Evaluating the Impact of VevoVitall and/or CRINA as Potential Porcine Epidemic Diarrhea Virus Mitigation Strategies as Determined by Polymerase Chain Reaction Analysis and Bioassay

Feed and feed ingredients have been shown to be potential vectors of porcine epidemic diarrhea virus (PEDV). Potential strategies to mitigate the risk of disease transmission via feed and feed ingredients would be valuable to the swine and feed milling industries. Therefore, the objective of this experiment was to determine the impact of VevoVitall (5,000 ppm; DSM Nutritional Products Inc., Parsipanny, NJ), CRINA (200 ppm; DSM Nutritional Products Inc., Parsipanny, NJ), and a combination of both products (COMBINATION; 5,000 ppm VevoVitall and 200 ppm CRINA) as feed additives with potential to mitigate the risk of PEDV, in swine gestation diet (FEED) and spraydried porcine plasma (SDPP) as determined by real time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyzed at seven sampling days post laboratory inoculation (d 0, 1, 3, 7, 14, 21, and 42) and bioassay. There was a marginally significant treatment × feed matrix × day interaction (P = 0.082), in which the cycle threshold (Ct) value increased over time in the diet when treated with the COMBINATION, whereas, there was no increase over time observed in SDPP. There was a highly significant (P \u3c 0.001) feed matrix × day interaction in which the Ct increased over time in FEED, whereas, there was very little increase over time observed in SDPP. Additionally, there was a marginally significant treatment × feed matrix interaction (P = 0.079). Overall, the COMBINATION was most effective at reducing the quantity of genetic material as detected by qRT-PCR (P \u3c 0.001). Virus shedding was observed in the d 7 post-inoculation SDPP COMBINATION treatment, as well as d 0 FEED COMBINATION treatment. No other treatment bioassay room had detectable RNA shed and detected in fecal swabs or cecal contents (d 1, 3, 7, 14, and 21 post-laboratory inoculation FEED, COMBINATION). In summary, the combination of CRINA and VevoVitall enhanced degradation of PEDV RNA in swine feed, but had no impact on RNA degradation in SDPP. Furthermore, both untreated feed and feed treated with the combination of CRINA and VevoVitall caused infection at d 0 post-laboratory inoculation; however, neither set of samples was infective at d 1 post-laboratory inoculation

Evaluation of the Effects of Flushing Feed Manufacturing Equipment with Chemically- Treated Rice Hulls on Porcine Epidemic Diarrhea Virus Cross Contamination During Feed Manufacturing

Various strategies have been proposed to mitigate potential risk of porcine epidemic diarrhea virus (PEDV) transmission via feed and feed ingredients. Wet decontamination has been found to be the most effective decontamination of feed mill surfaces; however, this is not practical on a commercial feed production-scale. Another potential mitigation strategy, easier to implement, would be using chemically-treated rice hulls flushed through the feed manufacturing equipment. The objective of this experiment was to determine the impact of MCFA- or formaldehyde-treated rice hull flush batches as potential PEDV mitigation strategies during feed manufacturing. Feed without evidence of PEDV RNA contamination was inoculated with PEDV. Based on PCR analysis, this feed had a Ct = 30.2 and was confirmed infective in bioassay. After manufacture of PEDV positive feed, untreated rice hulls, or rice hulls treated with Sal CURB, 2%, or 10% medium chain fatty acid blend (MCFA; 1:1:1 ratio of caproic, caprylic, and capric acid) were flushed through laboratory-scale mixers. For the untreated rice hulls, 3 of 6 samples had detectable PEDV RNA (avg. Ct = 41.4) while 1 of 6 Sal CURB treated rice hull flush samples and 2 of 6 of the 2% MCFA rice hull flush samples had detectable PEDV RNA. However, PEDV RNA was not detected in any of the 10% MCFA rice hull flush samples. Additionally, rice hulls treated with 10% MCFA were mixed and discharged through a production-scale mixer and bucket elevator following manufacturing of PEDV positive feed. In the production-scale system, no rice hull flush or feed samples from the mixer following chemically-treated rice hull flush had detectable PEDV RNA. However, one 10% MCFA rice hull sample collected from the bucket elevator discharge spout had detectable PEDV RNA. Dust collected following mixing of PEDV-contaminated feed had a large quantity of PEDV RNA (avg. Ct = 29.4). Dust collected immediately after the 10% MCFA rice hull flush batch had a reduced quantity of PEDV RNA (Ct = 33.7), and the subsequent feed following the 10% rice hull flush had no detectable PEDV RNA. Pigs inoculated with dust collected after manufacturing PEDV-positive feed were shedding PEDV RNA by 2 dpi and continued to have detectable RNA until necropsy. Dust collected from the 10% MCFA rice hull flush batch or the subsequent batch was not infective. Overall, the use of rice hull flushes effectively reduced the quantity of detectable RNA present after mixing a batch of PEDV-positive feed. Chemical treatment of rice hulls with Sal CURB and 10% MCFA provided additional reduction in detectable RNA present in the rice hull flush samples. Finally, dust collected after manufacturing PEDVinoculated feed contains a very high quantity of viral RNA and was found infective, demonstrating it has the potential to serve as a vector for PEDV transmission

