A. R. Huss

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.

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.

Effect of pelleting on survival of porcine epidemic diarrhea virus-contaminated feed

Abstract Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that has devastated the U.S. swine industry. Because of its heat sensitivity, we hypothesized that a steam conditioner and pellet mill mimicking traditional commercial thermal processing may mitigate PEDV infectivity. Pelleting, a common feed processing method, includes the use of steam and shear forces, resulting in increased temperature of the processed feed. Two thermal processing experiments were designed to determine if different pellet mill conditioner retention times and temperatures would impact PEDV quantity and infectivity by analysis of quantitative reverse transcription PCR and bioassay. In Exp. 1, a 3 · 3 · 2 factorial design was used with 3 pelleting temperatures (68.3, 79.4, and 90.6°C), 3 conditioning times (45, 90, or 180 s), and 2 doses of viral inoculation (low, 1 · 102 tissue culture infectious dose50 (the concentration used to see cytopathic effect in 50% of the cells)/g, or high, 1 · 104 tissue culture infectious dose50/g). Noninoculated and PEDV-inoculated unprocessed mash were used as controls. The low-dose PEDV–infected mash had 6.8 ± 1.8 cycle threshold (Ct) greater (P < 0.05) PEDV than the high-dose mash. Regardless of time or temperature, pelleting reduced (P < 0.05) the quantity of detectable viral PEDV RNA compared with the PEDV-inoculated unprocessed mash. Fecal swabs from pigs inoculated with the PEDV-positive unprocessed mash, regardless of dose, were clinically PEDV positive from 2 to 7 d (end of the trial) after inoculation. However, if either PEDV dose of inoculated feed was pelleted at any of the 9 tested conditioning time · temperature combinations, no PEDV RNA was detected in fecal swabs or cecum content. Based on Exp. 1 results, a second experiment was developed to determine the impact of lower processing temperatures on PEDV quantity and infectivity. In Exp. 2, PEDV-inoculated feed was pelleted at 1 of 5 conditioning temperatures (37.8, 46.1, 54.4, 62.8, and 71.1°C) for 30 s. The 5 increasing processing temperatures led to feed with respective mean Ct values of 32.5, 34.6, 37.0, 36.5, and 36.7, respectively. All samples had detectable PEDV RNA. However, infectivity was detected by bioassay only in pigs from the 37.8 and 46.1°C conditioning temperatures. Experiment 2 results suggest conditioning and pelleting temperatures above 54.4°C could be effective in reducing the quantity and infectivity of PEDV in swine feed. However, additional research is needed to prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step.

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.

Effect of Thermal Mitigation on Porcine Epidemic Diarrhea Virus (PEDV)- Contaminated Feed

Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. However, epidemiological evidence has shown that swine feed and ingredients may serve as potential vectors of transmission. Since it is known that PEDV is a heat-sensitive virus, we hypothesized that a conditioner and pellet mill mimicking commercial thermal processing would mitigate PEDV infectivity. To test this hypothesis, two experiments were designed to determine if different pellet mill conditioner retention times or temperatures would impact PEDV infectivity determined by polymerase chain reaction (PCR) analysis and bioassay. For the first study, a 3×3×2 factorial was utilized, with three pelleting temperatures (155, 175, or 195°F), three conditioning times (45, 90, or 180 s), and two levels of virus (low: 1×102 TCID50/g, or high: 1×104 TCID50/g). Non-inoculated and PEDV-inoculated unprocessed mash were used as controls. There was no PEDV RNA detected in the PEDV-free mash. The low-dose PEDV-infected mash was 6.8 ± 1.8 cycle threshold (Ct) greater (P < 0.01) than the high dose mash. Regardless of time or temperature, feed processing increased (P < 0.01) the Ct compared to the PEDV-inoculated unprocessed mash. As expected, fecal shedding of PEDV was not detected in rectal swabs from control pigs for the duration of the study. Fecal swabs from pigs fed the PEDV-inoculated unprocessed mash, regardless of dose, were PEDV-positive from 2 to 7 days post-inoculation, at which time the pigs were sacrificed. However, if either PEDV dose of inoculated feed was pelleted at any of the nine tested conditioning time × temperature combinations, no PEDV RNA was detected in fecal swabs or cecum content. Based on these results, a second experiment was developed to determine the impact of lower processing temperatures on PEDV infectivity. The pellet mill was heated for 1 hour at normal manufacturing conditions prior to simulating a plug by turning off the steam supply. This allowed the temperature of the mash feed to decrease below 100°F. The PEDV-inoculated feed was then pelleted at one of five conditioning temperatures (100, 115, 130, 145, or 160°F) for 30 s. This study was repeated three times on three separate days with complete decontamination between each experiment day. Again, non-inoculated and PEDV-inoculated mash were used as controls. The five increasing temperatures led to feed with respective mean Ct values of 32.5, 34.6, 37.0, 36.5, and 36.7. Even though all samples had detectable PEDV RNA in the feed, infectivity was only detected by bioassay in pigs from the 100 and 115°F conditioning treatments. In each of the other processing temperatures, no PEDV RNA was detected in fecal swabs or cecum contents. Our results suggest that processing feed through a conditioner and pellet mill similar to those used in commercial feed mills will be effective as a point-in-time mitigation step for PEDV as long as conditioning temperatures remain above 130°F. Any time feed is processed at temperatures below that level, such as during start-up or when the pellet mill die becomes plugged and the steam is consequently shut off, there is a risk that the feed can act as a vector for transferring infectious PEDV and lead to cross-contamination of post-pelleting handling equipment.

