Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing

Kansas Agricultural Experiment Station Research Reports, Nov 2016

Porcine Epidemic Diarrhea virus (PEDV) is a possible hazard in feed mills that could impact pig health. If the virus enters a feed mill, it quickly becomes widely distributed and is difficult to decontaminate from surfaces.6,7 The objective of this study was to evaluate a variety of liquid and dry chemical treatments that could be used as sanitizers to reduce the amount of PEDV found on feed manufacturing surfaces in mills. This experiment was replicated 3 times and was designed in a 5 × 10 factorial with main effects of 5 different feed manufacturing surfaces and 10 sanitizing treatments. Surfaces included stainless steel, plastic, rubber, woven polypropylene tote bag, and sealed concrete coupons (4 × 4 in). One mL (1×105 TCID50/mL) of stock PEDV was applied to each surface and allowed to dry completely for 60 min. Next, a mitigation treatment was applied for 15 min: 1) no sanitation treatment (control); 2) untreated rice hulls; 3) rice hulls treated with formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 4) liquid formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 5) dry commercial benzoic acid and probiotic blend (VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ); 6) liquid ammonium chloride, isopropanol, and hydrogen peroxide-based commercial food-grade sanitizer (DrySan Duo; Ecolab, St. Paul, MN); 7) liquid hydrogen peroxide commercial product (INTERvention; Virox Technologies Inc. Ontario, Canada); 8) liquid quaternary ammonium glutaraldehyde commercial product (Synergize; Preserve International, Reno NV); 9) liquid sodium hypochlorite commercial sanitizer (Bleach; Clorox, Oakland, CA); and 10) liquid medium chain fatty acid blend of caprylic, caproic, and capric acids. There were 3 replicates per treatment. The quantity of PEDV RNA was determined using qRT-PCR. All main effects, interaction, and comparisons were highly significant (P ≤ 0.001). Liquid Sal CURB and liquid bleach were the most effective chemical treatments to reduce the quantity of detectable PEDV RNA, but their application is limited due to their liquid state and potential corrosiveness. Additional research is necessary to identify the role of sanitizer on PEDV infectivity, even if RNA residue remains, and to develop dry sanitizers capable of removing PEDV RNA on swine feed manufacturing surfaces that are not corrosive.

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Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing

Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing M. Muckey 0 S. S. Dritz 0 J. C. Woodworth 0 C. R. Stark 0 0 Kansas State University , Manhattan , USA Part of the Large or Food Animal and Equine Medicine Commons, Other Animal Sciences Commons, and the Veterinary Infectious Diseases Commons Recommended Citation - See next page for additional authors Follow this and additional works at: http://newprairiepress.org/kaesrr 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 January 2016 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. K-State Research and Extension is an equal opportunity provider and employer. Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing Abstract Porcine Epidemic Diarrhea virus (PEDV) is a possible hazard in feed mills that could impact pig health. If the virus enters a feed mill, it quickly becomes widely distributed and is difficult to decontaminate from surfaces.6,7 The objective of this study was to evaluate a variety of liquid and dry chemical treatments that could be used as sanitizers to reduce the amount of PEDV found on feed manufacturing surfaces in mills. This experiment was replicated 3 times and was designed in a 5 × 10 factorial with main effects of 5 different feed manufacturing surfaces and 10 sanitizing treatments. Surfaces included stainless steel, plastic, rubber, woven polypropylene tote bag, and sealed concrete coupons (4 × 4 in). One mL (1×105 TCID50/mL) of stock PEDV was applied to each surface and allowed to dry completely for 60 min. Next, a mitigation treatment was applied for 15 min: 1) no sanitation treatment (control); 2) untreated rice hulls; 3) rice hulls treated with formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 4) liquid