Understanding the Reduction of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 RNA in Inoculated Feed and the Environment Following Thermal Processing

Pelleting of feed has been demonstrated to be an effective mitigation strategy for porcine epidemic diarrhea virus (PEDV) contaminated feed but has not been evaluated for other endemic swine viruses like porcine reproductive and respiratory syndrome virus (PRRSV) or Seneca Valley virus 1 (SVV1). Therefore, the objective of this experiment was to evaluate the efficacy of pelleting to inactivate PEDV, PRRSV, and SVV1 inoculated feed. Ten replicates were conducted in the Cargill Feed Safety Research Center at Kansas State University (K-State) using a pilot scale mixer, bucket elevator, pellet mill (including conditioner and die), and cooler. First, a virus negative batch of gestation feed was run through all equipment to simulate a commercial feed mill, then a positive batch of feed inoculated with all three viruses was run through all feed manufacturing equipment. Feed was conditioned to a minimum of 180°F with a 30 sec retention time; all feed was cooled for 10 min. Feed and environmental samples were taken from each piece of equipment following both the negative and positive batch. Samples were analyzed via PCR at the K-State Veterinary Diagnostic Laboratory. A four-room bioassay was conducted to evaluate the infectivity of the feed samples. Feed from the mixer and bucket elevator had greater quantities of SVV1, PEDV, and PRRSV RNA (P \u3c 0.05) than the other sampling locations. Similarly, environmental samples from the mixer and bucket elevator had greater SVV1 detection (P \u3c 0.05) than those collected from the conditioner, pellet die, and cooler. Pelleting reduced viral RNA (P \u3c 0.05) for all viruses in both feed and environmental samples. Although SVV1 and PEDV RNA were still detectable following pelleting, no pigs inoculated with the pelleted feed showed signs of SVV1 or PEDV clinical infection. Interestingly, PRRSV RNA was not detectable in pelleted feed samples. However, one pig showed signs of replicating PRRSV virus on d 7 of the bioassay which suggests a greater sensitivity when utilizing a bioassay compared to PCR alone. Overall, pelleting reduced the quantity of detectable viral RNA and reduced the risk of infectivity; yet small quantities of viral RNA remaining in the feed and environment following pelleting may increase the risk of re-contamination

Evaluating the Distribution of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 Inoculated Feed After the Use of Physical or Chemical Mitigants to Flush a Feed Manufacturing Facility

Contaminated feed is a route of virus transmission between feed mills and swine farms. To reduce the risk of transmission, an understanding of the virus distribution and mitigation strategies are needed. The objective of this study was to evaluate the distribution of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca Valley virus 1 (SVV1) inoculated feed in the environment and feed of a feed mill before and after the use of chemical mitigants. A 50-lb batch of feed was run through a mixer and bucket elevator followed by a batch inoculated with PEDV, PRRSV, and SVV1. Following the virus-inoculated batch, a flush treatment of either 1) ground corn (GC); 2) GC + 1.5% liquid formaldehyde (LF; SalCURB LF Liquid, Kemin, Des Moines, IA); 3) GC + 1.5% LF + 25% abrasive material (SalCURB; Shell & Bone Builder, Iowa Limestone Company, Urbandale, IA); 4) double flush – GC + 25% abrasive material followed by GC +1.5% LF (Shell & Bone Builder; SalCURB); or 5) dry formaldehyde (SalCURB F2 Dry, Kemin, Des Moines, IA) was utilized, followed by 3 virus-free batches of complete feed. Feed and environmental samples were collected from each piece of equipment following every batch. Dust samples were collected after manufacturing from the inoculated, flush, and final batches from non-feed contact surfaces. Non-feed contact surfaces were considered those where dust would accumulate during manufacturing but would not be included in the final diet. The surfaces included the grates of the mixer, the top of the discharge bin following the bucket elevator, and the floor surrounding the same discharge bin. Samples were analyzed via a triplex PCR at the Kansas State University Veterinary Diagnostic Laboratory. A treatment × batch × location interaction was not observed (P \u3e 0.05) in feed or the environment for any of the viruses. A flush treatment × batch interaction was observed for SVV1 where greater quantities of viral RNA (P \u3c 0.05) were present in the positive batches and the ground corn flush than in those batches which used chemical mitigants or the post-flush batches. A lower quantity of viral RNA (P\u3c 0.05) in dust was observed in the last batch of feed compared to the inoculated batch for all viruses; however, SVV1 RNA was still detectable in the dust following the last batch in all treatments. A batch effect (P \u3c 0.05) was observed in all sample matrices for PEDV and PRRSV as viral RNA decreased after the implementation of the flush regardless of treatment. The use of chemical mitigants and the implementation of a flush batch reduced the quantity of viral RNA for PEDV, PRRSV, and SVV1. However, viral presence was still observed in feed and the dust on non-feed contact surfaces which could be a source of contamination if re-introduced into finished feed