Effect of mixing and feed batch sequencing on the prevalence and distribution of African swine fever virus in swine feed

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double ‘X’ pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log10 genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log10 genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contamination

Prevalence and Distribution of African Swine Fever Virus in Swine Feed After Mixing and Feed Batch Sequencing

As the United States maintains trade with countries where African swine fever virus (ASFV) is endemic, it is critical to have methods that can detect and mitigate the risk of ASFV in potentially contaminated feed or ingredients. Therefore, the objectives of this study were to 1) evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and 2) determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University’s Biosafety Research Institute in Manhattan, KS. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for a final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a biohazard tote, 10 samples were collected in a double ‘X’ pattern. Samples were analyzed using a qPCR assay specific for the ASFV p72 gene to determine the cycle threshold (Ct) and log10 genomic copy number (CN)/g of feed. Batch of feed affected the qPCR Ct values (P \u3c 0.0001) and the log10 genomic CN/g (P \u3c 0.0001) content of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest (P \u3c 0.05) amounts of ASFV p72 DNA across all criteria. Quantity of ASFV p72 DNA decreased sequentially as additional batches of initially ASFV-free feed were manufactured, but it was still detectable after batch sequence 4, suggesting cross contamination between batches. This subsampling method was able to identify ASFV genetic material in feed samples using the PCR assay specific for the ASFV p72 gene. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients or feed can be accurately evaluated for ASFV contamination by collecting 10 evenly distributed subsamples, representing 0.05% of the volume of the container, using the sampling method described herein

Evaluating the distribution of African swine fever virus within a feed mill environment following manufacture of inoculated feed

11 Pág. Centro de Investigación en Sanidad Animal (CISA)It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.Funding for this work was obtained from the NBAF Transition Funds from the state of Kansas (JAR), the National Pork Board under award number 20-018 (CKJ), the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR), and the AMP Core of the NIGMS COBRE Center on Emerging and Zoonotic Infectious Diseases (CEZID) under award number P20GM13044 (JAR)Peer reviewe

Detection of African Swine Fever Virus in Feed and Feed Mill Environment Following Extended Storage

7 Pág.One way to mitigate risk of feed-based pathogens for swine diets is to quarantine feed ingredients before inclusion in complete diets. Data have been generated evaluating the stability of swine viruses in ingredients, but the stability of African swine fever virus (ASFV) in feed or in a feed manufacturing environment has not been well characterized. Therefore, this study aimed to determine the stability of ASFV DNA in swine feed and on mill surfaces over time. A pilot-scale feed mill was used to manufacture six sequential batches of feed consisting of a batch of ASFV-free feed, followed by a batch inoculated with ASFV (final concentration = 5.6 × 104 TCID50/g), and then four subsequent ASFV-free batches. After each batch, 10 feed samples were aseptically collected in a double “X” pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and allowed to collect dust during feed manufacturing. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. Three of each were randomly selected from storage on 3, 7, 14, 28, 60, 90, and 180 days after feed manufacturing and analyzed for ASFV DNA. For feed samples, there was evidence of a batch × day interaction (P ¼ 0:023) for the quantification of genomic copies/g of feed, indicating that the amount of ASFV DNA present was impacted by both the batch of feed and days held at room temperature. There were no differences of genomic copies/g in early batches, but quantity of detectable ASFV decreased with increasing storage time. In Batches 4–6, the greatest quantity of ASFV DNA was detected on the day of feed manufacturing. The lowest quantity was detected on Day 7 for Batch 4, Day 60 for Batch 5, and at 28 and 180 days for Batch 6. There was no evidence of ASFV degradation on environmental discs across holding times (P ¼ 0:433). In conclusion, the quarantining of feed may help reduce but not eliminate the presence of ASFV DNA in feed over time. Importantly, ASFV DNA was detectable on feed manufacturing surfaces for at least 180 days with no overt evidence of reduction, highlighting the importance of bioexclusion of ASFV within feed manufacturing facilities and the need for thorough/effective decontamination and other mitigation processes in affected areas.Funding for this work was obtained from the NBAF Transition Funds from the State of Kansas and by the National Pork Board (Award #20-018), the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006, and the AMP Core of the NIGMS COBRE Center on Emerging and Zoonotic Infectious Diseases (CEZID) under award number P20GM13044.Peer reviewe

Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern

ABSTRACTSince emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants

Coronal Heating as Determined by the Solar Flare Frequency Distribution Obtained by Aggregating Case Studies

Flare frequency distributions represent a key approach to addressing one of the largest problems in solar and stellar physics: determining the mechanism that counter-intuitively heats coronae to temperatures that are orders of magnitude hotter than the corresponding photospheres. It is widely accepted that the magnetic field is responsible for the heating, but there are two competing mechanisms that could explain it: nanoflares or Alfv\'en waves. To date, neither can be directly observed. Nanoflares are, by definition, extremely small, but their aggregate energy release could represent a substantial heating mechanism, presuming they are sufficiently abundant. One way to test this presumption is via the flare frequency distribution, which describes how often flares of various energies occur. If the slope of the power law fitting the flare frequency distribution is above a critical threshold, $\alpha=2$ as established in prior literature, then there should be a sufficient abundance of nanoflares to explain coronal heating. We performed $>$600 case studies of solar flares, made possible by an unprecedented number of data analysts via three semesters of an undergraduate physics laboratory course. This allowed us to include two crucial, but nontrivial, analysis methods: pre-flare baseline subtraction and computation of the flare energy, which requires determining flare start and stop times. We aggregated the results of these analyses into a statistical study to determine that $\alpha = 1.63 \pm 0.03$. This is below the critical threshold, suggesting that Alfv\'en waves are an important driver of coronal heating.Comment: 1,002 authors, 14 pages, 4 figures, 3 tables, published by The Astrophysical Journal on 2023-05-09, volume 948, page 7