formaldehydebased commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 5) dry commercial benzoic acid and probiotic blend (VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ); 6) liquid ammonium chloride, isopropanol, and hydrogen peroxide-based commercial food-grade sanitizer (DrySan Duo; Ecolab, St. Paul, MN); 7) liquid hydrogen peroxide commercial product (INTERvention; Virox Technologies Inc. Ontario, Canada); 8) liquid quaternary ammonium glutaraldehyde commercial product (Synergize; Preserve International, Reno NV); 9) liquid sodium hypochlorite commercial sanitizer (Bleach; Clorox, Oakland, CA); and 10) liquid medium chain fatty acid blend of caprylic, caproic, and capric acids. There were 3 replicates per treatment. The quantity of PEDV RNA was determined using qRT-PCR. All main effects, interaction, and comparisons were highly significant (P ≤ 0.001). Liquid Sal CURB and liquid bleach were the most effective chemical treatments to reduce the quantity of detectable PEDV RNA, but their application is limited due to their liquid state and potential corrosiveness. Additional research is necessary to identify the role of sanitizer on PEDV infectivity, even if RNA residue remains, and to develop dry sanitizers capable of removing PEDV RNA on swine feed manufacturing surfaces that are not corrosive. Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. Cover Page Footnote Appreciation is expressed to the National Pork Board for financial support (awards #15-208); Dr. Dick Hesse and Joe Anderson for technical support and laboratory use, Elizabeth Poulsen, and Rusty Ransbrough for technical support and laboratory use. K 2016 Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing1,2 M.B. Muckey, 3 S.S. Dritz, 4 J.C. Woodworth, C.R. Stark,3 J. Bia,4 J. Zhang,5 P.C. Gauger,5 R.G. Main,5 and C.K. Jones Summary Porcine Epidemic Diarrhea virus (PEDV) is a possible hazard in feed mills that could impact pig health. If the virus enters a feed mill, it quickly becomes widely distributed and is difficult to decontaminate from surfaces. 6,7 The objective of this study was to evaluate a variety of liquid and dry chemical treatments that could be used as sanitiz ers to reduce the amount of PEDV found on feed manufacturing surfaces in mills. This experiment was replicated 3 times and was designed in a 5 × 10 factorial with main effects of 5 different feed manufacturing surfaces and 10 sanitizing treatments. Sur faces included stainless steel, plastic, rubber, woven polypropylene tote bag, and sealed concrete coupons (4 × 4 in). One mL (1×10 5 TCID50/mL) of stock PEDV was applied to each surface and allowed to dry completely for 60 min. Next, a mitigation treat ment was applied for 15 min: 1) no sanitation treatment (control); 2) untreated rice hulls; 3) rice hulls treated with formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 4) liquid formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 5) dry commercial benzoic acid and probiotic blend (VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ); 6) 1 Appreciation is expressed to the National Pork Board for financial support (awards #15-208). 2 Appreciation is expressed to Dr. Dick Hesse and Joe Anderson for technical support and laboratory use, Elizabeth Poulsen, and Rusty Ransbrough for technical support and laboratory use. 3 Department of Grain Science and Industry, Kansas State University. 4 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University. 5 Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA. 6 Schumacher, L.L., R.A. Cochrane, C.E. Evans, J.R. Kalivoda, J.C. Woodworth, C.R. Stark, C.K. Jones, Q. Chen, R.G. Main, J. Zhang, P.C. Gauger, S.S. Dritz, and M.D. Tokach. 2016. Evaluating the effect of manufacturing porcine epidemic diarrhea virus (PEDV)-contaminated feed on subsequent feed mill environmental surface contamination. J. Anim. Sci. 99(E2)164. 