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.