Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P \u3c 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P \u3c 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Anderson, Joe W.

Bai, Jianfa

Dritz, Steve S.

Elijah, C. Grace

Gebhardt, Jordan T.

Jones, Cassandra K.

Nichols, Gage E.

Paulk, Chad B.

Poulsen Porter, Elizabeth G.

Singrey, Aaron

Woodworth, Jason C.

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P= 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P\u3c0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P\u3c0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Kansas State University

Kansas Agricultural Experiment Station Research Reports Volume 7 Issue 11 Swine Day Article 49 2021 Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed C. Grace Elijah Kansas State University, cgelijah@k-state.edu Gage E. Nichols Kansas State University, gage500@k-state.edu Jordan T. Gebhardt Kansas State University, jgebhardt@k-state.edu See next page for additional authors This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2021 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Follow this and additional works at: https://newprairiepress.org/kaesrr  Part of the Other Animal Sciences Commons Recommended Citation Elijah, C. Grace; Nichols, Gage E.; Gebhardt, Jordan T.; Jones, Cassandra K.; Woodworth, Jason C.; Dritz, Steve S.; Bai, Jianfa; Anderson, Joe W.; Poulsen Porter, Elizabeth G.; Singrey, Aaron; and Paulk, Chad B. (2021) "Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed," Kansas Agricultural Experiment Station Research Reports: Vol. 7: Iss. 11. https://doi.org/10.4148/2378-5977.8213 Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed Abstract Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants. Keywords Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, medium chain fatty acids, formaldehyde Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. Authors C. Grace Elijah, Gage E. Nichols, Jordan T. Gebhardt, Cassandra K. Jones, Jason C. Woodworth, Steve S. Dritz, Jianfa Bai, Joe W. Anderson, Elizabeth G. Poulsen Porter, Aaron Singrey, and Chad B. Paulk This section 4. feed safety research is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol7/iss11/49 1Kansas State University Agricultural Experiment Station and Cooperative Extension Service2021 ANIMAL Feed AND  PET FoOD RESEARCHFeed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in FeedC. Grace Elijah,1 Gage E. Nichols, Jordan T. Gebhardt,1 Cassandra K. Jones,2 Jason C. Woodworth,2 Steve S. Dritz,1 Jianfa Bai,1 Joe W. Anderson,1 Elizabeth G. Poulsen Porter,1 Aaron Singrey,3 and Chad B. Paulk Summary Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quan-titative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants. 1  Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University.2  Department of Animal Sciences and Industry, College of Agriculture, Kansas State University.3  Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD.22021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceIntroductionThe suspected mode of entry for PEDV into the United States during the 2013 and 2014 outbreak was in contaminated feed ingredients.4 Consequently, this taught us that feed biosecurity should be enhanced as part of standard farm biosecurity practices. While most of the work regarding feed as a potential vector for viral disease trans-mission has focused on PEDV, research has demonstrated that other viruses such as PRRSV, foot and mouth disease, and other foreign animal diseases could survive in feed under transatlantic shipping conditions.5 These findings highlight that while foreign trade can potentially transmit novel viruses across geographical borders, these findings also stress the importance of applying biosecurity