Pitchfork and Hopf bifurcation thresholds in stochastic equations with delayed feedback

The bifurcation diagram of a model stochastic differential equation with delayed feedback is presented. We are motivated by recent research on stochastic effects in models of transcriptional gene regulation. We start from the normal form for a pitchfork bifurcation, and add multiplicative or parametric noise and linear delayed feedback. The latter is sufficient to originate a Hopf bifurcation in that region of parameters in which there is a sufficiently strong negative feedback. We find a sharp bifurcation in parameter space, and define the threshold as the point in which the stationary distribution function p(x) changes from a delta function at the trivial state x=0 to p(x) ~ x^alpha at small x (with alpha = -1 exactly at threshold). We find that the bifurcation threshold is shifted by fluctuations relative to the deterministic limit by an amount that scales linearly with the noise intensity. Analytic calculations of the bifurcation threshold are also presented in the limit of small delay tau -> 0 that compare quite favorably with the numerical solutions even for tau = 1

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

With the global spread of African swine fever virus (ASFV) and evidence that feed and/or ingredients may be potential vectors for pathogen transmission, it is critical to understand the role the feed manufacturing industry may have in regard to potential distribution of this highly virulent virus. A pilot-scale feed mill consisting of a mixer, bucket elevator, and relevant spouting was constructed in the Biosafety Level-3 Ag animal room at the Biosecurity Research Institute at Kansas State University. A total of 18 different sites on the equipment and in the room were swabbed to evaluate environmental contamination before and after introduction of ASFV-inoculated feedstuff. First, a batch of feed was manufactured through the system to confirm the feed mill was ASFV negative; then a feedstuff inoculated with ASFV was added into the mixer and manufactured with other, non-infected ingredients. Ingredients were mixed and discharged through the bucket elevator. Subsequently, four additional ASFV-free batches of feed were manufactured. Environmental swabs were collected after each batch of feed was discharged with locations categorized into four zones: A) feed contact surface, B) non-feed contact surface but \u3c 3.2 feet away from feed, C) non-feed contact surface \u3e 3.2 feet from feed, and D) transient surfaces such as worker shoes. Environmental swabs were analyzed using qPCR analysis for the P72 ASFV gene in a BSL-3 laboratory setting to detect ASFV-specific DNA. Environmental swabs collected prior to ASFV inoculation of feed were negative for ASFV DNA. Environmental swabs collected after the manufacture of ASFV-inoculated feed resulted in contamination of zones A-D. Contamination levels with ASFV-DNA are reported as Ct value or genomic copy number (CN) per mL. In this setup, there was no evidence of sampling zone × batch interaction and no difference in the proportion of ASFV positive reactions between sampling location or batch of feed throughout the experiment. This indicates that once ASFV contamination entered the facility, the contamination quickly becomes widespread and persists on the environmental surfaces, even during manufacturing of subsequent batches of ASFV non-inoculated feed. Samples from transient surfaces (Zone D) had more detectable ASFV (a lower Ct value) compared to all other surfaces (P \u3c 0.05), indicating high level of ASFV contamination (high CN values). Samples collected after manufacturing sequence 3 had less detectable ASFV (a greater Ct value) compared to samples collected immediately following manufacture of the ASFV-inoculated batch of feed (P \u3c 0.05), indicating lower levels of ASFV contamination (low CN values) in subsequent repeats of the feed production process. 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 ASFV-inoculated batch of feed, a lower number of ASFV genomic copies/mL (higher Ct) was observed for swabs collected from non-feed contact surfaces \u3e 3.2 feet from feed (Zone C) compared to feed contact surfaces (zone A) (P \u3c 0.05), with other surfaces (zone B and D) having no evidence of a significant difference. Following manufacturing sequences 1, 2, and 3, samples collected from the transient surfaces (zone D) had a greater number of ASFV genomic copies/mL (low Ct) detected compared to other sampling locations (P \u3c 0.05). After manufacturing sequence 4, there was no evidence of a difference in the number of detected ASFV genomic copies/mL between sampling locations (P \u3e 0.05). In summary, once ASFV was experimentally introduced into a feed manufacturing environment, the virus became widely distributed throughout the facility with only minor changes in detection frequency as subsequent batches of feed were produced

N-(4-iodophenyl)-N′-(2-chloroethyl)urea as a microtubule disrupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice

