bioassay – Livestock and Poultry Environmental Learning Community

Manure management practices recycle nutrients in animal manures for crop production. Harmful microbes and viruses in the manure are generally reduced in the soil environment over time. The soil properties influencing how long animal virus persistence are poorly understood and may be specific even down to the type of microbe present. Recently, porcine epidemic diarrhea virus (PEDV), which causes nearly 100% mortality in newborn piglets, has become a serious challenge for swine production. An important concern is whether PEDV in manure applied to nearby farmland may be a source for herd reinfection. How long will PEDV persist in the soil and still be infectious? Are some soils better suited to reduce PEDV risk?

What did we do?

A laboratory study was conducted to mimic a standard manure application practice (manure slurry application into soil) to determine if it reduced the potential for PEDV reinfection. In our study, we tested a range of soil types spiked with PEDV-positive manure slurry and evaluated how PEDV detection and potential infectious risk was affected by soil type. Quantitative PCR and live swine bioassays were used to enumerate PEDV and to determine whether manure and soil samples contained infectious PEDV (Stevens et al., 2018).

What have we learned?

Manure Slurry/Soil Incubations. PEDV genomes declined at different rates depending upon the type of soil tested (Figure 1). While PEDV declined rapidly and was not detected by PCR in Soil #1, #2, and #5 in just 24 hours, PEDV genomes in Soil #6 and #7 decreased more slowly the other soils. Soils #3 and #4 displayed an intermediate rate of decline and reached our detection limit at 48 hours. Soil is an important factor on PEDV persistence.

Figure 1. Porcine epidemic diarrhea virus genomes in the manure slurry/soil incubation determined by reverse-transcriptase quantitative polymerase chain reaction.

Swine Bioassay. Several of the samples tested positive for infections PEDV (Table 2) even when PCR indicated no virus was present; PCR molecular detection of the virus did not produce a complete picture of PEDV survival. For instance, the PCR method indicated no virus in soil #1 or #2 at 24 hours, yet the soil-manure mixture caused disease in a swine bioassay test—the gold standard test for infectious PEDV.

Table 2. Outcome of Swine Bioassay
	Manure-slurry Soil Composite
Time (hours)	#1	#2	#3	#4	#5	#6	#6
24	Pos	Pos	Neg	Pos	Neg	Neg	Pos
48	Pos	Neg	Neg	Neg	Pos	Neg	Pos

†Animals inoculated by oral gavage of 10 mL of phosphate buffer-diluted sample. A porcine epidemic diarrhea virus positive (Pos) or negative (Neg) score is based on fecal swab molecular diagnostic test (reverse transcriptase quantitative polymerase chain reaction).

Are there any soil environmental factors that can help predict whether/how long infectious PEDV lasts in soils? Anything that would damage or disrupt the membrane or proteins on the outside of PEDV would render the virus non-infectious. Theoretically moist soils with lots of active bacteria would release enzymes to chew up PEDV proteins or alkaline (high pH) soils may denature PEDV proteins and damage membranes to inactivate PEDV. On the other hand, soils where manure rapidly dries would help preserve PEDV. None of these hypotheses could explain the PCR or swine bioassay results. Only one factor seemed related to PEDV persistence—high soil phosphorous seemed to protect the virus. No single factor seemed to destroy the virus.

Future Plans

Additional studies are underway determining where PEDV is found within three production sites and the surrounding environment immediately after an outbreak of PEDV. The sites will be monitored over 18 months to signs of PEDV re-emergence.

Authors

Corresponding author: Dan Miller, Research Microbiologist, USDA Agriculture Research Service; email: Dan.miller@ars.usda.gov

Other authors: Erin Stevens (Department of Animal Science, University of Nebraska – Lincoln); Amy Schmidt (Department of Biological Systems Engineering, University of Nebraska – Lincoln); Sarah Vitosh-Sillman and J. Dustin Loy (School of Veterinary Medicine and Biomedical Sciences, University of Nebraska – Lincoln).

Additional information

Stevens EE, Miller DN, Brittenham BA, Vitosh-Sillman SJ, Brodersen BW, Jin VL, et al. Alkaline stabilization of manure slurry inactivates porcine epidemic diarrhea virus. Journal of Swine Health and Production. 2018;26(2):95-100.

Acknowledgements

Funding for this research was provided by the National Pork Board and USDA Agriculture Research Service operational funds. USDA is an equal opportunity provider and employer.

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Purpose

The porcine epidemic diarrhea virus (PEDv) outbreak in North America has substantially impacted swine production, causing nearly 100% mortality in infected newborn piglets. Because manure may remain a source of reinfection, proper manure management practices to limit outbreaks need to be developed and evaluated. Two laboratory studies simulating manure pit treatment with increasing amounts of quicklime were conducted to determine PEDv susceptibility to increasing pH. Additionally, two laboratory soil incubation studies contrasting manure liming, multiple soil types, and two antecedent soil moistures were conducted over several months with incubation conditions mimicking the climates in Minnesota, Missouri, and Oklahoma to determine whether current manure application practices reduce the potential for PEDv reinfection via manure-amended soil. Quantitative PCR and live swine bioassays were used to enumerate PED virus and to determine whether manure and soil samples contained infectious PEDv.

What did we do?

