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.

An experiment was conducted to evaluate the effect of dietary medium-chain fatty acid (MCFA) addition on nursery pig growth performance, fecal microbial composition, and mitigation of porcine epidemic diarrhea virus (PEDV) following storage. A total of 360 pigs (DNA 400 × 200, Columbus, NE; initially 6.7 ± 0.07 kg) were randomized to pens (5 pigs per pen) on the day of weaning (approximately 20 d of age), allowed a 6-d acclimation, blocked by BW, and randomized to dietary treatment (9 pens per treatment). All MCFA (Sigma-Aldrich, St. Louis, MO) were guaranteed ≥98% purity, including hexanoic (C6:0), octanoic (C8:0), and decanoic (C10:0) acids. Treatment diets were formulated in 2 phases (7 to 11 and 11 to 23 kg BW) and formulated to meet or exceed NRC requirement estimates. Treatments (n = 8) were a dose response including 0%, 0.25%, 0.5%, 1.0%, and 1.5% added MCFA blend (1:1:1 ratio C6:0, C8:0, and C10:0), as well as treatments with individual additions of 0.5% C6:0, C8:0, or C10:0. Fecal samples were collected from pigs fed control and 1.5% MCFA blend diets on days 0 and 14 and analyzed using 16s rDNA sequencing. Following feed manufacture, feed was stored in bags at barn temperature and humidity for 40 d before laboratory inoculation with PEDV. Subsamples of retained feed were inoculated with PEDV to achieve a titer of 104 TCID50/g and separate sample bottles were analyzed on 0 and 3 d post-inoculation (dpi). Overall, ADG and ADFI were increased (linear, P ≤ 0.010) and feed efficiency (G:F) improved (linear, P = 0.004) with increasing MCFA blend. Pigs fed 0.5% C8:0 had greater (P = 0.038) ADG compared with pigs fed the control diet, and G:F was improved (P ≤ 0.024) when pigs were fed 0.5% C6:0, 0.5% C8:0, or 0.5% C10:0 compared with control. An inclusion level × day interaction was observed (quadratic, P = 0.023), where PEDV Ct values increased (quadratic, P = 0.001) on 0 dpi with increasing levels of MCFA blend inclusion and also increased on 3 dpi (linear, P < 0.001). Fecal microbial diversity and composition were similar between control and 1.5% MCFA blend. In summary, the use of MCFA in nursery pig diets improves growth performance, provides residual mitigation activity against PEDV, and does not significantly alter fecal microbial composition.

Assessing the effects of medium-chain fatty acids and fat sources on PEDV infectivity.

The overall objective of this study was to compare the efficacy of medium-chain fatty acids (MCFA) to other common fat sources to minimize the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination in a pig bioassay. Treatments were feed with mitigants inoculated with PEDV after application and were: 1) positive control with no chemical treatment; 2) 0.325% commercially available formaldehyde-based product; 3) 1% blend of 1:1:1 caproic (C6), caprylic (C8), and capric acids (C10) and applied with an aerosolizing nozzle; 4) treatment 3 applied directly into the mixer without an aerosolizing nozzle; 5) 0.66% caproic acid; 6) 0.66% caprylic acid; 7) 0.66% capric acid; 8) 0.66% lauric acid; 9) 1% blend of 1:1 capric and lauric acids; 10) 0.3% commercially available dry C12 product; 11) 1% canola oil; 12) 1% choice white grease; 13) 2% coconut oil; 14) 1% coconut oil; 15) 2% palm kernel oil; 16) 1% palm kernel oil; 17) 1% soy oil and four analysis days (0, 1, 3, and 7 post inoculation) as well as 1 treatment of PEDV-negative feed without chemical treatment. There was a treatment × day interaction (P < 0.002) for detectable PEDV RNA. The magnitude of the increase in Ct value from d 0 to 7 was dependent upon the individual treatments. Feed treated with individual MCFA, 1% MCFA blend, or commercial-based formaldehyde had fewer (P < 0.05) detectable viral particles than all other treatments. Commercial-based formaldehyde, 1% MCFA, 0.66% caproic, 0.66% caprylic, and 0.66% capric acids had no evidence of infectivity 10-d old pig bioassay, while there was no evidence the C12 commercial product or longer chain fat sources inhibited PEDV infectivity. Interestingly, pigs given the coconut oil source with the highest composition of caprylic and capric only showed signs of infectivity on the last day of bioassay. These data suggest some MCFA have potential for reducing post feed manufacture PEDV contamination.

