The purpose of this project was to determine the appropriate time-temperature combinations required for inactivation of the porcine epidemic diarrhea virus (PEDv) in composting material as a basis for evaluation of composting for disposal of swine mortalities and/or other PEDv-positive biological material.
What did we do?
In vitro propagation of PEDv for laboratory survivability assays was conducted using a cell culture-adapted isolate received from APHIS-NVSL (Ames, IA) that was free of extraneous agents (5th passage Colorado 2013 PEDv 1303). Propagation was conducted by infection of confluent VERO cell monolayers at a multiplicity of infection (MOI) of 0.1 with a concentration of 5 µg/mL TPCK trypsin. Virus stocks were be amplified following a 2-4 day incubation period on cell monolayers, frozen and thawed, centrifuged, and culture supernatants containing virus were harvested. Virus concentration was calculated and standardized to 1×105-1×106 TCID50/mL using immunocytochemistry and indirect fluorescent antibody assay (IFA) using a PEDV specific mouse monoclonal antibody (MedGene Labs).
The effect of temperature on survivability of PEDv in compost material was evaluated by inoculating compost material and subjecting the material to temperatures of 50°C (122°F), 55°C (131°F), 60°C (140°F), 65°C (149°F), and 70°C (158°F) for 0, 24, 48, 72, 96 h, and 120 h. Sawdust was acquired from a commercial source, autoclaved to eliminate existing microbes, oven dried and used to simulate compost material. One gram of prepared sawdust was placed in each of 140 1-mL centrifuge tubes. Cell culture supernatant containing infectious PEDv was added to phosphate buffered saline and added to each tube achieve a moisture content of 50% w.b. Tubes were randomly assigned to laboratory incubators at the five temperature treatment levels. At each sampling point, four tubes were removed from each incubator and tested to determine virus survivability.
PEDv survivability was determined via two independent assay methods. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and sensitive method that was used by the Nebraska Veterinary Diagnostic Center to quantify the amount of virus RNA genome in the samples. To validate results from the RT-qPCR in laboratory assays, sawdust simulated compost matrix was spiked with known concentrations of PEDv target RNA and compared to known standards to ensure no inhibition was present and that proper extraction methods were being used. An alternative method using virus isolation was also conducted to determine whether viable virus was present in tubes at a smaller subset of time points. To do this, Vero cell monolayers were infected with filter sterilized aliquots of compost exudate, blindly passaged once after seven days, and examined for virus presence using IFA with a PED specific monoclonal antibody. At specific time points, RT-qPCR Ct values and Virus Isolation were run in parallel to ensure sensitivity of testing and to evaluate correlation of the testing modalities under the simulated testing conditions and matrices.
What have we learned?
At the time of proceedings submission, results were not available for inclusion in this report. Results will be presented during the scheduled oral seminar at the conference.
Results of this laboratory study will be used to evaluate appropriate time-temperature combinations necessary during swine mortality composting to inactivate the PEDv virus and determine the feasibility of on-farm mortality composting as a biosecure disposal method for PEDv-infected pigs. Following this laboratory study, mortality composting was initiated using PEDv-positive piglets to confirm the inactivation of PEDv during composting.
Results of this and the full-scale composting study will be used to recommend appropriate swine mortality disposal methods for swine producers with losses due to PEDv as part of their farm biosecurity plan. Additional swine enteric corornaviruses will likely be studied to confirm similar requirements for disposal of mortalities caused by these viruses.
Amy Millmier Schmidt, Assistant Professor and Livestock Bioenvironmental Engineer, University of Nebraska – Lincoln firstname.lastname@example.org
J. Dustin Loy, Assistant Professor, Clayton Kelling, Professor, Judith Galeota, Virology Laboratory Manager, and Sarah Vitosh, Graduate Research Assistant, 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.
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