to the feed supply chain to control the spread of endemic viruses like PRRSV and PEDV. In general, if contaminated feed is a concern for any swine production system, there are multiple solutions to this problem but the most common is use of feed additives to reduce virus viability. These feed additives have been shown to be successful with mitigating risk in feed experimentally inoculated with PEDV,6 but there is a lack of data concerning feed mitigant efficacy in the presence of more than one virus in feed. Therefore, the objective of this study was to evaluate the efficacy of two feed additives to increase Ct values of feed when inoculated with PEDV or PRRSV or inoculated with both of these viruses. Materials and Methods GeneralA corn and soybean meal-based swine gestation diet was used. No evidence for contam-ination was observed in the test diet for PEDV and PRRSV using quantitative real time reverse transcription PCR (qRT-PCR) prior to the study. The treatments for this study included feed inoculated with PEDV alone, PRRSV alone, or co-inoculated with both viruses; and feed additives included no treatment, 0.33% commercial formalde-hyde-based product (Sal Curb, Kemin Industries, Des Moines, IA), or 0.50% MCFA blend (MCFA; 1:1:1 ratio of C6:C8:C10, Sigma Aldrich, St. Louis, MO). Chemical treatments were added to 100-g batches of feed and mixed for 15 min in a mason jar mixer (Central Machine Shop, Purdue University, West Lafayette, IN). For treatments consisting of a single virus inoculate, 22.5 g of treated feed was placed into three sepa-rate polyethylene bottles (250 mL Nalgene bottle, square wide-mouth high-density polyethylene; Thermo Fisher Scientific, Waltham, MA). For treatments containing the co-inoculate, 20 g of feed was placed into three separate 250 mL polyethylene bottles to ensure equal virus titer levels for the individual virus across all treatments. All treat-ments had three replicates for this study. Polyethylene bottles were stored at room temperature for 24 h before inoculation. 4  USDA-APHIS. United States Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services (2015). Swine enteric coronavirus introduction to the United States: Root cause investigation report. Accessed February 24, 2021.5  Dee, S. A., Bauermann, F. V., Niederwerder, M. C., Singrey, A., Clement, T., de Lima, M., ... & Petrovan, V. (2018). Survival of viral pathogens in animal feed ingredients under transboundary shipping models. PloS ONE, 13(3). doi:10.1371/journal.pone.0194509.6  Gebhardt, J.T., J.C. Woodworth, M.D. Tokach, J.M. DeRouchey, R.D. Goodband, C.K. Jones, and S.S. Dritz. 2020. Effect of dietary medium-chain fatty acids on nursery pig growth performance, fecal microbial composition, and mitigation properties against porcine epidemic diarrhea virus following storage. J. Anim. Sci. 98(1):1-11. doi:10.1093/jas/skz358. 32021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceInoculationThe samples were inoculated at the Kansas State University Veterinary Diagnostic Laboratory (KSVDL), Manhattan, KS, with PEDV, PRRSV, or both viruses with an initial concentration of 107 TCID50/mL. All treatments were inoculated by pipetting 2.5 mL of each viral inoculum into each bottle as required for the treatment to result in a final viral concentration of 106 TCID50/g of feed. Bottles were then shaken for 15 s to distribute the virus through the feed. Laboratory analysisBottles were held at room temperature for 24 h after inoculation. Then, 100 mL of phosphate buffered saline (PBS) was added to each inoculated bottle and shaken to ensure even mixing. Bottles were placed in a refrigerator at 39°F for 24 h to allow feed to settle. Supernatant was collected and placed into a 96 well plate for qRT-PCR. Quantitative real time reverse transcription PCR