7 Bowman, A. S., Nolting, J. M., Nelson, S. W., Bliss, N., Stull, J. W., Wang, Q., and Premanandan, C. 2015. Effects of disinfection on the molecular detection of porcine epidemic diarrhea virus. Veterinary Microbiology, 179(3), 213-218. liquid ammonium chloride, isopropanol, and hydrogen peroxide-based commercial food-grade sanitizer (DrySan Duo; Ecolab, St. Paul, MN); 7) liquid hydrogen peroxide commercial product (INTERvention; Virox Technologies Inc. Ontario, Canada); 8) liquid quaternary ammonium glutaraldehyde commercial product (Synergize; Preserve International, Reno NV); 9) liquid sodium hypochlorite commercial sanitizer (Bleach; Clorox, Oakland, CA); and 10) liquid medium chain fatty acid blend of caprylic, caproic, and capric acids. There were 3 replicates per treatment. The quantity of PEDV RNA was determined using qRT-PCR. All main effects, interaction, and comparisons were highly significant (P ≤ 0.001). Liquid Sal CURB and liquid bleach were the most effective chemical treatments to reduce the quantity of detectable PEDV RNA, but their application is limited due to their liquid state and potential corrosiveness. Addi tional research is necessary to identify the role of sanitizer on PEDV infectivity, even if RNA residue remains, and to develop dry sanitizers capable of removing PEDV RNA on swine feed manufacturing surfaces that are not corrosive. Introduction The swine feed mill may be a potential vector for Porcine Epidemic Diarrhea virus (PEDV) transmission into swine herds. 8,9,10 Recent studies have demonstrated the potential for PEDV to be introduced to the feed mill through ingredients, vehicles, and employees.11 Regardless of the method of entry, viral contamination becomes wide spread once within the manufacturing environment due to dust contamination. 6,12 There are limited options to decontaminate feed mills once viral RNA has become established. Thermal processing inactivates the virus at 130 °F.13 However, it does not prevent re-contamination from PEDV-contaminated dust or residue on feed manu facturing equipment surfaces prior to loadout. Chemical sanitizers are typically used 8 Schumacher, L.L., Cochrane, R.A., Evans, C.E., Kalivoda, J.R., Woodworth, J.C., Stark, C.R., Jones, C.K., Main, R.G., Zhang, J., Dritz, S.S. and Gauger, P.C., 2015. Evaluating the Effect of Manufacturing Porcine Epidemic Diarrhea Virus (PEDV)-Contaminated Feed on Subsequent Feed Mill Environmental Surface Contamination. Kansas Agricultural Experiment Station Research Reports, 1(7), p. 4. 9 Greiner, Laura L. 2016. Evaluation of the likelihood of detection of porcine epidemic diarrhea virus or porcine delta coronavirus ribonucleic acid in areas within feed mills. Journal of Swine Health and Production. 24.4 198-204. 10 Pasick, J., Berhane, Y., Ojkic, D., Maxie, G., Embury ‐Hyatt, C., Swekla, K., and Alexandersen, S. 2014. Investigation into the Role of Potentially Contaminated Feed as a Source of the First ‐Detected Out breaks of Porcine Epidemic Diarrhea in Canada. Transboundary and emerging diseases, 61(5), 397-410. 11 Cochrane, R.A., Dritz, S.S., Woodworth, J.C., Stark, C.R., Huss, A.R., Cano, J.P., Thompson, R.W., Fahrenholz, A.C. and Jones, C.K., 2016. Feed mill biosecurity plans: A systematic approach to prevent biological pathogens in swine feed. Journal of Swine Health and Production 24.3: 154-164. 12 Gebhardt J. T., Woodworth J. C., Jones C. K., Gauger P. C., Tokach, M. D., DeRouchey J. M., Good band, R. D., Muckey M., Cochrane R. A., Stark C. R., Bai J., Chen Q., Zhang J., Ramirez A., Derscheid R. J., Main R. G., and Dritz S. S. 2016. Evaluation of the effects of flushing feed manufacturing equipment with chemically treated rice hulls on likelihood of porcine epidemic diarrhea virus (PEDV) transmission by swine feed and feed manufacturing equipment. In Kansas State University Swine Day 2016. Kansas Agricultural Experiment Station Research Reports. 13 Cochrane, R. A., L. L. Schumacher, S. S. Dritz, J. C. Woodworth, A. R. Huss, C. R. Stark, J. M. DeR ouchey, M. D. Tokach, R. D. Goodband, J. Bai, Q. Chen, Jianqiang Zhang, P. C. Gauger, R. G. Main, and C. K. Jones. 