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

Abstract 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.

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.

Mitigation of Salmonella on Pet Food Kibbles by Using Liquid and Powdered 3-Hydroxy-3-Methylbutyric Acid

In recent years, several pet food recalls have been attributed to Salmonella contamination. In addition to the negative impacts on animal health, Salmonella-contaminated pet foods have been linked to infection in humans. With that in mind, the U.S. Food and Drug Administration has set forth a zero-tolerance policy for Salmonella in pet foods. Typically, pet foods are extruded or processed at high temperatures that are sufficient to reduce pathogenic bacteria. However, the possibility for postextrusion contamination still exists. One potential method to reduce the risk of postextrusion contamination of pet foods with Salmonella is through the addition of a chemical additive coating. The objective of this research was to evaluate the ability of β-hydroxy-β-methylbutyrate (HMB), in either free acid (HMBFA) or calcium salt (CaHMB) form, to reduce postextrusion contamination of dry extruded dog kibble with Salmonella. Three trials were conducted with HMBFA and CaHMB coated onto the kibbles at levels of 0, 0.1, 0.3, 0.5, 0.9, and 1.5% (w/w). The coated kibbles were then inoculated with Salmonella enterica subsp. enterica Enteritidis (ATCC 13076), with enumeration done on days 0, 1, 2, 7, and 14 postinoculation. Subsamples on each day were serially diluted, spread plated to xylose lysine deoxycholate agar, and incubated at 37°C for 24 h. Salmonella colonies were then counted and log CFU per gram was calculated. The 1.5% HMBFA reduced counts by 4.9 ± 0.2 log units on day 1, whereas the positive control only decreased 2.2 ± 0.1 log units (P < 0.0001). The 1.5% CaHMB level decreased counts by 7.1 ± 0.04 log units by day 7 compared with the control decrease of 2.1 ± 0.1 log units (P < 0.0001). All HMBFA and CaHMB treatments resulted in the elimination of detectable Salmonella counts by day 14 (P < 0.0001 versus controls). In conclusion, HMB coating was effective at reducing Salmonella artificially inoculated to dog kibbles in a model of postextrusion contamination.

Assessing the Effects of Medium Chain Fatty Acids and Fat Sources on Porcine Epidemic Diarrhea Virus Viral RNA Stability and Infectivity

