The feed supply chain can be a significant risk of pathogen transfer. While historically, farms have evolved to have biosecurity limiting farm-to-farm interaction, there continues to be gaps in swine biosecurity because the feed mill serves as a central point of cross-contact. These biosecurity gaps can lead to pathogens entering the feed supply chain. If a pathogen enters the feed manufacturing environment, it has the potential to be distributed. The objective of this experiment was to evaluate the prevalence and distribution of African swine fever virus (ASFV) if it enters a feed mill as a contaminated ingredient. To meet this objective, a pilot-scale feed mill was built inside a biosafety level-3Ag room in the Kansas State University Biosecurity Research Institute in Manhattan, KS. Equipment included a pilot-scale 50-kg mixer and a bucket elevator with downspout with 74 buckets and discharge rate of 4.5 kg/min. Initially, ASFV-negative ingredients were batched into the mixer, mixed for 5 minutes, and discharged through the bucket elevator and downspout into biohazard totes. Next, a batch of feed was manufactured that contained an ingredient spiked with ASFV. Finally, four subsequent batches of feed were manufactured using ingredients that did not contain ASFV. Between batches, ten samples of feed were collected after discharging into the biohazard tote in a double X pattern. Additionally, environmental samples were collected from feed contact surfaces, non-feed contact surfaces nearby the manufacturing location, non-feed contact surfaces further away from the manufacturing location (> 1 m), and transient surfaces that moved between zones, such as the shoes and clothing of personnel. Results indicated that ASFV was detected in all batches of feed after it was initially introduced into the manufacturing equipment. For example, there was no ASFV detected in feed samples in batch 1, but all ten samples collected in batches 2, 3, 4, 5, and 6 contained detectable levels of ASFV, even though batches 3, 4, 5, and 6 were manufactured with ASFV-free ingredients. This is supported by environmental samples, which demonstrated the interior of manufacturing equipment remained contaminated, even after manufacturing batches of feed with ASFV-free ingredients in an attempt to flush out the contamination. Contamination was distributed throughout the room, with 100% of transient surfaces containing ASFV after batch 2 was manufactured and staying contaminated through batch 6. In summary, this research demonstrates that once introduced to a feed mill, African swine fever virus is widely and relatively uniformly distributed. Manufacturing subsequent batches of feed helped reduce the quantity of ASFV, but some level of contamination remained, even after 4 batches of ASFV-free ingredients. This research indicates that feed mills should prioritize prevention of ASFV entry into the facility because once it enters, the contaminant can impact multiple batches and surfaces. Questions regarding this research or its implications can be directed to the Principal Investigator, Dr. Cassie Jones, at

Key Findings:
• Contamination with ASFV was rapid and widespread after introduction through inoculated feed and presence of ASFV-specific DNA minimally changed with each subsequent batch.
• Sequencing with four batches of feed can decrease overall ASFV contamination within feed samples but not eliminate it entirely
• Collecting 10 evenly distributed samples allows for detection of ASFV under the conditions of the current investigation
• If there is viral contamination within the feed mill environment, it can be found with environmental swabs
• Transient surfaces play an important role in the spread of virus through the feed mill. Moving objects like people, PPE, and trucks should be taken in account when designing feed biosecurity protocols and feed/feed mill surveillance could be pivotal in maintaining appropriate feed biosecurity.