Proof-of-concept method to sanitize a feed mill contaminated with Porcine Epidemic Diarrhea Virus

Citation: Huss, A. R., Schumacher, L. L., Cochrane, R. A., Poulsen, E., Bai, J. F., Woodworth, J. C., . . . Jones, C. K. (2016). Proof-of-concept method to sanitize a feed mill contaminated with Porcine Epidemic Diarrhea Virus. Journal of Animal Science, 94, 102-103. doi:10.2527/msasas2016-217Porcine Epidemic Diarrhea Virus (PEDV) has been linked to 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 PEDV RNA in feedstuffs, and has not addressed potential residual contamination within the manufacturing facility that may lead to continuous cross-contamination of finished feeds. The objective 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 5 decontamination steps: 1) baseline before 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 h. The feed mill was contaminated and decontaminated 3 separate times for a total of 3 replications. Collected swabs were analyzed via RT-qPCR and categorized by surface (plastic, rubber, concrete, and metal), type (equipment and structural), and zone (1, 2, and 3). 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 direct feed contact surfaces (zone 1) contained PEDV RNA after production of the contaminated feed. Additionally, all swabs collected directly adjacent to direct feed contact surfaces (zone 2) were positive following production of the contaminated feed. Of the remaining swabs collected (zone 3), outside of zones 1 and 2, 88.9% had detectable RNA, emphasizing the potential role dust plays in cross-contamination of pathogens throughout a manufacturing facility. Application of the cleaner, sanitizer, and heat were effective at reducing PEDV RNA (P < 0.05), but did not completely eliminate it. Specifically, 29.6%, 14.8%, and 7.4% of zone 1 swabs had detectable PEDV RNA after decontamination with the cleaner, sanitizer and heat, respectively, during only replication 2. Due to this, decontamination was repeated with no PEDV RNA detected from subsequent swab collection. These findings do provide a method for facility decontamination of PEDV, however, the use of liquid cleaners, sanitizers, and/or facility heat-up may not be applicable for most commercial feed manufacturing facilities

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

Citation: Huss AR, Schumacher LL, Cochrane RA, Poulsen E, Bai J, Woodworth JC, et al. (2017) Elimination of Porcine Epidemic Diarrhea Virus in an Animal Feed Manufacturing Facility. PLoS ONE 12(1): e0169612. doi:10.1371/journal.pone.0169612Porcine 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

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome

The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla[subscript CMY-2], bla[subscript CTX-M], tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins

Experimental infection of conventional nursing pigs and their dams with \u3ci\u3ePorcine deltacoronavirus\u3c/i\u3e

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi

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

Citation: Cochrane, R. A., Schumacher, L. L., Dritz, S. S., Woodworth, J. C., Huss, A. R., Stark, C. R., . . . Jones, C. K. (2017). Effect of pelleting on survival of porcine epidemic diarrhea virus-contaminated feed. Journal of Animal Science, 95(3), 1170-1178. doi:10.2527/jas2016.0961Porcine 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 x 3 x 2 factorial design was used with 3 pelleting temperatures (68.3, 79.4, and 90.6 degrees C), 3 conditioning times (45, 90, or 180 s), and 2 doses of viral inoculation (low, 1 x 102 tissue culture infectious dose(50) (the concentration used to see cytopathic effect in 50% of the cells)/g, or high, 1 x 10(4) tissue culture infectious dose(50)/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 x 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 degrees 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 degrees C conditioning temperatures. Experiment 2 results suggest conditioning and pelleting temperatures above 54.4 degrees 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 the Impact of Presence of Organic Matter on Environmental Samples and Sample Processing Technique on RNA Detection of PEDV

Environmental sampling has become a commonly accepted diagnostic sampling technique for a means of identifying breaks in biosecurity. However, environmental samples have yet to be validated for reverse transcriptase real-time PCR (qRT-PCR) analysis and there is no standardization for environmental sample processing. Therefore, the objective of this project was to evaluate different types of environmental samples, and whether processing the samples prior to qRT-PCR analysis would impact results. Steel coupons were inoculated with PEDV in different types of environmental conditions, then were environmentally swabbed using cotton gauze. Treatments were arranged as a 5 × 4 factorial with five treatments for the different types of contamination and four treatments for the types of sample processing. Samples were processed in four different ways: no pre-qRT-PCR processing, centrifuging, syringe filtering, and centrifuging then syringe filtering to determine if pre-sample processing impacted the cycle threshold (Ct) value. Once samples were processed, they were submitted for PEDV qRT-PCR analysis. Results were reported as proportion of qRT-PCR positive and the resulting Ct value. If samples had no detectable RNA, they were assigned a Ct value of 45. For the Ct values, there was an inoculated surface × sample processing (P \u3c 0.0001) interaction indicating that the type of environmental sample and the way the sample was processed impacted the Ct value of the sample. For pure virus and virus with PBS, there was no difference in Ct values between different sample processing techniques (PP \u3c 0.05). For virus and fecal contamination, samples that were not processed or samples that were processed with centrifuging only had greater amounts of PEDV RNA detected compared to syringe filtered samples or centrifuged and syringe filtered samples (P \u3c 0.05). For virus and organic matter contamination, samples that were centrifuged had greater amounts of PEDV RNA detected compared to all other sample processing techniques (P \u3c 0.05). Main effects of inoculated surface (P \u3c 0.0001) and sample processing (P \u3c 0.0001) were also significant. For surface inoculation type, pure virus inoculation and virus with PBS inoculation had greater amounts of PEDV RNA compared to virus with feces inoculation or virus with organic matter inoculation, while virus with dirt was intermediate. For sample processing type, centrifuged samples had the greatest amount of PEDV RNA compared to syringe filtered and centrifuged then syringe filtered samples with unprocessed samples being intermediate. In summary, if environmental samples are particularly dirty, processing prior to qRT-PCR analysis will impact the results

Clinical and Immunopathological Features of Moyamoya Disease

Background: Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive stenosis or occlusion of the terminal portion of internal carotid arteries and the formation of a vascular network at the base of the brain. The pathogenesis of MMD is still unclear. Methodology/Principal Findings: We retrospectively analyzed clinical data for 65 consecutive patients with MMD in our institutions and evaluated the histopathological and immunohistochemical findings of intracranial vessels from 3 patients. The onset age distribution was found to have 1 peak at 40–49 year-old age group, no significant difference was observed in the female-to-male ratio (F/M = 1.2). Intracranial hemorrhage was the predominant disease type (75%). Positive family history was observed in 4.6 % of patients. Histopathological findings were a narrowed lumen due to intimal fibrous thickening without significant inflammatory cell infiltration, and the internal elastic lamina was markedly tortuous and stratified. All 3 autopsy cases showed vacuolar degeneration in the cerebrovascular smooth muscle cells. Immunohistochemical study showed the migration of smooth muscle cells in the thickened intima, and aberrant expression of IgG and S100A4 protein in vascular smooth muscle cells. The Complement C3 immunoreactivity was negative. Conclusion/Significance: This study indicated that aberrant expression of IgG and S100A4 protein in intracranial vascular wall of MMD patients, which suggested that immune-related factors may be involved in the functional and morphologica

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P \u3e 0.10). An interaction between disinfectant × surface impacted (P \u3c 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P \u3e 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P \u3e 0.10). An interaction between disinfectant × surface impacted (P \u3c 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P \u3e 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces

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