R. A. Cochrane

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.

Evaluating Chemical Mitigation of Porcine Epidemic Diarrhea Virus (PEDV) in Swine Feed and Ingredients

Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. Research has confirmed swine feed or ingredients as potential vectors of transmission, so strategies are needed to mitigate PEDV in feed. The objective of this experiment was to evaluate the effectiveness of various chemical additives to prevent or mitigate post-processing PEDV contamination in swine feed and ingredients. Treatments were arranged in a 7 × 4 factorial with seven chemical treatments and four feed matrices. The chemical treatments included: negative control with no chemical addition, 0.3% commercial formaldehyde product, 1% sodium bisulfate, 1% sodium chlorate, 3% custom organic acid blend (OA), 2% custom essential oil blend (EO), and 2% custom medium chain fatty acid blend (MCFA). The four matrices included a complete swine diet, blood meal, meat and bone meal, and spray-dried animal plasma. Matrices were first chemically treated, then inoculated with PEDV, stored at room temperature, and analyzed by RT-PCR on d 0, 1, 3, 7, 14, 21, and 42 post inoculation. Formaldehyde, MCFA, EO, and OA addition each decreased RNA concentration of PEDV compared to the control (P < 0.05), with formaldehyde being the most effective on d 0. Feed matrix appears important in PEDV retention, as RNA concentrations were lower in the swine diet and blood meal than meat and bone meal or spray-dried animal plasma on d 0 (P < 0.05). Additionally, PEDV stability over time was influenced by matrix as RNA concentrations were greater by d 42 for spray-dried animal plasma and meat and bone meal than the complete swine diet and blood meal. In summary, time, formaldehyde, MCFA, EO, and OA all enhance the RNA degradation of PEDV in swine feed and ingredients as measured by RT-PCR, but their effectiveness varies within matrix. Notably, the MCFA was equally as successful at mitigating PEDV as a commercially-available formaldehyde product.

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.

Evaluating the Effect of Manufacturing Porcine Epidemic Diarrhea Virus (PEDV)-Contaminated Feed on Subsequent Feed Mill Environmental Surface Contamination

This study aimed to utilize the only known pilot feed mill facility approved for pathogenic feed agent use in the United States to evaluate the effect of manufacturing Porcine Epidemic Diarrhea Virus (PEDV)contaminated feed on subsequent feed mill environmental surface contamination. In this study, PEDV inoculated feed was manufactured and conveyed on equipment along with four subsequent batches of PEDV-free feed. Equipment and environmental surfaces were sampled using swabs and analyzed for the presence of PEDV RNA by PCR. The experiment was replicated three times with decontamination of the feed mill and all equipment between replications. Overall, environmental swabs indicated widespread surface contamination of the equipment and work area after a PEDV contaminated batch of feed was processed. There was little difference in environmental sample cycle threshold (Ct) values after manufacturing each of the subsequent PEDV-negative feed batches. In summary, introduction of PEDVinfected feed into a feed mill will likely result in widespread contamination of equipment and surfaces, even after several batches of PEDV-free feed are produced. Eliminating the PEDV RNA from the feed mill environment was challenging and required procedures that are not practical to apply on a regular basis in a feed mill. This data suggests that it is extremely important to prevent the introduction of PEDVcontaminated feed, ingredients, or other vectors of transmission to minimize PEDV-risk. More research should be conducted to determine if contaminated surfaces can lead to PEDV infectivity and to determine the best feed mill PEDV-decontamination strategies.

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.

Effects of dietary supplementation of formaldehyde and crystalline amino acids on gut microbial composition of nursery pigs

Formaldehyde-based feed additives are approved in the US for Salmonella control and reducing bacterial contamination in animal feed. However, we hypothesize formaldehyde inclusion in swine diets may influence gut microbial composition due to its antimicrobial properties which might negatively influence microbial populations and pig growth performance. Also, formaldehyde inclusion in diets is known to reduce the dietary availability of amino acids. Therefore, our study was conducted to characterize if the effects of feed formaldehyde-treatment are due to influences on microbial population or diet amino acid (AA) sources. Dietary treatments were arranged in a (2 × 2) + 1 factorial with formaldehyde treatment (none vs. 1000 ppm formaldehyde) and crystalline AA inclusion (low vs. high) with deficient AA content plus a positive control diet to contain adequate AA content without dietary formaldehyde. Treating diets with formaldehyde reduced growth rate (P = 0.001) while the AA inclusion had no evidence of impact. Formaldehyde reduced feed bacterial content and altered fecal microbial communities (P < 0.05). Therefore, we conclude that the negative influence on growth was due to the impact on the fecal microbial community. Implications are that strategies for feed pathogen control need to take into account potential negative impacts on the gut microbial community.

Animal Feed Mill Biosecurity: Prevention of Biological Hazards

Abstract To maintain the sustainable production of animal agriculture to feed the growing human population, animal feed manufacturers must use a variety of feed ingredients. These ingredients can be derived from plant- and animal-based sources, and both can present challenges regarding contamination with biological hazards. Biological hazards in animal feed can be categorized as transmissible spongiform encephalopathies and microbiological contaminants, with a more comprehensive draft list published by the FDA in 2006. More recently, feed has been identified as a vector for the transmission of viral particles, specifically porcine epidemic diarrhea virus. With the majority of animal feed being produced at commercial feed mills, these facilities play an important role in preventing the introduction of biological hazards from contaminating finished feeds. Although the complete elimination of biological hazards in finished animal feeds is not obtainable, multiple interventions can be implemented to reduce the risk. The following is a brief overview of animal feed ingredients and how they are derived and processed into finished feeds, associated biological hazards with various animal feed ingredients, recent regulatory guidelines regarding biological hazards, and an outline of the steps required to implement a biosecurity plan at a feed mill.