2015. Effect of Thermal Mitigation on Porcine Epidemic Diarrhea Virus (PEDV)-Contam inated Feed. Kansas State University Swine Day 2015. Kansas Agricultural Experiment Station Research Reports. Vol. 1: Iss. 7. for such purposes in human food manufacturing and have shown some promise on reducing PEDV RNA on trailer surfaces. Current industry practices include the use of heat, sodium hypochloride, or quaternary ammonium/glutaraldehyde combinations to sanitize swine farm surfaces contaminated with PEDV. However, there is limited infor mation regarding their success on reducing viral RNA on feed manufacturing surfaces. Even if there were successful options, there may be limited application of liquid sanitiz ers due to the inherent dry nature of ingredients and feed. The introduction of water, even in the form of a liquid sanitizer, may actually increase the quantity of other biolog ical hazards if they are not targeted by the sanitizer. Furthermore, ideal sanitizers would be safe for use in both the animal feed and on the equipment surface. The objective of this study was to evaluate the ability of a variety of liquid and dry chemical sanitizers to reduce the quantity of detectable PEDV RNA. Procedures The experimental treatments were arranged as a 5 × 10 factorial with 5 different feed manufacturing surfaces and 10 chemical treatments. Each combination was replicated 3 times. Surfaces included: 1) stainless steel (stainless steel type 316; Built-So-Well Manhattan, KS); 2) plastic (Dura Bucket National Oats Co. Collinsville, Ill.); 3) rub ber (Maxi-Lift Inc. Addison, TX); 4) woven polypropylene tote bag (The MegaSack Corp. Magnolia, AR); and 5) sealed concrete (Quikrete Co. Atlanta, GA). Chemical treatments included: 1) no sanitation treatment (control); 2) untreated rice hulls; 3) rice hulls treated with formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA; 30% formaldehyde and 10% propionic acid/methanol blend); 4) liquid formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 5) dry commercial benzoic acid and probiotic blend (VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ; 96% benzoic acid and 4% probiotic blend); 6) liquid commercial food-grade sanitizer (DrySan Duo; Ecolab, St. Paul, MN; 10.98% isopropyl alcohol, 0.045% hydrogen peroxide, 0.016% alkyl dimethyl benzyl ammonium chloride, 0.007% dodecyl dimethyl ammonium chloride, and 0.005% dioctyl dimethyl ammonium chloride); 7) 3% dilution of liquid hydrogen peroxide commercial product (INTERvention; Virox Technologies Inc. Ontario, Canada; 4.25% hydrogen peroxide); 8) 0.39% dilution of liquid quaternary ammonium glutaral dehyde commercial product (Synergize; Preserve International, Reno NV; 26.0% alkyl dimethyl benzyl ammonium chloride and 7% glutaraldehyde); 9) 10% dilution of liquid sodium hypochlorite commercial sanitizer (Bleach; The Chlorox Company, Oakland, CA; 5 to 10% sodium hypochlorite); and 10) liquid medium chain fatty acid blend of caprylic, caproic, and capric acids (1:1:1 custom blend 11). A 4 × 4 in. coupon of each surface was prepared, inoculated, and treated with chemical as previously described. 7 Briefly, surfaces were sanitized, rinsed, and autoclaved. Next, 1 mL of PEDV (USA/IN/2013/19338; 1×105 TCID50/ml) was applied to the surfaces and spread using cell spreader to cover the entire area. Surfaces were allowed to dry for 60 min. After drying of PEDV, respective treatment was applied to coupon surface for 15 min. Surfaces were then swabbed to determine residual PEDV contamination using premoistened environmental swabs in 5 mL of neutralizing broth (World Bioproducts LLC., Mundelein, IL). Swabs were vortexed and PEDV was quantified using qRTPCR. Results were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC). A preplanned contrast included the comparison of dry vs. liquid chemical treatments. Significance was considered at P ≤ 0.05 and marginally significant from P > 0.05 to P ≤ 0.10. Results and Discussion All main effects and interactions were highly significant ( P ≤ 0.001; Table 1 and 2). Rubber belting obtained from a bucket elevator retained the most PEDV RNA of any tested surface, while the polyethylene tote bag retained the least ( P < 0.05; 28.0 vs. 31.4 CT for rubber vs. tote bag, respectively). Concentrated liquid Sal CURB was the most effective sanitizer at removing PEDV RNA across surfaces, followed by liquid bleach ( P < 0.05; 42.9 vs. 35.2 CT for Sal CURB vs. bleach, respectively). The liquid Sal CURB prevented detection of PEDV RNA (> 45 CT) on plastic, polyethylene tote bag, rub ber, and stainless steel. Cement still contained residual PEDV RNA, even after liquid formaldehyde application, but the sanitizer was still more effective than other treat ments (P < 0.05; 36.7 CT). Liquid bleach was most effective at reducing PEDV RNA on the polyethylene tote bag (43.0 CT), followed by stainless steel, rubber, and plastic (37.1, 35.6, and 