was conducted using methods previ-ously described by Gebhardt et al.7 Briefly, supernatant was extracted using a Kingfisher 96 magnetic particle processor (Fisher Scientific, Pittsburgh, PA) and the MagMAX-96 Viral RNA Isolation kit (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions with one modification, reducing the final elution volume to 60 μL. One negative extraction control consisting of all reagents except the sample was included in each extraction. Samples inoculated with PEDV or PRRSV alone were analyzed using a qRT-PCR assay designed for PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed by the KSVDL to independently detect both PEDV and PRRSV nucleic acid within a single reaction. All responses from the qRT-PCR reactions were reported in Ct value. Statistical analysisStatistical analysis for this study was performed using the PROC GLIMMIX procedure in SAS (v. 9.4, SAS Institute, Inc., Cary, NC). This study was a completely randomized design with the effect of mitigant type (none, formaldehyde, and MCFA) analyzed for each inoculation type (PEDV alone, PRRSV alone, or both viruses). The experimental unit was each 250 mL polyethylene container. If inoculated samples had no detect-able RNA, a value of 45.0 was assigned to the sample for statistical analysis. A Tukey multiple comparison adjustment was incorporated when appropriate. Results were considered significant at P ≤ 0.05 and marginally significant at P ≤ 0.10.Results and DiscussionFor PEDV alone, there was marginally significant evidence that feed additives resulted in differences in Ct value (P = 0.052; Table 1), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed.7  Gebhardt, J. T., J. C. Woodworth, C. K. Jones, M. D. Tokach, P. C. Gauger, R. G. Main, J. Zhang, Q. Chen, J. M. DeRouchey, R. D. Goodband, C. R. Stark, J. R. Bergstrom, J. Bai, and S. S. Dritz. 2018. Determining the impact of commercial feed additives as potential porcine epidemic diarrhea virus mitigation strategies as determined by polymerase chain reaction analysis and bioassay. Transl. Anim. Sci. 3:93-102. doi: 10.1093/tas/txy100. 42021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceIn summary, formaldehyde effectively increased Ct values in feed when contaminated with PRRSV alone, while both mitigants effectively increased Ct values in feed when co-inoculated with PRRSV and PEDV. Both mitigants were marginally effective in feed when contaminated with PEDV alone. This study also supplied evidence that the co-inoculation model can effectively evaluate mitigants. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. Persons using such products assume responsibility for their use in accordance with current label directions of the manufacturer.Table 1. Cycle threshold values for PEDV and PRRSV when inoculated either alone or together following application of feed additives1Virus AssayMitigantSEM P =Untreated MCFA2 Formaldehyde3PEDV PEDV 31.2 33.5 34.2 0.69 0.052PRRSV PRRSV 30.0b 34.2b 42.0a 1.78 0.009Co-inoculatedPEDV quantification Duplex 30.8b 31.9a 32.5a 0.23 0.006PRRSV quantification Duplex 30.0b 33.8a 34.7a 0.85 0.0191 Swine feed samples were inoculated with 106 TCID50/g of PEDV, PRRSV, or PEDV and PRRSV co-inoculation then analyzed using one of three quantitative real time reverse transcription polymerase chain reaction assays, including an assay detecting PEDV only, an assay detecting PRRSV only, or an assay detecting and independently reporting quantification of both PEDV and PRRSV (duplex).2 Medium chain fatty acid blend (MCFA; 1:1:1 ratio of C6:C8:C10; Sigma Aldrich, St. Louis, MO) at 0.50% inclusion.3 Commercial formaldehyde-based feed additive (SalCURB; Kemin Industries, Des Moines, IA) at 0.33% inclusion.a,b,c Means within row lacking common superscripts differ, P < 0.05.

Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P\u3c0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P\u3c0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Kansas Agricultural Experiment Station Research Reports Volume 7 Issue 10 Animal Feed and Pet Food Research Report Article 11 2021 Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed C. Grace Elijah Kansas State University, cgelijah@k-state.edu Gage E. Nichols Kansas State University, gage500@ksu.edu Jordan T. Gebhardt Kansas State University, Manhattan, jgebhardt@k-state.edu See next page for additional authors This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2021 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Follow this and additional works at: https://newprairiepress.org/kaesrr  Part of the Other Animal Sciences Commons, and the Veterinary Infectious Diseases Commons Recommended Citation Elijah, C. Grace; Nichols, Gage E.; Gebhardt, Jordan T.; Jones, Cassandra K.; Woodworth, Jason C.; Dritz, Steve S.; Bai, Jianfa; Anderson, Joe W.; Poulsen Porter, Elizabeth G.; Singrey, Aaron; and Paulk, Chad B. (2021) "Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed," Kansas Agricultural Experiment Station Research Reports: Vol. 7: Iss. 10. https://doi.org/10.4148/2378-5977.8149 Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed Abstract Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants. Keywords Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, Medium Chain Fatty Acids, Formaldehyde Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. Authors C. Grace Elijah, Gage E. Nichols, Jordan T. Gebhardt, Cassandra K. Jones, Jason C. Woodworth, Steve S. Dritz, Jianfa Bai, Joe W. Anderson, Elizabeth G. Poulsen Porter, Aaron Singrey, and Chad B. Paulk This feed safety research is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol7/iss10/11 1Kansas State University Agricultural Experiment Station and Cooperative Extension Service2021 ANIMAL Feed AND  PET FoOD RESEARCHFeed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in FeedC. Grace Elijah,1 Gage E. Nichols, Jordan T. Gebhardt,1 Cassandra K. Jones,2 Jason C. Woodworth,2 Steve S. Dritz,1 Jianfa Bai,1 Joe W. Anderson,1 Elizabeth G. Poulsen Porter,1 Aaron Singrey,3 and Chad B. Paulk Summary Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quan-titative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants. 1  Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University.2  Department of Animal Sciences and Industry, College of Agriculture, Kansas State University.3  Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD.22021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceIntroductionThe suspected mode of entry for PEDV into the United States during the 2013 and 2014 outbreak was in contaminated feed ingredients.4 Consequently, this taught us that feed biosecurity should be enhanced as part of standard farm biosecurity practices. While most of the work regarding feed as a potential vector for viral disease trans-mission has focused on PEDV, research has demonstrated that other viruses such as PRRSV, foot and mouth disease, and other foreign animal diseases could survive in feed under transatlantic shipping conditions.5 These findings highlight that while foreign trade can potentially transmit novel viruses across geographical borders, these findings also stress the importance of applying biosecurity to the feed supply chain to control the spread of endemic viruses like PRRSV and PEDV. In general, if contaminated feed is a concern for any swine production system, there are multiple solutions to this problem but the most common is use of feed additives to reduce virus viability. These feed additives have been shown to be successful with mitigating risk in feed experimentally inoculated with PEDV,6 but there is a lack of data concerning feed mitigant efficacy in the presence of more than one virus in feed. Therefore, the objective of this study was to evaluate the efficacy of two feed additives to increase Ct values of feed when inoculated with PEDV or PRRSV or inoculated with both of these viruses. Materials and Methods GeneralA corn and soybean meal-based swine gestation diet was used. No evidence for contam-ination was observed in the test diet for PEDV and PRRSV using quantitative real time reverse transcription PCR (qRT-PCR) prior to the study. The treatments for this study included feed inoculated with PEDV alone, PRRSV alone, or co-inoculated with both viruses; and feed additives included no treatment, 0.33% commercial formalde-hyde-based product (Sal Curb, Kemin Industries, Des Moines, IA), or 0.50% MCFA blend (MCFA; 1:1:1 ratio of C6:C8:C10, Sigma Aldrich, St. Louis, MO). Chemical treatments were added to 100-g batches of feed and mixed for 15 min in a mason jar mixer (Central Machine Shop, Purdue University, West Lafayette, IN). For treatments consisting of a single virus inoculate, 22.5 g of treated feed was placed into three sepa-rate polyethylene bottles (250 mL Nalgene bottle, square wide-mouth high-density polyethylene; Thermo Fisher Scientific, Waltham, MA). For treatments containing the co-inoculate, 20 g of feed was placed into three separate 250 mL polyethylene bottles to ensure equal virus titer levels for the individual virus across all treatments. All treat-ments had three replicates for this study. Polyethylene bottles were stored at room temperature for 24 h before inoculation. 