Effects of medium chain fatty acids as a mitigation or prevention strategy against porcine epidemic diarrhea virus in swine feed.

Feed has been shown to be a vector for viral transmission. Four experiments were conducted to: 1) determine if medium chain fatty acids (MCFA) are effective mitigants when applied to feed both pre- and post-porcine epidemic diarrhea virus (PEDV) inoculation measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), 2) evaluate varying levels and combinations of MCFA measured by qRT-PCR, and 3) evaluate selected treatments in bioassay to determine infectivity. In exp. 1, treatments were arranged in a 2 × 2 + 1 factorial with main effects of treatment (0.3% commercial formaldehyde [CF] product, Sal CURB [Kemin Industries, Inc.; Des Moines, IA], or 1% MCFA blend (Blend) of 1:1:1 C6:C8:C10 [PMI, Arden Hills, MN]) and timing of application (pre- or post-inoculation with PEDV) plus a positive control (PC; feed inoculated with PEDV and no treatment). All combinations of treatment and timing decreased detectable PEDV compared with the PC (P < 0.05). Pre-inoculation treatment elicited decreased magnitude of PEDV detection (cycle threshold value) compared with post-inoculation (P = 0.009). Magnitude of PEDV detection was decreased for CF compared with Blend (P < 0.0001). In exp. 2, pre-inoculation treatments consisted of: 1) PC, 2) 0.3% CF, 3 to 5) 0.125% to 0.33% C6:0, 6 to 8) 0.125% to 0.33% C8:0, 9 to 11) 0.125% to 0.33% C10:0, and 12 to 15) 0.125% to 0.66% C5:0. Treating feed with 0.33% C8:0 resulted in decreased (P < 0.05) PEDV detection compared with all other treatments. Increasing concentration of each individual MCFA decreased PEDV detectability (P < 0.042). In exp. 3, pre-inoculation treatments consisted of: 1) PC, 2) 0.3% CF, 3 to 7) 0.25% to 1% Blend, 8 to 10) 0.125% to 0.33% C6:0 + C8:0, 11 to 13) 0.125% to 0.33% C6:0 + C10:0, and 14 to 16) 0.125% to 0.33% C8:0 + C10:0. Treating feed with CF, 0.5% Blend, 0.75% Blend, 1% Blend, all levels of C6:0+C8:0, 0.25% C6:0 + 0.25% C10:0, 0.33% C6:0 + 0.33% C10:0, 0.25% C8:0 + 0.25% C10:0, or 0.33% C8:0 + 0.33% C10:0 elicited decreased detection of PEDV compared with PC (P < 0.05). Increasing concentration of each MCFA combination decreased PEDV detectability (linear, P < 0.012). In exp. 4, feed was treated pre-inoculation with: 1) no treatment (PC), 2) 0.3% CF, 3) 0.5% Blend, or 4) 0.3% C8:0 and analyzed via qRT-PCR and bioassay. Adding 0.5% Blend or 0.3% C8:0 resulted in decreased PEDV compared with PC and only PC resulted in a positive bioassay. Therefore, MCFA can decrease detection of PEDV in feed. Further, inclusion of lower levels of MCFA than previously evaluated are effective against PEDV.

Feed batch sequencing to decrease the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination during feed manufacturing.

