PEDv has caused significant losses in the Nebraska pork industry and mortality can approach 100%. Disposal of these carcasses is a challenge as they serve as a source of tremendous amounts of infectious virus. Current alternative methods of disposal include rendering, incineration and burial. Rendering trucks may serve as a farm-to-farm vector. Incineration is not feasible for the significant number of mortalities and burial may enable long-term survival of virus in soil and may cause re-infection after disease elimination. Therefore, composting may serve as an ideal solution for disposal and mortalities this would provide a biosecure, safe, and cost-effective method to mitigate on-farm sources of virus. The overall objective of this study was to determine the efficacy of composting as a mortality disposal method following death loss from the porcine epidemic diarrhea virus (PEDv). Validation of time-temperature combinations for PEDv inactivation in mortality compost piles was the primary intended outcome of this project.
PEDv virus challenge protocol modeled one that has shown previous success using weanling pigs (Hesse et al., 2013). Twenty-seven animals (approximately 21-day-old weaned piglets) were sourced from a high-health commercial source that had no history of PEDv and with dams that tested negative for the presence of PEDv-specific antibodies and were negative for fecal virus shedding as determined by PCR. Experimental groups were housed in pens and maintained at appropriate temperature and in accordance with national animal care space requirements. Pigs were given five days of acclimation and maintained on commercial nursery pig diets. Following acclimation, each pig was inoculated orally with 5 mL of virus inoculum (NE 9282) supplemented with gentamicin that had been diluted to a real time PCR assay cycle threshold (Ct) 22. Inocula (feces/intestinal contents) from a natural outbreak of PEDv were used. Pigs were evaluated twice daily for evidence of infection: temperature, pulse, respiration, dehydration, and diarrhea. Fecal samples were collected daily for evaluation of fecal shedding of PEDv. When significant clinical signs of enteric disease were present or pigs became sufficiently ill that the attending veterinarian determined euthanasia was appropriate, animals were humanely euthanized and samples taken for necropsy.
Following necropsy, carcasses from infected and euthanized pigs were composted inside biosecure rooms in the Veterinary and Biomedical Sciences Research Facility at the University of Nebraska – Lincoln. Three compost piles were constructed using commercial sawdust and wood shavings at a target moisture content of 50% w.b. For each pile, an insulated platform with internal dimensions of 121.92 cm (W) x 152.4 cm (L) (48 in x 60 in) was used to contain piles. Platforms were constructed of an outer layer of plywood and an inner layer of PolyBoard sheeting with foam board insulation in between to simulate the linear continuation of the pile and the insulative properties of a compacted soil base. Compost piles were constructed by placing a layer of wood shavings on each base to a depth of 60 cm (24 in), followed by placement of five carcasses in a single layer in the center of the pile followed by a 15 cm (6 in) layer of pile material and a second layer of four carcasses in a single layer. Additional sawdust was placed over and around the carcasses to achieve 60 cm of coverage on the top of the pile. Rooms were maintained at approximately 21°C (70°F) and 25% RH throughout the duration of the project.
Temperature was monitored at ten locations within each pile using Apresys in-transit digital temperature recorders (Apresys, Inc., Duluth, GA) beginning at establishment of the piles and continuing at a 20-min sampling frequency (duration of primary compost cycle not established at time of proceedings submission). Temperature within each pile was also monitored manually using a thermometer at 0, 24, 48, 96 h, and 168 h, and then weekly for the duration of the compost cycle to confirm success of the heating process.
Following completion of the primary compost cycle, temperature loggers will be recovered and each pile will be mixed, sampled for analysis of survivability of PEDv at five locations, moisture will be added, and piles will be re-established for a secondary composting cycle with temperature loggers placed as previously described. At the completion of the secondary composting cycle, piles will again be sampled for analysis of survivability of PEDv (5 samples per pile) and temperature loggers will be recovered.
PEDv survivability will be determined via two independent assay methods. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and sensitive method that will be used to quantify the amount of virus RNA genome in the samples. The Nebraska Veterinary Diagnostic Center currently has a validated RT-qPCR test to assay for the presence of PEDv in manure sample matrices. To validate results from the RT-qPCR in laboratory assays, sawdust simulated compost matrix will be spiked with known concentrations of PEDv target RNA and compared to known standards to ensure no inhibition is present and that proper extraction methods are being used. An alternative method using virus isolation will also be conducted to determine whether viable virus is present in flasks at a smaller subset of time points. To do this, Vero cell monolayers will be infected with filter sterilized aliquots of compost exudate, blindly passaged once after seven days, and examined for virus p resence using IFA with a PED specific monoclonal antibody. At specific time points, RT-qPCR Ct values and Virus Isolation will be run in parallel to ensure sensitivity of testing and to evaluate correlation of the testing modalities under the simulated testing conditions and matrices. If these testing methods show agreement, and/or no virus is isolated, RT-qPCR testing will be utilized to facilitate rapid and consistent assessment of virus persistence during the majority of experimental time points.
Biosecurity is essential to controlling the spread of PEDv and any facility that is currently positive for PEDv should work diligently to prevent contamination of neighboring facilities. Vehicle transport has been shown as a high-risk activity that may facilitate spread of PEDv (Lowe 2014) and mortalities that are positive for PEDv may be rejected by renderers to protect them from liability for transmitting the disease. Burial of mortalities can be detrimental to water quality (Bartelt-Hunt et al., 2013) and it is unknown how long the PEDv can remain active in the cool, dark, moist environment that accompanies land burial of carcasses, but extrapolation of available data suggests virus may persist for months. Therefore, we believe composting is likely to provide an effective, biosecure, economically viable and environmentally compatible option for disposal of PEDv mortalities. This research will validate the effectiveness of composting through controlled mortality composting trials subsequent to experimental infections. With the completion of this research, our expectation is that we will know what operating parameters are required to ensure inactivation of PEDv during composting of PEDv mortalities.
Using the information generated from this research, we will deliver extension programming and outreach materials to swine producers, veterinarians, and stakeholders within and beyond Nebraska to promote biosecure disposal of PEDv-infected mortalities.
Amy Millmier Schmidt, Assistant Professor and Livestock Bioenvironmental Engineer, University of Nebraska - Lincoln firstname.lastname@example.org
J. Dustin Loy, Assistant Professor, Veterinary & Biomedical Sciences, University of Nebraska - Lincoln
Dr. Amy Millmier Schmidt
The authors would like to acknowledge the Nebraska Pork Producers Association and the National Pork Board for providing funding for this research. Special thanks to Jared Korth for helping with laboratory activities on this project and construction of mortality composting platforms.
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