Evaluating the Distribution of African Swine Fever Virus Within a Evaluating the Distribution of African Swine Fever Virus Within a Feed Mill Environment Following Manufacture of Inoculated Feed Feed Mill Environment Following Manufacture of Inoculated Feed

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Introduction
African swine fever virus (ASFV) is a significant concern for United States (US) pork producers, and a research priority for the swine industry due to a lack of vaccine or cure for the disease exists.Both North and South America are currently free from ASFV, but China and its neighboring countries are endemic for the disease since 2018-2019; this has the US concerned about ASFV-contaminated feed being shipped from these countries. 6Currently, research has evaluated AFSV survival during transboundary shipping7 and determined the infectious dose for pigs of ASFV in feed and liquid consumption. 8owever, it is unknown how ASFV may be distributed within a feed manufacturing facility if introduced.Porcine Epidemic Diarrhea Virus (PEDV) is the only virus that Swine Day 2020 has been extensive researched in feed manufacturing environments.We have previously reported that PEDV has the ability to remain in feed manufacturing environments, on feed and non-feed surfaces, even for subsequent batches. 9Additional PEDV research has also proven that once the virus enters the feed mill, it is difficult and labor-intensive to decontaminate the facility. 10Together, these same factors could elevate the risk for ASFV contamination of feed and ingredients originating in contaminated mills, but to our knowledge, no research is available to confirm the environmental risk associated with ASFV contamination of a feed ingredient.Therefore, the objective of this study was to evaluate the effect of using ASFV-contaminated feed on the feed mill environment and subsequent feed batches.