Feed has been identified as a vector of transmission for porcine epidemic diarrhea virus (PEDV). The objective of this study was to determine if feed batch sequencing methods could minimize PEDV cross-contamination. Porcine epidemic diarrhea virus-free swine feed was manufactured to represent the negative control. A 50 kg feed batch was mixed in a pilot scale feed mill for 5 min, sampled, then discharged for 10 min into a bucket elevator and sampled again upon exit. Next, a pathogenic PEDV isolate was used to inoculate 49.5 kg of PEDV-free feed to form the positive control. The positive control was mixed, conveyed and sampled similar to the negative control. Subsequently, 4 sequence batches (sequence 1 to 4) were formed by adding a 50 kg batch of PEDV-negative feed to the mixer after the prior batch was mixed and conveyed; all sequences were mixed, conveyed, and sampled similar to the negative and positive control batches. None of the equipment was cleaned between batches within a replicate. This entire process was replicated 3 times with cleaning the feed mill between replicates. Feed was then analyzed for PEDV RNA by real-time reverse transcriptase semiquantitative polymerase chain reaction (rRT-PCR) as measured by cycle threshold (Ct) and for infectivity by bioassay. Sequence 1 feed had higher (P ˂ 0.05) rRT-PCR Ct values than the positive batch and sequence 2 feed had higher (P ˂ 0.05) Ct values than sequence 1, regardless of sampled location. Feed sampled from the mixer from sequence 2, 3, and 4 was rRT-PCR negative whereas feed sampled from the bucket elevator was rRT-PCR negative from sequence 3 and 4. Bioassay was conducted using 66 mixed sex 10-d-old pigs confirmed negative for PEDV allocated to 22 different rooms. Pigs were initially 10-d old. Control pigs remained PEDV negative for the study. All pigs from the mixer positive batch (9/9) and bucket elevator positive batch (3/3) were rRT-PCR positive on fecal swabs by the end of the study. One replicate of pigs from mixer sequence 1 was rRT-PCR positive (3/3) by 7 dpi. One replicate of mixer pigs from sequence 2 was rRT-PCR positive (3/3) by 7 dpi although no detectable PEDV RNA was found in the feed. The results demonstrate sequenced batches had reduced quantities of PEDV RNA although sequenced feed without detectible PEDV RNA by rRT-PCR can be infectious. Therefore, a sequencing protocol can reduce but not eliminate the risk of producing infectious PEDV carryover from the first sequenced batch of feed.

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 disinfection 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 would be using chemically treated rice hulls flushed through the feed manufacturing equipment. Therefore, the objective of this study was to determine the effects of medium-chain fatty acids (MCFA) or formaldehyde-treated rice hull flush batches as potential chemical mitigation strategies for PEDV during feed manufacturing. Feed without evidence of PEDV RNA contamination was inoculated with PEDV. Based on polymerase chain reaction analysis, this feed had a cycle threshold (Ct) = 30.2 and was confirmed infective in bioassay. After manufacturing the PEDV-positive feed, untreated rice hulls, formaldehyde-treated rice hulls, 2% MCFA- (a 1:1:1 blend of hexanoic, octanoic, and decanoic acid) treated rice hulls, or 10% MCFA-treated rice hulls were flushed through laboratory scale mixers. For the untreated rice hulls, 3 of 6 samples had detectable PEDV RNA, whereas 1 of 6 formaldehyde-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. Then, rice hulls treated with 10% MCFA were mixed and discharged through a production scale mixer and bucket elevator following PEDV-positive feed. No rice hull flush or feed samples from the mixer following chemically treated rice hull flush had detectible PEDV RNA. However, one 10% MCFA rice hull sample collected from the bucket elevator discharge spout had detectible PEDV RNA. Dust collected following mixing of PEDV contaminated feed had detectable PEDV RNA (Ct = 29.4) and was infectious. However, dust collected immediately after the 10% MCFA rice hull flush batch had a reduced quantity of PEDV RNA (Ct = 33.7) and did not cause infection. Overall, the use of rice hull flushes effectively reduced the quantity of detectible RNA present after mixing a batch of PEDV-positive feed. Chemical treatment of rice hulls with formaldehyde or 10% MCFA provided additional reduction in detectible RNA. Finally, dust collected after manufacturing PEDV-inoculated feed has the potential to serve as a vector for PEDV transmission.