The antitumoral profile of the microtubule disrupter N-(4-iodophenyl)-N′-(2-chloroethyl)urea (ICEU) was characterised in vitro and in vivo using the CT-26 colon carcinoma cell line, on the basis of the drug uptake by the cells, the modifications of cell cycle, and β-tubulin and lipid membrane profiles. N-(4-iodophenyl)-N′-(2-chloroethyl)urea exhibited a rapid and dose-dependent uptake by CT-26 cells suggesting its passive diffusion through the membranes. Intraperitoneally injected ICEU biodistributed into the grafted CT-26 tumour, resulting thus in a significant tumour growth inhibition (TGI). N-(4-iodophenyl)-N′-(2-chloroethyl)urea was also observed to accumulate within colon tissue. Tumour growth inhibition was associated with a slight increase in the number of G2 tetraploid tumour cells in vivo, whereas G2 blockage was more obvious in vitro. The phenotype of β-tubulin alkylation that was clearly demonstrated in vitro was undetectable in vivo. Nuclear magnetic resonance analysis showed that cells blocked in G2 phase underwent apoptosis, as confirmed by an increase in the methylene group resonance of mobile lipids, parallel to sub-G1 accumulation of the cells. In vivo, a decrease of the signals of both the phospholipid precursors and the products of membrane degradation occurred concomitantly with TGI. This multi-analysis established, at least partly, the ICEU activity profile, in vitro and in vivo, providing additional data in favour of ICEU as a tubulin-interacting drug accumulating within the intestinal tract. This may provide a starting point for researches for future efficacious tubulin-interacting drugs for the treatment of colorectal cancers

Involvement of TLR2 in Recognition of Acute Gammaherpesvirus-68 Infection

Toll-like receptors (TLRs) play a crucial role in the activation of innate immunity in response to many viruses. We previously reported the implication of TLR2 in the recognition of Epstein-Barr virus (EBV) by human monocytes. Because murine gammaherpesvirus-68 (MHV-68) is a useful model to study human gammaherpesvirus pathogenesis in vivo, we evaluated the importance of mouse TLR2 in the recognition of MHV-68.In studies using transfected HEK293 cells, MHV-68 lead to the activation of NF-κB reporter through TLR2. In addition, production of interleukin-6 (IL-6) and interferon-α (IFN-α) upon MHV-68 stimulation was reduced in murine embryonic fibroblasts (MEFs) derived from TLR2-/- and MyD88-/- mice as compared to their wild type (WT) counterpart. In transgenic mice expressing a luciferase reporter gene under the control of the mTLR2 promoter, MHV-68 challenge activated TLR2 transcription. Increased expression levels of TLR2 on blood granulocytes (CD115(-)Gr1(+)) and inflammatory monocytes (CD115(+)Gr1(+)), which mobilized to the lungs upon infection with MHV-68, was also confirmed by flow cytometry. Finally, TLR2 or MyD88 deficiency was associated with decreased IL-6 and type 1 IFN production as well as increased viral burden during short-term challenges with MHV-68.TLR2 contributes to the production of inflammatory cytokines and type 1 IFN as well as to the control of viral burden during infection with MHV-68. Taken together, our results suggest that the TLR2 pathway has a relevant role in the recognition of this virus and in the subsequent activation of the innate immune response

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

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

Persistence of African Swine Fever Virus in Feed and Feed Mill Environment over Time after Manufacture of Experimentally Inoculated Feed

To reduce the risk of disease from harmful feed-based pathogens, some feed manufacturers quarantine high-risk ingredients prior to their inclusion in feed. Data exist that confirms this practice is effective, but to our knowledge there is no information about porcine pathogen survival in mill environments. The objective of this study was to determine survival of African swine fever virus (ASFV) in swine feed and on mill surfaces after manufacture of experimentally inoculated swine feed. A pilot-scale feed mill was placed within a biosecurity level (BSL) 3 facility to manufacture batches of feed. The priming batch, Batch 1, was ASFV-free feed and was followed with Batch 2 which was experimentally inoculated with ASFV (5.6 × 104 TCID50/gram). Four subsequent ASFV-free batches were then manufactured (Batch 3-6). After each batch of feed, 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 feed dust was allowed to settle onto them overnight. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. On the day of (day 0) and d 3, 7, 14, 28, 60, 90, and 180 after feed manufacturing, feed samples and 3 steel coupons were randomly selected, taken out of storage, and analyzed for ASFV DNA. For feed samples there was a statistically significant (P = 0.023) batch × day interaction for log10 genomic copies per gram of feed, and a marginal statistical significance (P = 0.072) for batch × day interaction for cycle threshold (Ct) values. This indicates that the batch of feed and days held at room temperature impacted the amount of the detectable ASFV DNA in feed samples. There was no evidence (P = 0.433) of ASFV degradation on environmental coupons over the 180-d storage period. This study found that quarantine time can help reduce, but not eliminate ASFV DNA in feed over time. Surprisingly, ASFV DNA is detectable on feed manufacturing surfaces for at least 180 days

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

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