Procedures
The study was conducted at the Biosecurity Research Institute (BRI) in Manhattan, KS, with approval by the Kansas State University Institutional Biosafety Committee (project approval #1427.1).The workspace was prepared within a BSL-3Ag large animal room.The room was cleaned and disinfected in compliance with BRI protocol prior to the start of the study.One 55-pound batch of uninfected swine gestation diet in meal form (Table 1) was mixed in a 110-pound capacity stainless steel mixer (H.C. Davis Sons Manufacturing, model # SS-L1; Bonner Springs, KS), conveyed through a pilot scale bucket elevator, and distributed into double-lined bags.Environmental swabs were collected from various predetermined locations relative to their position to the feed (Table 2).Upon completion of priming the system with the initial batch of ASFVdevoid feed, 530 mL of Armenia/07 African swine fever virus (1×10 5 TCID 50 mL) was then mixed with 10.5 pounds of diet in an 11-pound stainless steel mixer to make 11.6 pounds of contaminated inoculum, which was subsequently added to 44 pounds of diet, in a 110 pound stainless steel mixer, and mixed to make the final inoculated batch of feed.The feed was then conveyed and discharged into a double-lined bag.Following discharge of the inoculated batch of feed and collection of appropriate environmental swabs, the process of mixing and discharging 55 pound batches of feed was repeated four additional times using ASFV-devoid diet.
Environmental swabs were cotton gauze squares, 0.4 × 0.4 inch, pre-moistened with 5 mL of PBS and individually stored in a 50-mL conical tube prior to usage.All swabs collected prior to inoculation with ASFV were PCR negative, as expected.Within the analysis of Ct and proportion of PCR positive samples, the negative control data were excluded as the primary research question was the effect of zones and batch of feed on detection of ASFV on environmental surfaces following controlled inoculation.Data were analyzed as 4 × 5 factorial with 4 sampling surfaces, and 5 batches of feed, not including the initial negative control samples.The individual sample collected from a surface for a specific batch was considered the experimental unit.Environmental swabs were used for the negative control, positive control, and sequences 1-4.Negative control samples were taken prior to the usage of ASFV-inoculated feed, positive Swine Day 2020 control samples were taken after the usage of ASFV-inoculated feed, and sequences 1-4 samples were taken after each batch was mixed after the positive control.Locations for environmental sampling were chosen based off proximity to feed (Table 2).Zone A locations were the mixer ribbon, mixer barrel, mixer discharge, bucket elevator bucket, bucket elevator belt, and bucket elevator discharge.Zone B locations were wall close to mixer, wall close to bucket elevator, floor close to mixer, floor close to bucket elevator, and ceiling close to mixer.Zone C locations were wall far from mixer, floor far from mixer, floor far from bucket elevator, and ceiling far from mixer.Zone D locations were the boot soles of researchers A, B, and C. To collect samples, a clean pair of disposable gloves were worn and the cotton gauze square was aseptically opened from a 50-mL conical tube.The chosen location was swabbed, environmental swab placed back in the conical tube, and gloves were changed.Once the experiment was concluded, environmental swabs were placed in a primary container, the outside of the container decontaminated, then placed in a secondary container, then the secondary container decontaminated, then placed in a tertiary container, the surface decontaminated again, then transported to the BSL-3 laboratory.Any excess or unused feed was spread on the floor, watered down, and washed down the drain with lots of water.Equipment was disassembled, wet-cleaned, and surface decontaminated with Virkon.Then the room was turned over to BRI staff for final decontamination per BRI standard operating procedures.
Environmental swabs were processed in a BSL-3 laboratory within the BRI by adding 1-2 mL of sterile PBS, incubated overnight at room temperature, vortexed, then held upright for 5 minutes.Approximately 1-2 mL was recovered and stored at -112°F for further processing at a later time.Samples were then tested by qPCR using the ASFVspecific qPCR assay for detecting the ASFV P72 gene.The current data do not include measures of viral infectivity.However, ongoing investigations aim to evaluate infectivity characteristics.Data reported include Ct values and number of genomic copies/mL of solution recovered from swab sample.If no ASFV DNA was identified, samples were assigned a Ct value of 45, which was the threshold for cutoff for detection.
Visualization on data was performed using the ggplot2 package using the RStudio environment (Version 1.2.1335,RStudio, Inc., Boston, MA) using R programming language [Version 3.6.1 (2019-07-05), R Core Team, R Foundation for Statistical Computing, Vienna, Austria].The proportion of PCR reactions positive for ASFV DNA are reported as: (# of qPCR positive reactions/total # of qPCR reactions).The proportion of PCR reactions having detectable ASFV DNA was fit using the glmer function in the lme4 package using a binomial distribution with the fixed effects of sampling zone, batch of feed, and the associated interaction, with a random effect of environmental swab to indicate the appropriate level of experimental replication given the duplicate qPCR analysis of environmental swabs.
Cycle threshold and genomic copies/mL data were analyzed using a linear mixed model fit with the lme function in the nlme package using similar fixed effects.Results of Ct and genomic copy number/mL data are reported as least squares means ± standard error of the mean.Samples not containing detectable ASFV DNA were assigned a value of 45 because that was the greatest number of cycles the qPCR assay performed before concluding a sample did not have detectable ASFV DNA.Analysis of genomic copies/ mL included PCR-negative reactions using a value of 0 for the quantified genomic Swine Day 2020 copies/mL.All statistical models were evaluated using visual assessment of studentized residuals, and models accounting for heterogeneous residual variance were used when appropriate.A Tukey multiple comparison adjustment was incorporated when appropriate.Results were considered significant at P ≤ 0.05 and marginally significant between P > 0.05 and P ≤ 0.10.