4  USDA-APHIS. United States Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services (2015). Swine enteric coronavirus introduction to the United States: Root cause investigation report. Accessed February 24, 2021.5  Dee, S. A., Bauermann, F. V., Niederwerder, M. C., Singrey, A., Clement, T., de Lima, M., ... & Petrovan, V. (2018). Survival of viral pathogens in animal feed ingredients under transboundary shipping models. PloS ONE, 13(3). doi:10.1371/journal.pone.0194509.6  Gebhardt, J.T., J.C. Woodworth, M.D. Tokach, J.M. DeRouchey, R.D. Goodband, C.K. Jones, and S.S. Dritz. 2020. Effect of dietary medium-chain fatty acids on nursery pig growth performance, fecal microbial composition, and mitigation properties against porcine epidemic diarrhea virus following storage. J. Anim. Sci. 98(1):1-11. doi:10.1093/jas/skz358. 32021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceInoculationThe samples were inoculated at the Kansas State University Veterinary Diagnostic Laboratory (KSVDL), Manhattan, KS, with PEDV, PRRSV, or both viruses with an initial concentration of 107 TCID50/mL. All treatments were inoculated by pipetting 2.5 mL of each viral inoculum into each bottle as required for the treatment to result in a final viral concentration of 106 TCID50/g of feed. Bottles were then shaken for 15 s to distribute the virus through the feed. Laboratory analysisBottles were held at room temperature for 24 h after inoculation. Then, 100 mL of phosphate buffered saline (PBS) was added to each inoculated bottle and shaken to ensure even mixing. Bottles were placed in a refrigerator at 39°F for 24 h to allow feed to settle. Supernatant was collected and placed into a 96 well plate for qRT-PCR. Quantitative real time reverse transcription PCR was conducted using methods previ-ously described by Gebhardt et al.7 Briefly, supernatant was extracted using a Kingfisher 96 magnetic particle processor (Fisher Scientific, Pittsburgh, PA) and the MagMAX-96 Viral RNA Isolation kit (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions with one modification, reducing the final elution volume to 60 μL. One negative extraction control consisting of all reagents except the sample was included in each extraction. Samples inoculated with PEDV or PRRSV alone were analyzed using a qRT-PCR assay designed for PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed by the KSVDL to independently detect both PEDV and PRRSV nucleic acid within a single reaction. All responses from the qRT-PCR reactions were reported in Ct value. Statistical analysisStatistical analysis for this study was performed using the PROC GLIMMIX procedure in SAS (v. 9.4, SAS Institute, Inc., Cary, NC). This study was a completely randomized design with the effect of mitigant type (none, formaldehyde, and MCFA) analyzed for each inoculation type (PEDV alone, PRRSV alone, or both viruses). The experimental unit was each 250 mL polyethylene container. If inoculated samples had no detect-able RNA, a value of 45.0 was assigned to the sample for statistical analysis. A Tukey multiple comparison adjustment was incorporated when appropriate. Results were considered significant at P ≤ 0.05 and marginally significant at P ≤ 0.10.Results and DiscussionFor PEDV alone, there was marginally significant evidence that feed additives resulted in differences in Ct value (P = 0.052; Table 1), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed.7  Gebhardt, J. T., J. C. Woodworth, C. K. Jones, M. D. Tokach, P. C. Gauger, R. G. Main, J. Zhang, Q. Chen, J. M. DeRouchey, R. D. Goodband, C. R. Stark, J. R. Bergstrom, J. Bai, and S. S. Dritz. 2018. Determining the impact of commercial feed additives as potential porcine epidemic diarrhea virus mitigation strategies as determined by polymerase chain reaction analysis and bioassay. Transl. Anim. Sci. 3:93-102. doi: 10.1093/tas/txy100. 42021 ANIMAL Feed AND PET FOOD RESEARCHKansas State University Agricultural Experiment Station and Cooperative Extension ServiceIn summary, formaldehyde effectively increased Ct values in feed when contaminated with PRRSV alone, while both mitigants effectively increased Ct values in feed when co-inoculated with PRRSV and PEDV. Both mitigants were marginally effective in feed when contaminated with PEDV alone. This study also supplied evidence that the co-inoculation model can effectively evaluate mitigants. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. Persons using such products assume responsibility for their use in accordance with current label directions of the manufacturer.Table 1. Cycle threshold values for PEDV and PRRSV when inoculated either alone or together following application of feed additives1Virus AssayMitigantSEM P =Untreated MCFA2 Formaldehyde3PEDV PEDV 31.2 33.5 34.2 0.69 0.052PRRSV PRRSV 30.0b 34.2b 42.0a 1.78 0.009Co-inoculatedPEDV quantification Duplex 30.8b 31.9a 32.5a 0.23 0.006PRRSV quantification Duplex 30.0b 33.8a 34.7a 0.85 0.0191 Swine feed samples were inoculated with 106 TCID50/g of PEDV, PRRSV, or PEDV and PRRSV co-inoculation then analyzed using one of three quantitative real time reverse transcription polymerase chain reaction assays, including an assay detecting PEDV only, an assay detecting PRRSV only, or an assay detecting and independently reporting quantification of both PEDV and PRRSV (duplex).2 Medium chain fatty acid blend (MCFA; 1:1:1 ratio of C6:C8:C10; Sigma Aldrich, St. Louis, MO) at 0.50% inclusion.3 Commercial formaldehyde-based feed additive (SalCURB; Kemin Industries, Des Moines, IA) at 0.33% inclusion.a,b,c Means within row lacking common superscripts differ, P < 0.05.

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Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

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