Detection and Investigation of Atypical Porcine Pestivirus within a Breed-to-Finish Farm and Off-Site Nursery and Finisher Locations

Atypical porcine pestivirus (APPV) has been associated with congenital tremors (CT) and splay leg (SL) in piglets of infected dams. The major cost of this virus is the increased pre-weaning mortality due to CT or SL interfering with the piglet’s ability to nurse and move around the farrowing stall. A commercial farrow-to-finish farm with replacement gilts coming from an off-site genetic multiplier farm, and semen delivery from a commercial boar stud began to see an increase of CT and SL in the farrowing room in early 2020. Diagnostics on clinically affected pigs’ samples identified APPV RNA and no other suspected pathogen. At this point, the origin of the virus and means of introduction into the farm was unknown since the farm had no previous clinical cases of CT or SL prior to this investigation. The two hypothesized routes were the introduction of replacement gilts or incoming semen doses. Therefore, the objectives of this investigation were to determine the prevalence of clinical APPV cases at the farrow-to-finish farm, understand the route of introduction of APPV into the farrowto- finish farm, and understand the prevalence of APPV viremia within a population of offspring from a gilt multiplication farm through an off-site nursery and finisher barn. Farrowing records from the farm were analyzed for the presence of CT or SL and parities of females with affected litters. Blood samples were collected at two different times from the new group of replacement gilts and maternal barrows at the isolation nursery barn. Serum and oral fluids were collected from the same pigs at an off-site finisher barn to determine APPV persistence. The APPV sequencing was conducted on a serum sample from a gilt housed at the isolation nursery intended as a replacement gilt for the farrow-to-finish farm, semen dose utilized at the farrow-to-finish farm, and serum of a clinically affected piglet in the farrowing room of the farrow-to-finish farm. Overall, the prevalence of affected litters within batch farrowing groups ranged from 0 to 31%. The prevalence of APPV within samples pooled by pens (5 pigs) ranged from 37.5 to 77.5%, while individual prevalence ranged from 20 to 40%. When followed to the finisher, the same group of pigs had an APPV prevalence in serum ranging from 0 to 26%, while oral fluid prevalence was 100%. Sequencing results indicated that the virus circulating in clinically affected piglets was the most similar to an incoming semen dose. In summary, introduction of APPV into a naïve herd is associated with an increase in clinical CT and SL. While APPV is present in herds previously exposed to APPV, the APPV RNA remains detectable in serum and oral fluids with no clinical disease. To decrease the chance of infection to a naïve herd, quarantines should be implemented for all introductions. Additionally, semen should be screened for APPV presence if there is a recent onset of clinically affected piglets with CT or SL with no other explanation. The APPV RNA was detected in group oral fluids, suggesting the technique may be used to screen incoming animals

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

Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P \u3c 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P \u3c 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Quantification of Semi-Truck Cab Decontamination