Results and Discussion
As expected, no ASFV DNA was identified by Ct (Figure 1), or genomic copies (Figure 2) in environmental swabs collected prior to ASFV inoculation of feed.Environmental swabs collected after the manufacture of the ASFV-inoculated feed showed contamination of all zones, with 38% to 100% of qPCR reactions resulting in detectable ASFV DNA, depending on the contact surface (Table 3).There was no evidence of a sampling zone × batch of feed interaction for prevalence of PCR reactions detecting ASFV DNA (P = 0.912) or Ct value (P = 0.519).Additionally, there was insufficient evidence to conclude that the proportion of ASFV-qPCR-detectable reactions was affected by sampling zone (P = 0.701) or batch of feed (P = 1.000).This indicates that once ASFV contamination entered the facility, the contamination quickly became widespread and persisted on all tested environmental surfaces even after manufacturing subsequent 'clean' batches of feed.
The respective batch of feed influenced the concentration of detectable ASFV DNA, with the samples collected after feed batch Sequence 3 having less detectable ASFV (a greater Ct) than samples collected immediately after manufacture of the ASFV-inoculated batch of feed (Table 4; P < 0.05), with samples collected after all other batches of feed post ASFV-inoculation being intermediate.The Ct value from samples collected from transient surfaces (soles of worker boots) was lower than all other surfaces, indicating these surfaces contained a greater quantity of detectable ASFV DNA (P < 0.05).
There was evidence of a sampling zone × batch interaction for the number of genomic copies/mL (P = 0.002).For samples collected after manufacture of the inoculated batch of feed, fewer genomic copies/mL were observed for swabs collected from zone C compared to zone A (P < 0.05), with zone B intermediate.The number of genomic copies/mL in zone D was numerically greater than other surfaces following the inoculated batch of feed, but a high degree of variability resulted in no evidence of statistical differences compared to the other surfaces at this sampling point.After Sequences 1, 2, and 3, samples collected from the transient surfaces had more genomic copies/mL detected compared to other sampling locations (P < 0.05).After Sequence 4, there was no evidence of a difference in the number of detected genomic copies/mL between sampling locations (P > 0.05).
Batch order impacted the number of genomic copies/mL (P = 0.045), but mean separation using a Tukey multiple comparison adjustment to control Type I error rate did not result in evidence of pairwise differences (P > 0.05).The non-feed contact surfaces (both < 3.2 feet and > 3.2 feet) had fewer genomic copies/mL compared to the transient surfaces (P < 0.05), with the feed contact surface being intermediate.
In summary, once ASFV was introduced into a controlled feed manufacturing environment, the virus became widely distributed throughout the facility.We observed Swine Day 2020 minimal evidence of a change in the amount of ASFV DNA Ct as subsequent ASFVfree batches of feed were manufactured, indicating that ASFV DNA remains detectable on production surfaces for a period of time after manufacture of ASFV-contaminated feed.We also observed that the spread of ASFV is greatly influenced by transient surfaces, indicating that people play a huge role in the transmission of ASFV through fomites.Additional work needs to be completed to understand the infectivity of feed manufactured in a contaminated environment.
Brand names appearing in this publication are for product identification purposes only.No endorsement is intended, nor is criticism implied of similar products not mentioned.Persons using such products assume responsibility for their use in accordance with current label directions of the manufacturer. 1Each lb of premix contains 33 g Fe, 33 g Zn, 10 g Mn, 5 g Cu, 90 mg I, and 90 mg Se.

Figure 1 .
Figure1.Cycle threshold value using ASFV P72 encoding gene assay for environmental swabs collected following the manufacture of African swine fever virus (ASFV) inoculated swine feed.Batch 1 was used to prime the feed manufacturing equipment prior to manufacture of batch 2, which was inoculated with ASFV at 2 × 10 3 TCID 50 /gram inoculated feed.Batches 3 through 6 were subsequent batches of feed that were manufactured within the system and were initially devoid of ASFV.Zone A = surfaces that are feed contact surface; Zone B = non-feed contact surfaces < 3.2 feet away from feed contact surface; Zone C = non-feed contact surfaces > 3.2 feet away from feed contact surfaces; Zone D = transient surface.

Figure 2 .
Figure2.Genomic copies per mL using ASFV P72 encoding gene assay for environmental swabs collected following the manufacture of African swine fever virus (ASFV) inoculated swine feed.Batch 1 was used to prime the feed manufacturing equipment prior to manufacture of batch 2, which was inoculated with ASFV at 2 × 10 3 TCID 50 /gram inoculated feed.Batches 3 through 6 were subsequent batches of feed that were manufactured within the system and were initially devoid of ASFV.Zone A = feed contact surface; Zone B = non-feed contact surfaces < 3.2 feet away from feed contact surface; Zone C = non-feed contact surfaces > 3.2 feet from feed contact surface; Zone D = transient surface.

Table 2 .
Location of environmental swabs and grouping by zone.

Table 3 .
1,teractive effect of feed batch and contact surface on detection of African swine fever virus (ASFV) during manufacture of virus-inoculated feed1,2