35.0 CT, respectively). Liquid bleach was least effective on cement ( 0.05; 25.4 CT). All other sanitizers did not influence the detection of PEDV RNA on any surface compared to that detected on the untreated control ( P > 0.05). Due to the performance of liquid Sal CURB and liquid bleach, liquid sanitizers were substantially more effective at reducing the quantity of detectable PEDV RNA compared to dry sanitizers ( P < 0.05). P < In summary, liquid Sal CURB and liquid bleach were the most effective chemical treat ments to reduce the quantity of detectable PEDV RNA, but their application is limited due to their liquid state and potential corrosiveness. Additional research is necessary to identify the role of sanitizer on PEDV infectivity, even if RNA residue remains, and to develop dry sanitizers capable of removing PEDV RNA on swine feed manufacturing surfaces that are not corrosive. Kansas State University Agricultural Experiment Station and Cooperative Extension Service 3% dilution of liquid hydrogen peroxide commercial product 5 0.39% dilution of liquid quaternary ammonium/glutaraldehyde commercial product 6 10% dilution of liquid sodium hypochlorite commercial sanitizer 7 Concentrated liquid medium chain fatty acid blend 8 P = Surface Treatment SEM Surface × treatment Dry vs. liquid treatment Surface Treatment Surface × treatment PEDV, CT 30.0ab 28.5bc 31.4a 28.0c 28.9bc 26.2c 26.7c 26.2c 42.9a 27.9c 26.2c 26.5c 28.4c 35.2b 27.4c 1 This experiment was conducted in a 5 × 10 factorial with 3 replicates per treatment. 2 Sal CURB; Kemin Inc., Des Moines, IA; 30% formaldehyde and 10% propionic acid/methanol blend. 3 VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ; 96% benzoic acid and 4% probiotic blend. 4 DrySan Duo; Ecolab, St. Paul, MN; 10.98% isopropyl alcohol, 0.045% hydrogen peroxide, 0.016% alkyl dimethyl benzyl ammonium chloride, 0.007% dodecyl dimethyl ammonium chloride, and 0.005% dioctyl dimethyl am monium chloride. 5 INTERvention; Virox Technologies Inc. Ontario, Canada; 4.25% hydrogen peroxide. 6 Synergize; Preserve International, Reno NV; 26.0% alkyl dimethyl benzyl ammonium chloride and 7% glutaral dehyde. 7 Bleach; The Chlorox Company, Oakland, CA; 5 to 10% sodium hypochlorite. 8 Caprylic, caproic, and capric acids in 1:1:1 custom blend described by Cochrane et al., 2015, 2016. abc Means with different superscripts differ (P < 0.05). Chemical treatment Untreated control Untreated rice hulls Commercial formaldehyde-treated rice hulls (2 kg/ton) 2 Concentrated commercial formal dehyde2 Concentrated dry commercial benzoic acid and probiotic blend Ready-to-use liquid commercial food-grade sanitizer 4 3% dilution of liquid hydrogen peroxide commercial product 5 0.39% dilution of liquid quater nary ammonium/glutaraldehyde commercial product 6 10% dilution of liquid sodium hypochlorite commercial sanitizer 7 Concentrated liquid medium chain fatty acid blend 8 P = SEM 0.001 1.91 Stainless steel 24.6ij 24.5ij 24.5ij 45.0a 26.3ghij 26.0ghij 27.2fghij 27.3fghij 37.1b 26 . 0ghij 1 This experiment was conducted in a 5 × 10 factorial with 3 replicates per treatment. 2 Sal CURB; Kemin Inc ., Des Moines , IA; 30% formaldehyde and 10% propionic acid/methanol blend. 3 VevoVitall and CRINA; DSM Nutritional Products Inc ., Parsippany, NJ; 96% benzoic acid and 4% probiotic blend. 4 DrySan Duo; Ecolab, St . Paul, MN; 10 .98% isopropyl alcohol, 0 .045% hydrogen peroxide, 0 .016% alkyl dimethyl benzyl ammonium chloride, 0.007% dodecyl dimethyl ammonium chloride, and 0.005% dioctyl dimethyl ammonium chloride. 5 INTERvention; Virox Technologies Inc . Ontario, Canada; 4 .25% hydrogen peroxide. 6 Synergize; Preserve International, Reno NV ; 26 . 0% alkyl dimethyl benzyl ammonium chloride and 7% glutaraldehyde. 7 Bleach; The Chlorox Company , Oakland, CA; 5 to 10% sodium hypochlorite. 8 Caprylic, caproic and capric acids in 1:1:1 custom blend described by Cochrane et al ., 2015 , 2016 . abcdefghijkl Means with different superscripts differ (P < 0 .05).


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M. Muckey, S. S. Dritz, J. C. Woodworth, C. R. Stark, J. Bai, Jianqiang Zhang, Phillip Charles Gauger, Rodger G. Main, C. K. Jones. Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing, Kansas Agricultural Experiment Station Research Reports, 2016,