Evidence suggests that the inside of vehicle cabs used for feed delivery may serve as a potential source for disease, yet there are no standardized protocols or scientific evidence for methods of their disinfection. Therefore, the objective of this project was to evaluate commercially available disinfectants and disinfection application methods against PEDV and PRRSV on various surfaces within semi-truck cabs. Three different surface types common in vehicle cabs (fabric, plastic, and rubber) were cut into 4 × 4 inch coupons and inoculated with either PEDV or PRRSV. Once inoculated, surfaces were placed in one of 3 semi-truck cabs and the disinfectant treatment was applied. Disinfectant treatments were as follows: 1) no-disinfectant, 2) hurricane fumigation with 1:256 dilution of Synergize, 3) hurricane fumigation with 1:64 dilution of Intervention, 4) pump sprayer with 1:256 dilution of Synergize, 5) pump sprayer with 1:64 dilution of Intervention, 6) pump sprayer with 10% bleach, 7) no chemical with 10 hr downtime, and 8) gaseous fumigation over a 10 hr period with water-based chlorine dioxide. Once a disinfectant treatment was applied, the coupons were environmentally swabbed and submitted for qPCR duplex analysis for PEDV and PRRSV. There was a significant disinfectant × surface interaction (P \u3c 0.0001) indicating that the disinfectant treatment efficacy differed based on surface. Within rubber surfaces, 10% bleach had a greater Ct value compared to all other treatments (P \u3c 0.05), with the exception of Intervention with hurricane fumigation application, which was intermediate. In both fabric and plastic surfaces, there was no evidence (P \u3e 0.05) of a difference in Ct value between any of the treatments. Additionally, for the no-disinfectant treatment, the Ct value was greater on fabric surfaces compared to plastic and rubber (P \u3c 0.05); fabric was greater than plastic in the Intervention with pump sprayer application treatment (P \u3c 0.05), fabric and rubber greater than plastic in the 10% bleach treatment (P \u3c 0.05); and fabric greater than plastic and rubber in the 10 hr downtime and gaseous fumigation treatments (P \u3c 0.05). There was a significant main effect of disinfectant treatment (P = 0.016), where 10% bleach had a greater Ct value compared to both the control treatment, 10 hr downtime treatment, and Intervention applied using the pump sprayer (P \u3c 0.05). There was a main effect of surface (P \u3c 0.0001) where rubber had a greater Ct value compared to plastic (P \u3c 0.05), and fabric had a greater Ct value compared to both rubber and plastic (P \u3c 0.05). Finally, the Ct value for PRRSV was greater than PEDV (P \u3c 0.0001) when averaged across all surfaces and disinfectant treatments. In summary, these data highlight that it is important to consider the surface of interest when implementing disinfectant protocols. In general, most disinfectant applications were only able to reduce the quantity of detectable virus, but not completely eliminate it from surface. However, additional research is necessary to understand the viability of residual virus on disinfected surfaces

Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P \u3c 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P \u3c 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

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

Complete Genome Sequences of Chop, DelRio, and GrandSlam, Three Gordonia Phages Isolated from Soil in Central Arkansas

Chop, DelRio, and GrandSlam are phage with a Siphoviridae morphotype isolated from soil in Arkansas using the host Gordonia terrae 3612. All three are temperate, and their genomes share at least 96% nucleotide identity. These phage are assigned to cluster DI based on gene content similarity to other sequenced actinobacteriophage

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

Determining factors of intent for sustainability implementation: An exploratory study of family business successors

Purpose – This study aims to dive into the minds of family business successors to identify factors that drive their intent to implement sustainable practices and introduce a framework that would best illustrate the emerging factors. Design/methodology/approach – This exploratory research incorporates the theory of planned behavior (Ajzen, 1985) as a conceptual model and grounded theory as the methodological approach (Glaser & Strauss, 1967) to determine the factors that influence the intent of family business successors to implement sustainable practices. Data was gathered by interviewing fifteen second-generation family business successors. Findings – Eight factors emerged that directly influence successors’ intent for sustainability implementation: Environment, Future Orientation, Business Improvement, Family Relations, Employees, Customers, Values and Mindset, and Community Development. Additionally, three moderating variables were also identified: Personal Resistance and Concerns, External Environmental Factors, and Financial Expense. Research limitations/implications – The study only extends to observable factors regarding the intent of successors to implement sustainable practices and does not consider whether participating respondents will implement such practices. The researchers also acknowledge that social desirability bias may have been present during the interview phase of the research. Additionally, the research is focused on proposing an emerging framework and utilizes a qualitative approach which leaves an opportunity for future research to validate and assign weights to the identified factors through quantitative means. Practical and social implications – Understanding the numerous factors that attribute to the implementation of sustainable practices is relevant to family businesses as it may serve as a guide on how these businesses would position themselves in the current economic and social landscape. Originality/value – This novel study provides a lens through which it assesses the role of successors in the implementation of sustainable business practices