Date Full Report Received

02/25/2016

Date Abstract Report Received

02/25/2016

Investigation

Institution:
Primary Investigator:

This project was conducted in an effort to determine the transmission risk for PEDv in stored manure and manure-amended soil. Specific objectives of this project were to: 1) determine the survivability of the PED virus over time in two common soils at two moisture regimes treated with PEDv-positive swine slurry and held at temperatures representing three climates (southern Minnesota, northern Missouri, and central Oklahoma); and 2) determine the impact of lime application to manure on PEDv survivability.

PEDv Survivability in Soil
The manure+soil incubation experiment investigated how PEDv survived in two manure-amended soils (silty clay loam and loamy fine sand) at two initial moisture conditions (10 and 30% water holding capacity) and under three different winter climate conditions (southern Minnesota, northern Missouri, and central Oklahoma). PEDv-positive manure slurry was divided into two 3-L quantities of manure. Quick lime (30 g) was added to one 3-L portion of manure (equivalent to 80 lbs. quick lime per 1000 gallons of slurry) to achieve a final pH of 12. After 24 h, 10 mL of slurry from the two sources (un-limed or limed) were added to 50 mL conical tubes containing 30 g of soil under the two soil types and two moisture conditions. Tubes were loosely capped and placed into one of three incubators operated independently throughout the trial to simulate soil temperatures between November 1 and May 1 at one of three geographic locations: southern Minnesota, northern Missouri, and central Oklahoma. A set of four replicated samples for each treatment combination was removed on days 0, 30, 60, 90, 120 and 150 of the incubation and immediately transferred to a -80°C freezer for storage. After the 150-day incubation, RNA was extracted from each soil sample using the MO BIO RNA PowerSoil kit. PEDv was detected in samples by reverse transcription and quantitative polymerase chain reaction (RT-qPCR) using a standard prepared from extracted PEDv RNA.

Based upon other studies conducted concurrently, only samples collected on days 0 and 30 were analyzed by qPCR. The other manure+soil samples (days 60, 90, 120, and 150) are still available for analysis, but were not analyzed because they likely do not contain any infectious PEDv. Soil receiving un-limed manure had a pH of 7.45 to 7.96 immediately following application of manure (day 0) and 8.15 to 8.74 at day 30. The pH of soil receiving limed manure was 8.60 to 9.27 at day 0 and 8.15 to 8.74 at day 30. Results revealed that abundance of PEDV RNA decreased immediately to background levels following manure addition to soil, regardless of  whether the manure was limed or un-limed. No differences in abundance of PEDv RNA were observed based on soil type, initial soil moisture, or incubation condition. From this data, it was concluded that a soil pH of 7.5 or greater is sufficient to inactivate the PED virus. When utilizing manure from a PEDv-positive site as a soil amendment, the pH of the receiving soil should be tested. If the pH is 7.5 or greater, the soil is not expected to serve as a vector for the virus. Soils with a pH below 7.5 may still serve as a vector for the virus and lime addition to the soil or manure is recommended to ensure that the virus does not survive in the soil environment.

Impact of Lime Application on PEDv Survivability
Three separate studies were conducted under this objective. In the first study, PEDv-positive manure (RT-qPCR Ct value of 25) was collected from the pit of a commercial farm. Three samples (250-mL) at initial pH 7.54 were placed into glass beakers and quick lime was added in 0.25-g increments until pH 12 was achieved. This experiment revealed that 2.5 g of quick lime per 250 mL of slurry achieved a final manure pH of 11.9. This equates to 83.5 lbs of quick lime per 1000 gallons of manure at an initial pH of 7.54.

A second study was conducted to determine how quickly the PED virus is inactivated when exposed to manure with pH 12. Manure slurry from a commercial farm (RT-qPCR Ct value of 25 and pH 7.54) was treated with quick lime to achieve pH 12 (n=3) and compared to non-limed slurry (n=3). Both treatments were sampled at time = 0, 1, 12, and 24 h and analyzed for the presence of the PED virus RNA. Results revealed that limed manure at t = 1, 12, and 24 h no longer contained detectable PED virus.

A third study was then conducted to determine if a lower pH could be effective at inactivating the virus. Three treatments were compared: manure limed to achieve pH 10, manure limed to achieve pH 12, and manure with no lime addition. Three replicates were utilized per treatment. Samples (250-mL) of pooled PEDv-positive manure (RT-qPCR Ct value of 23) collected from swine that had been experimentally infected with PEDv strain CO13 was distributed among nine beakers (n=3 per treatment). Specimens were sampled at time = 0 and then lime was added to three beakers to achieve pH 10 and to three additional beakers to achieve pH 12. Sub-samples (10 mL) were collected from each beaker at 1 and 10 h following lime addition, neutralized, and stored at -80°C. PCR analysis of samples revealed that PEDv RNA was not detected in any of the limed samples (pH 10 or 12 with exposure times of 1 or 12 h), while untreated manure contained detectable virus.

To confirm that conditions yielding a PCR-negative result truly inactivated the PED virus and rendered the manure non-infectious, a live pig bioassay was conducted with the limed and non-limed manure slurry samples. Fifteen pigs, approximately 21 days old, were sourced from a high-health facility with dams testing negative for PEDv antibodies and virus by PCR. Piglets were tested for PEDv upon arrival and confirmed negative. Piglets were randomly assigned to individual housing in BSL-2 animal rooms: control (3 piglets), pH 10 (6 piglets), and pH 12 (6 piglets), and allowed to acclimate for three days. Each pig was then administered a 10 mL oral gavage of manure slurry: three piglets in the control room received one of the three un-limed slurry samples; six pigs in the pH 10 room received one of the six limed (pH 10) slurry samples (three limed for 1 h and three limed for 10 h); and six pigs in the pH 12 room received one of the six limed (pH 12) slurry samples (three limed for 1 h and three limed for 10 h). Pigs were monitored for fecal shedding of PEDv for four days until control animals began to demonstrate clinical signs of PEDv infection, at which time all piglets were humanely euthanized. Fecal swabs, and duodenum, ileum, jejunum, and cecum tissue samples were collected from each animal and fixed in formalin. All fecal and tissue samples were analyzed for the presence of detectable PED virus by immunohistochemistry and PCR. All pigs receiving limed manure (pH 10 or 12 maintained for 1 or 10 h) demonstrated no clinical signs of PEDv infection while control pigs (un-limed treatment) all tested positive for PEDv infection.

Conclusions garnered from this project include:
1. Lime addition to manure to achieve pH 10 for at least 1 h is sufficient to inactivate the PED virus.
2. For stored manure slurry at an initial pH of 7.5 or greater, addition of 50 pounds of quick lime per 1000 gallons of manure slurry is required to achieve a final pH of 10.
3. Addition of lime to manure in a storage pit is not recommended due to significant precipitation of solids resulting from the lime addition, which can create a thick sludge that is difficult to remove from the storage. Likewise, ammonia volatilizes more rapidly as pH increases so liming of stored manure can create significant odor and a potentially harmful concentration of ammonia gas. Liming of manure in a tank wagon prior to transport of manure to a land application site is the recommended alternative.
4. Application of PEDv-positive swine manure to soil with pH 7.5 or greater does not present a risk for survival of the virus in the soil.

Contact Information: Dr. Amy Millmier Schmidt, University of Nebraska – Lincoln, aschmidt@unl.edu, (402) 472-0877