Elimination of Porcine Epidemic Diarrhea Virus in an Animal Feed Manufacturing Facility.

Porcine Epidemic Diarrhea Virus (PEDV) was the first virus of wide scale concern to be linked to possible transmission by livestock feed or ingredients. Measures to exclude pathogens, prevent cross-contamination, and actively reduce the pathogenic load of feed and ingredients are being developed. However, research thus far has focused on the role of chemicals or thermal treatment to reduce the RNA in the actual feedstuffs, and has not addressed potential residual contamination within the manufacturing facility that may lead to continuous contamination of finished feeds. The purpose of this experiment was to evaluate the use of a standardized protocol to sanitize an animal feed manufacturing facility contaminated with PEDV. Environmental swabs were collected throughout the facility during the manufacturing of a swine diet inoculated with PEDV. To monitor facility contamination of the virus, swabs were collected at: 1) baseline prior to inoculation, 2) after production of the inoculated feed, 3) after application of a quaternary ammonium-glutaraldehyde blend cleaner, 4) after application of a sodium hypochlorite sanitizing solution, and 5) after facility heat-up to 60°C for 48 hours. Decontamination step, surface, type, zone and their interactions were all found to impact the quantity of detectable PEDV RNA (P < 0.05). As expected, all samples collected from equipment surfaces contained PEDV RNA after production of the contaminated feed. Additionally, the majority of samples collected from non-direct feed contact surfaces were also positive for PEDV RNA after the production of the contaminated feed, emphasizing the potential role dust plays in cross-contamination of pathogen throughout a manufacturing facility. Application of the cleaner, sanitizer, and heat were effective at reducing PEDV genomic material (P < 0.05), but did not completely eliminate it.

Characterizing the rapid spread of porcine epidemic diarrhea virus (PEDV) through an animal food manufacturing facility.

New regulatory and consumer demands highlight the importance of animal feed as a part of our national food safety system. Porcine epidemic diarrhea virus (PEDV) is the first viral pathogen confirmed to be widely transmissible in animal food. Because the potential for viral contamination in animal food is not well characterized, the objectives of this study were to 1) observe the magnitude of virus contamination in an animal food manufacturing facility, and 2) investigate a proposed method, feed sequencing, to decrease virus decontamination on animal food-contact surfaces. A U.S. virulent PEDV isolate was used to inoculate 50 kg swine feed, which was mixed, conveyed, and discharged into bags using pilot-scale feed manufacturing equipment. Surfaces were swabbed and analyzed for the presence of PEDV RNA by quantitative real-time polymerase chain reaction (qPCR). Environmental swabs indicated complete contamination of animal food-contact surfaces (0/40 vs. 48/48, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05) and near complete contamination of non-animal food-contact surfaces (0/24 vs. 16/18, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05). Flushing animal food-contact surfaces with low-risk feed is commonly used to reduce cross-contamination in animal feed manufacturing. Thus, four subsequent 50 kg batches of virus-free swine feed were manufactured using the same system to test its impact on decontaminating animal food-contact surfaces. Even after 4 subsequent sequences, animal food-contact surfaces retained viral RNA (28/33 positive samples/total samples), with conveying system being more contaminated than the mixer. A bioassay to test infectivity of dust from animal food-contact surfaces failed to produce infectivity. This study demonstrates the potential widespread viral contamination of surfaces in an animal food manufacturing facility and the difficulty of removing contamination using conventional feed sequencing, which underscores the importance for preventing viruses from entering and contaminating such facilities.