Quicklime-Manure Slurry Incubations: An initial short-term manure slurry study was conducted on fresh PEDv-positive manure slurry collected in 2015 from the shallow pit of a commercial swine facility in southeast Nebraska. Manure was sampled prior to treatment (0 h) and then distributed among glass beakers (250 mL) to accommodate triplicates of three treatments: liming to pH 10, liming to pH 12, and unlimed manure. Following pH adjustment, aliquots of each sample were collected at 1 and 10 h, immediately neutralized with 10 mM HCl and stored at -80°C for subsequent analysis. In a second manure slurry incubation, triplicate PEDv-positive manure samples collected from a commercial swine operation in south central Nebraska site in December 2016 were mixed in equal portion (w:v) with distilled water to mimic manure slurry consistency observed in swine production pit storages. Quicklime was added stepwise (0.25 g addition) to each manure slurr! y sample with continuous stirring to gradually increase manure slurry pH. After each addition of quicklime, pH was measured and an aliquot of manure slurry was collected for subsequent quantitative PCR PEDv enumeration and infectivity in a pig bioassay.

Long-term manure and soil incubation. Initial tests determined appropriate initial soil moisture contents (representing a ‘dry’ and ‘moist’ soil condition) and manure:soil ratios (1 g slurry:3 g soil) to best represent the manure:soil within an injection furrow when slurry is injected into soil, and appropriate liming source (ag lime vs. quicklime). PEDv-positive manure slurry collected from a commercial swine operation in southeast Nebraska was divided between two 3-L containers, one for limed treatment (LIME) and the other for the control, or no-lime, treatment (CNL). Quicklime (30 g) was added to one 3 L portion (equivalent to an application of 80 lbs. quicklime per 1000 gallons of slurry) to achieve a final pH of 12. Both treated and untreated slurry stocks were incubated at room temperature for 24 hours. Distilled water was added to two soils, a silty clay loam (pH 7.0) and a loamy fine sand (pH 6.9), to attain 10% and 30% water holding capacity! (dry and moist soil condition). Thirty grams (dry weight) of soil was apportioned to multiple 50 mL screw top conical tubes and a cavity was made in the center of the soil by pressing a 10 mL pipet tip into the soil. Ten mL of slurry (LIME or CNL) were then added to each soil tube via pipet. Four replicate tubes were immediately frozen at -80°C for each combination of soil, moisture, and manure treatment to represent initial soil application (day 0). The 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 (Figure 1). Twenty replicate tubes were created for each combination of soil, moisture, incubation, and manure treatment, and a set of four tubes were collected for each treatment combination on days 30, 60, 90, 120 and 150 of the incubation and immediately transfer! red to a -80°C freezer for storage.

Molecular detection and quantification of PEDv. Prior to analysis, soil and manure samples were removed from -80°C storage and allowed to thaw at room temperature. The RNA in each sample was extracted using the RNA PowerSoil Total RNA Isolation kit (Mo Bio, Carlsbad, CA). PEDv was detected in samples by reverse transcription and quantitative polymerase chain reaction (RT-qPCR).

Swine bioassay. To confirm that conditions yielding a PCR negative result actually 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 from the initial short-term manure slurry incubation (quicklime addition). Fifteen pigs, approximately 21 days old, were sourced from a high-health facility whose dams tested 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 rooms at the University of Nebraska-Lincoln Life Sciences Annex as follows: 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 piglets in the pH 10 room received one of the six limed (pH 10) sl! urry samp les (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). Piglets 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 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.

PEDv, log # g soil

What have we learned?

Manure Slurry Incubation: Manure limed to pH 10 and pH 12 for 1 and 10 h yielded no detectable PEDv RNA. Live swine bioassay results confirmed that these samples were not infective while control samples resulted in PEDv infection of piglets. These results indicate that a final manure slurry pH of 10 (equivalent to 50 lbs. of quicklime added to 1000 gallons manure slurry) is sufficient to reduce PEDv RNA to an undetectable concentration after 1 hour of contact time. All pigs receiving limed manure (pH 10 or 12 maintained for 1 or 10 h) during the live swine bioassay tested negative for PEDv infection while control pigs (un-limed treatment) all tested positive for PEDv infection (Figure 1). The pig bioassay results confirmed that the PCR assay is a reliable predictor for the presence of infectious PEDv in these matrices and that lime addition to achieve pH 10 for just one hour is sufficient to deactivate the virus in stored manure.

Soil Incubations: At the completion of the long-term (150-day) soil incubation, a subset of the frozen samples (LIME and CON soil samples collected on day 0 and 30) was selected for RNA extraction and qPCR analysis. The qPCR results from days 0 and 30 yielded no detectable PEDv RNA in either the limed or un-limed manure-amended soils (Figure 1). Furthermore, manure-amended soils did not differ from soil-only controls even though PEDv RNA was still detectable in the original manure slurry at high concentrations. No differences in PEDv abundance were detected on either day when initial soil moisture (10% vs 30% water holding capacity), incubation condition (MN vs. MO vs. OK), or soil type (silty clay loam and loamy fine sand) were varied. For these soils, the concentration of PEDv in limed or un-limed manure decreased immediately to a non-detectable level. These results indicate that manure-amended soil with pH 6.9 or greater is not a vector for transmission of the PED virus.

A consistent finding from all of the studies is that pH of media (slurry or soil) strongly influences PED virus survival.

Future Plans

Additional studies are underway to identify the lowest pH at which the PED virus is rendered non-infectious in slurry manure.

Corresponding author, title, and affiliation

Amy Millmier Schmidt, Assistant Professor, Departments of Biological Systems Engineering and Animal Science, University of Nebraska – Lincoln

Corresponding author email

aschmidt@unl.edu

Other authors

Stevens, E., A. Schmidt, D. Miller, J.D. Loy and V. Jin

Additional information

Dr. Amy Millmier Schmidt, corresponding author, can also be reach at (402) 472-0877.

Acknowledgements

Funding for this research was provided by the National Pork Board. Gratitude is extended to Ashley Schmit for assistance with laboratory activities and animal care. Special thanks to the Nebraska pork producers who granted access to their farms for collection of PEDv-positive manure.