Research has confirmed that chemical treatments, such as medium chain fatty acids (MCFA) and commercial formaldehyde, can be effective to reduce the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination in feed. However, the efficacy of individual MCFA levels are unknown. The objective of this study is to compare the efficacy of commercially-available sources of MCFA and other fat sources versus a synthetic custom blend of MCFA to minimize the risk of PEDV cross-contamination as measured by qRT-PCR and bioassay. Treatments were arranged in a 17 × 4 plus 1 factorial with 17 chemical treatments: 1) Positive control with PEDV and no chemical treatment, 2) 0.3% Sal CURB, 3) 1% medium chain fatty acid blend [caproic, caprylic, and capric acids; 1:1:1] (aerosolized), 4) 1% medium chain fatty acid blend [caproic, caprylic, and capric acids; 1:1:1] (non-aerosolized), 5) 0.66% caproic acid, 6) 0.66% caprylic acid, 7) 0.66% capric acid, 8) 0.66% lauric acid, 9) 1% capric and lauric acid mixture (1:1 ratio), 10) FRA C12, 11) 1% choice white grease, 12) 1% soy oil, 13) 1% canola oil, 14) 2% palm kernel oil, 15) 1% palm kernel oil, 16) 2% coconut oil, and 17) 1% coconut oil; 4 analysis days of 0, 1, 3, and 7 post inoculation; and 1 treatment of PEDV negative, untreated feed. Matrices were first chemically treated, then inoculated with PEDV, and stored at room temperature until being analyzed by qRT-PCR. The analyzed values represent threshold cycle (CT), at which a higher CT value represents less detectable RNA. All main effects and interactions were significant (P < 0.002). The interaction of treatment × day indicated that over time the MCFA treatments, either as a mixture or as individual fatty acids, and Sal CURB had the greatest effect of reducing detectable PEDV RNA, which follows the same trend as the main effect of treatment and the bioassay results. Feed treated with individual synthetic MCFA, MCFA mixture, or Sal CURB had fewer (P < 0.05) detectable viral particles than all other treatments. Day also had a significant impact on quantification of viral RNA, and CT increased from 29.5 to 34.6 CT from day 0 to 7, respectively. In summary, time, Sal CURB, 1% MCFA, 0.66% caproic, 0.66% caprylic, and 0.66% capric acids enhance the RNA degradation of PEDV in swine feed. Notably, the MCFA was equally as successful at mitigating PEDV as a commercially-available formaldehyde product in the complete swine diet at 1% inclusion and as individual fatty acids.

Evaluating the Inclusion Level of Medium Chain Fatty Acids to Reduce the Risk of Porcine Epidemic Diarrhea Virus in Complete Feed and Spray-Dried Animal Plasma

Research has confirmed that chemical treatments, such as medium chain fatty acids (MCFA) and commercial formaldehyde, can be effective to reduce the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination in feed. However, the efficacy of MCFA levels below 2% inclusion is unknown. The objective of this experiment was to evaluate if a 1% inclusion of MCFA is as effective at PEDV mitigation as a 2% inclusion or formaldehyde in swine feed and spray-dried animal plasma (SDAP). Treatments were arranged in a 4 × 2 × 7 plus 2 factorial with 4 chemical treatments: 1) PEDV positive with no chemical treatment, 2) 0.325% commercial formaldehyde, 3) 1% MCFA, and 4) 2% MCFA. The 2 matrices were: 1) complete swine diet and 2) SDAP; with 7 analysis days: 0, 1, 3, 7, 14, 21, and 42 post inoculation; and 1 treatment each of PEDV negative untreated feed and plasma. Matrices were first chemically treated, then inoculated with PEDV, and stored at room temperature until being analyzed by RTqPCR. The analyzed values represent threshold cycle (CT), at which a higher CT value represents less detectable RNA. All main effects and interactions were significant (P < 0.009). Feed treated with MCFA, regardless of inclusion level, had fewer (P < 0.05) detectable viral particles than feed treated with formaldehyde. However, the SDAPtreated with either 1% or 2% MCFA had similar (P > 0.05) concentrations of detectable PEDV RNA as the untreated SDAP, while the SDAP treated with formaldehyde had fewer detectable viral particles (P < 0.05). The complete feed had a lower (P < 0.05) quantity of PEDV RNA than SDAP (39.5 vs. 35.0 for feed vs. SDAP, respectively) (P < 0.05). Analysis day also decreased (P < 0.05) the quantity of detectable viral particles from d 0 to 42, (33.2 vs. 44.0, respectively). In summary, time, formaldehyde, and MCFA all appear to enhance RNA degradation of PEDV in swine feed and ingredients; however, their effectiveness varies within matrix. The 1% inclusion level of MCFA was as effective as 2% in complete feed, but neither were effective at reducing the magnitude of PEDV RNA in SDAP.