L. L. Schumacher

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.

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.

Evaluation of the minimum infectious dose of porcine epidemic diarrhea virus in virus-inoculated feed

OBJECTIVE To determine the minimum infectious dose of porcine epidemic diarrhea virus (PEDV) in virus-inoculated feed. ANIMALS 30 crossbred 10-day-old pigs. PROCEDURES Tissue culture PEDV was diluted to form 8 serial 10-fold dilutions. An aliquot of stock virus (5.6 × 10(5) TCID50/mL) and each serial PEDV dilution were mixed into 4.5-kg batches of feed to create 9 PEDV-inoculated feed doses; 1 virus-negative dose of culture medium in feed was also created. Pigs were challenge exposed via oral administration of PEDV-inoculated feed, and fecal swab specimens were collected. All pigs were euthanized 7 days after challenge exposure; fresh tissues were collected and used for PCR assay, histologic examination, and immunohistochemical analysis. RESULTS The PCR cycle threshold (Ct) decreased by approximately 10 when PEDV was added to feed, compared with results for equivalent PEDV diluted in tissue culture medium. Pigs became infected with PEDV when challenge exposed with the 4 highest concentrations (lowest concentration to cause infection, 5.6 × 10(1) TCID50/g; Ct = 27 in tissue culture medium and 37 in feed). CONCLUSIONS AND CLINICAL RELEVANCE In this study, PEDV in feed with detectable Ct values of 27 to 37 was infective. The Ct was 37 for the lowest infective PEDV dose in feed, which may be above the limit of detection established for PEDV PCR assays used by some diagnostic laboratories. Overall, results indicated 5.6 × 10(1) TCID50/g was the minimum PEDV dose in feed that can lead to infection in 10-day-old pigs under the conditions of this study.

Determining the Minimum Infectious Dose of Porcine Epidemic Diarrhea Virus (PEDV) in a Feed Matrix

Understanding the magnitude of transmissible risk Porcine Epidemic Diarrhea Virus (PEDV)-infected feed imposes and establishing the minimum infectious dose of PEDV in a feed matrix are important components in strengthening virus prevention and control methods. In this study, an experiment was performed involving 30 crossbred, 10-d-old pigs that were used as a bioassay model for the minimum infectious dose of PEDV in feed. The PEDV was first diluted using tissue culture media to form 8 serial 10-fold dilutions. An aliquot of the original stock virus at 5.6 x 105 tissue culture infectious dose/ml (TCID50/ml), each serial PEDV dilution, and one virus-negative culture medium were mixed into separate 4.5 kg batches of swine diet to form 10 experimental treatments. The feed was then subsequently evaluated for infectivity using bioassay. Fecal swabs were collected at 0, 2, 4, 6, and 7 d after challenge for PCR testing. At 7 d after challenge, all pigs were necropsied. Cecum contents, ileum and jejunum were collected for PCR, histologic and immunohistochemistry (IHC) evaluation. Overall, the results indicate 5.6 × 101 TCID50/g was the minimum PEDV dose in which infection was detected. This feed had a corresponding PCR cycle threshold (Ct) of 37. This is a relatively low dose. To illustrate, using this dose, approximately 1 g of PEDV-infected baby piglet feces could contaminate up to 500 tons of feed. The data confirm that detectable Ct values in feed can result in pig infection. Our results also illustrate that the Ct in feed that was detected as infectious can be above the detection threshold used by some diagnostic laboratories.

Pyogranulomatous panophthalmitis with systemic coronavirus disease in a domestic ferret (Mustela putorius furo).

Abstract A 15‐month‐old spayed female ferret (Mustela putorius furo) presented for lethargy and weight loss of 2 weeks duration. Upon physical examination, a 2‐mm‐diameter focal area of opacity was noted in the left cornea. In addition, the ferret was quiet, in poor body condition, and dehydrated. A complete blood count and plasma biochemistry revealed a severe nonregenerative anemia, azotemia, hyperproteinemia, hypoalbuminemia, and mild hyperphosphatemia and hyperchloremia. Urinalysis revealed hyposthenuria. Whole body radiographs showed multifocal thoracic nodular disease, splenomegaly, and renomegaly. Abdominal ultrasonography confirmed bilaterally enlarged kidneys, hypoechoic liver and spleen, and a caudal abdominal hypoechoic mobile nodule. The ferret was humanely euthanized, and a postmortem examination with subsequent histopathology showed multifocal necrotizing pyogranulomas in the lung, spleen, kidneys, mesenteric lymph nodes, and serosa of the duodenum. Pyogranulomatous panophthalmitis was diagnosed in the left eye. The multisystemic granulomatous lesions were suggestive of ferret systemic coronavirus (FRSCV). The presence of coronavirus in the left eye was confirmed by positive immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT‐PCR) on formalin fixed paraffin embedded tissue from the lung, spleen, and kidney was negative for FRSCV and positive for ferret enteric coronavirus (FRECV). Systemic coronavirus disease in ferrets closely resembles feline infectious peritonitis (FIP) in domestic cats, which can manifest with anterior uveitis, chorioretinitis, optic neuritis, and retinal detachment. To the authors’ knowledge, this is the first report of ocular lesions in a ferret with systemic coronavirus disease, suggesting that ferrets presented with similar ocular lesions should also be evaluated for evidence of coronavirus infection.

SOLITARY T-CELL HEPATIC LYMPHOMA WITH LARGE GRANULAR LYMPHOCYTE MORPHOLOGY IN A CAPTIVE CHEETAH (ACINONYX JUBATUS).

A 13-yr-old male cheetah (Acinonyx jubatus) presented for an acute history of lateral recumbency and anorexia. Upon physical examination under general anesthesia, severe icterus was noted. A serum biochemical profile confirmed markedly elevated total bilirubin and alanine transaminase. Based on ultrasound-guided liver aspirates and cytology, a presumptive diagnosis of large granular lymphocyte hepatic lymphoma was reached. Abdominal and thoracic radiographs did not assist in reaching an antemortem diagnosis. Postmortem examination and histopathology provided a definitive diagnosis of hepatic lymphoma with acute massive hepatocelluar necrosis and hemorrhage, as well as concurrent lesions of gastric ulcers, ulcerative and sclerosing enteritis, myocardial hypertrophy, and splenic myelolipomas. Immunohistochemistry of the liver yielded CD-3 positive and CD-20 negative results, confirming lymphocytes of a T-cell lineage. Due to concern for possible retrovirus-associated disease, enzyme-linked immunosorbent assays for feline leukemia virus and feline immunodeficiency virus were performed retrospectively on a banked serum sample and yielded negative results, thus diminishing concern for the male conspecific housed in the same exhibit.

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.

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.