Evaluating Chemical Mitigation of Salmonella Typhimurium ATCC 14028 in Animal Feed Ingredients.

Salmonella Typhimurium is a potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella spp. during rendering is effective but does not eliminate the potential for cross-contamination. Therefore, the objective of this experiment was to evaluate the effectiveness of chemicals to mitigate postrendering Salmonella Typhimurium ATCC 14028 contamination in rendered proteins over time. Treatments were arranged in a 6 × 4 factorial with six chemical treatments and four rendered protein meals. The chemical treatments included (i) control without chemical treatment, (ii) 0.3% commercial formaldehyde product, (iii) 2% essential oil blend, (iv) 2% medium chain fatty acid blend, (v) 3% organic acid blend, and (vi) 1% sodium bisulfate. The four rendered protein meals included (i) feather meal, (ii) blood meal, (iii) meat and bone meal, and (iv) poultry by-product meal. After matrices were chemically treated, they were inoculated with Salmonella Typhimurium ATCC 14028, stored at room temperature, and enumerated via plate counts on days 0, 1, 3, 7, 14, 21, and 42 postinoculation. The Salmonella concentration in ingredients treated with medium chain fatty acid and commercial formaldehyde were similar to one another (P = 0.23) but were 2 log lower than the control (P < 0.05). Ingredients treated with organic acids and essential oils also had lower Salmonella concentrations than the control (P < 0.05). Time also played a significant role in Salmonella mitigation, because all days except days 14 and 21 (P = 0.92) differed from one another. Rendered protein matrix also affected Salmonella stability, because concentrations in meat and bone meal and blood meal were similar to one another (P = 0.36) but were greater than levels in feather meal and poultry by-product meal (P < 0.05). In summary, chemical treatment and time both mitigated Salmonella Typhimurium ATCC 14028, but their effectiveness was matrix dependent. Time and chemical treatment with medium chain fatty acids or a commercial formaldehyde product were most effective at mitigating Salmonella Typhimurium ATCC 14028 in rendered protein meals.

Evaluation of a Biological Pathogen Decontamination Protocol for Animal Feed Mills.

Animal feed and ingredients are potential vectors of pathogenic bacteria. Contaminated ingredients can contaminate facility equipment, leading to cross-contamination of other products. This experiment was conducted to evaluate a standardized protocol for decontamination of an animal feed manufacturing facility using Enterococcus faecium (ATCC 31282) as an indicator. A pelleted swine diet inoculated with E. faecium was manufactured, and environmental samples (swabs, replicate organism detection and counting plates, and air samples) were collected (i) before inoculation (baseline data), (ii) after production of inoculated feed, (iii) after physical removal of organic material using pressurized air, (iv) after application of a chemical sanitizer containing a quaternary ammonium-glutaraldehyde blend, (v) after application of a chemical sanitizer containing sodium hypochlorite, (vi) after facility heat-up to 60 8 C for 24 h, (vii) for 48 h, and (viii) for 72 h. Air samples collected outside the facility confirmed pathogen containment; E. faecium levels were equal to or lower than baseline levels at each sample location. The decontamination step and its associated interactions were the only variables that affected E. faecium incidence (P < 0.0001 versus P > 0.22). After production of the inoculated diet, 85.7% of environmental samples were positive for E. faecium. Physical cleaning of equipment had no effect on contamination (P = 0.32). Chemical cleaning with a quaternary ammonium-glutaraldehyde blend and sodium hypochlorite each significantly reduced E. faecium contamination (P < 0.0001) to 28.6 and 2.4% of tested surfaces, respectively. All samples were negative for E. faecium after 48 h of heating. Both wet chemical cleaning and facility heating but not physical cleaning resulted in substantial E. faecium decontamination. These results confirmed both successful containment and decontamination of biological pathogens in the tested pilot-scale feed mill.