheter valve with enough rubber tubbing to reach the bottom of each well. A 60cc plastic syringe was then used to create the suction to collect the water from the respective well. The first water well collection attempt was made day five post windrow formation and continued once weekly until the completion of the study.
Compost treatment:
In the windrow section on the opposite end of the known PRRSV and PEDV positive feeder pig carcasses, a compost treatment to support microbial digestion was applied. As seen in Figure 1, each windrow had three sections. One end section with the known positive viral surrogate pigs and no compost treatment, one end section with a compost treatment for encouraging microbial digestion but no confirmed viral positive pigs, and a middle section of compost acted as a buffer area between the treated and untreated areas. In the treated segment of each windrow, three products were applied via a hand pump sprayer in equal volumes that are traditionally used to encourage liquid manure digestion. The application was over the processed carcass material before the top layer of biomass was added to complete the windrow. The products used were the digestive microbe blends Pit Accelerator (ProfitPro, LLC, Albert Lea, MN), and Microbial Manure Master™ (ProfitPro, LCC, Albert Lea, MN) and the liquid biocatalyst Eubio-NBS (Eubio Tec, Albert Lea, MN).
Compost sample collection:
A sample was collected after the grinding was complete from each biomass type from the virus surrogate carcass section. Compost samples were initially collected daily for the first five days after windrow formation then weekly. PCR testing was performed on all compost samples for the presence of PRRSV and PEDV. Weekly compost sampling continued until two consecutive negative results were obtained from each windrow. At each collection point after windrow formation, two compost samples collected by a careful deconstruction of the biomass cover layer of the pile until the processed material was reached. At least a 10g grab sample from the shallow outer layer of processed material and a sample from at least 0.91m deep into the processed material was collected. Samples were collected wearing a shoulder-length plastic obstetrician sleeve, changing sleeve between each windrow type, and placing each 10g sample into a sterile Whirl-Pak bag (Nasco, Fort Atkinson, WI, US) and stored at -80°C until testing.
Compost temperature and weather monitoring:
This study targeted a start in February in the upper Midwest when winter conditions in Minnesota average negative 11°C (-11°C) for the state to assess cold weather effects on the grind and compost method (https://arcgis.dnr.state.mn.us/ewr/climatetrends/#). A local research weather monitoring station gathered the daily high and low temperatures during the study, along with the daily precipitation and snowfall (https://swroc.cfans.umn.edu/weather). Compost temperatures were monitored with 0.91m long temperature probes placed at two locations in each pile, one in the area treated with products to encourage microbial digestion and one in the untreated compost area in the site of the known surrogate positive pigs. Compost temperatures readings were taken daily the first five days following windrow formation and then collected weekly to the completion of the study.
Environmental testing of for Swine DNA:
All air elution samples and all PCR negative PRRSV and PEDV compost samples from days 0-5 post windrow formation were tested for the presence of porcine (Sus scrofa ) DNA (all viral positive compost samples saved for bioassay testing). In weeks 2 and 3, an additional compost sample from each windrow type was collected at the 0.91m depth specifically for swine DNA testing. Testing was performed using a commercially available real-time PCR kit on the processed samples according to kit directions (RapidFinder™ Pork ID Kit, ThermoFisher Scientific, Waltham, MA, USA). Swine DNA testing was performed at the Iowa State University Veterinary Diagnostic Laboratory.
Bioassay:
The infectivity of PRRSV and PEDV PCR positive results of the compost samples were tested via swine bioassay. Thirty (30) pigs approximately three weeks in age and negative for PRRSV and PEDV were used. Pigs were housed in a BSL2 research facility and divided into ten pens with three pigs/pen. Pen design prevented any physical or manure contact between the pigs of different pens. Challenge material came from the PCR positive processed compost samples. On day 0, pigs were challenged with 2ml intramuscularly and 2ml orally with a specific PCR positive processed compost sample. Pigs were tested following exposure on day three and day seven post-challenge via individual serum and fecal swab for PRRSV and PEDV PCR, respectively. Pigs were also monitored for signs of diarrhea after challenge administration. After the bioassay, all pigs were humanely euthanized.