Effect of Benzoic Acid and Essential Oil Blends on Viral Load in Swine Feed and Vitamin Premix

Feed has been shown to harbor viable virus of interest to swine producers over an extended period of time. The use of mitigants and kill steps have been investigated with variable results. This study investigated the use of benzoic acid (BA) and an essential oil blend (EO) to mitigate the presence of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Senecavirus A (SVA) in a complete diet (Exp. 1) and a vitamin premix (Exp. 2). Four treatments consisting of 0.5% BA; 0.5% BA and 200 ppm EO; 0.3% BA and 120 ppm EO; and 0.25% BA and 100 ppm EO were used in the complete feed, in addition to a control with no feed additive to test the mitigant’s effect on PEDV, PRRSV, and SVA detection. For Exp. 2, a vitamin premix without chemical treatment acted as the control and the other treatment was the vitamin premix treated with 2.68% EO, with both used to determine PEDV detection. The inoculated feed or premix was stored for up to 15 d with sampling points at 2, 5, and 15 d post-inoculation. Samples were analyzed using a triplex qRT-PCR to detect changes in RNA quantities for all three viruses. A significant treatment × day interaction was observed in the feed for both PEDV (P = 0.008) and SVA (P \u3c 0.001). Per the decreased cycle threshold (Ct) value, the 0.5% BA treatment had higher (P \u3c 0.05) measurements of detectible PEDV on d 2 and 5, and lower amounts of detectible PEDV on d 15, as compared to the control. The 0.5% BA treated feed had lower (P \u3c 0.05) detectable SVA on d 2 but higher detectible SVA on d 15 compared to the control. There was no evidence of difference in detectable PRRSV between treatments. During this experiment, PEDV and SVA showed a degradation over time with rates of degradation varying between treatments. Increasing time from d 2 to 15 decreased (quadratic, P = 0.038) detectable PRRSV. The use of the EO in the vitamin premix had no evidence of a treatment × day interaction, treatment effect, or degradation over time. In conclusion, the use of 0.5% BA had an increased PEDV Ct on d 15 compared to the control (33.8 vs. 32.7 Ct, respectively). However, the use of BA and EO mitigant in this model did not provide consistent evidence for increased viral degradation, but viral load was reduced in the feed matrix over time

Effect of Benzoic Acid and Essential Oil Blends on Viral Load in Swine Feed and Vitamin Premix

Feed has been shown to harbor viable virus of interest to swine producers over an extended period of time. The use of mitigants and kill steps have been investigated with variable results. This study investigated the use of benzoic acid (BA) and an essential oil blend (EO) to mitigate the presence of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Senecavirus A (SVA) in a complete diet (Exp. 1) and a vitamin premix (Exp. 2). Four treatments consisting of 0.5% BA; 0.5% BA and 200 ppm EO; 0.3% BA and 120 ppm EO; and 0.25% BA and 100 ppm EO were used in the complete feed, in addition to a control with no feed additive to test the mitigant’s effect on PEDV, PRRSV, and SVA detection. For Exp. 2, a vitamin premix without chemical treatment acted as the control and the other treatment was the vitamin premix treated with 2.68% EO, with both used to determine PEDV detection. The inoculated feed or premix was stored for up to 15 d with sampling points at 2, 5, and 15 d post-inoculation. Samples were analyzed using a triplex qRT-PCR to detect changes in RNA quantities for all three viruses. A significant treatment × day interaction was observed in the feed for both PEDV (P = 0.008) and SVA (P \u3c 0.001). Per the decreased cycle threshold (Ct) value, the 0.5% BA treatment had higher (P \u3c 0.05) measurements of detectible PEDV on d 2 and 5, and lower amounts of detectible PEDV on d 15, as compared to the control. The 0.5% BA treated feed had lower (P \u3c 0.05) detectable SVA on d 2 but higher detectible SVA on d 15 compared to the control. There was no evidence of difference in detectable PRRSV between treatments. During this experiment, PEDV and SVA showed a degradation over time with rates of degradation varying between treatments. Increasing time from d 2 to 15 decreased (quadratic, P = 0.038) detectable PRRSV. The use of the EO in the vitamin premix had no evidence of a treatment × day interaction, treatment effect, or degradation over time. In conclusion, the use of 0.5% BA had an increased PEDV Ct on d 15 compared to the control (33.8 vs. 32.7 Ct, respectively). However, the use of BA and EO mitigant in this model did not provide consistent evidence for increased viral degradation, but viral load was reduced in the feed matrix over time

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

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P \u3e 0.10). An interaction between disinfectant × surface impacted (P \u3c 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P \u3e 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P \u3e 0.10). An interaction between disinfectant × surface impacted (P \u3c 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P \u3e 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent 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

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

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1–3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5–16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained. • A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections; • High-concentration target does not have apparent effect on detecting low-concentration targets; • Detection sensitivity were similar between nasal swab and lung tissue samples

Effect of general anaesthesia on functional outcome in patients with anterior circulation ischaemic stroke having endovascular thrombectomy versus standard care: a meta-analysis of individual patient data

Background: General anaesthesia (GA) during endovascular thrombectomy has been associated with worse patient outcomes in observational studies compared with patients treated without GA. We assessed functional outcome in ischaemic stroke patients with large vessel anterior circulation occlusion undergoing endovascular thrombectomy under GA, versus thrombectomy not under GA (with or without sedation) versus standard care (ie, no thrombectomy), stratified by the use of GA versus standard care. Methods: For this meta-analysis, patient-level data were pooled from all patients included in randomised trials in PuMed published between Jan 1, 2010, and May 31, 2017, that compared endovascular thrombectomy predominantly done with stent retrievers with standard care in anterior circulation ischaemic stroke patients (HERMES Collaboration). The primary outcome was functional outcome assessed by ordinal analysis of the modified Rankin scale (mRS) at 90 days in the GA and non-GA subgroups of patients treated with endovascular therapy versus those patients treated with standard care, adjusted for baseline prognostic variables. To account for between-trial variance we used mixed-effects modelling with a random effect for trials incorporated in all models. Bias was assessed using the Cochrane method. The meta-analysis was prospectively designed, but not registered. Findings: Seven trials were identified by our search; of 1764 patients included in these trials, 871 were allocated to endovascular thrombectomy and 893 were assigned standard care. After exclusion of 74 patients (72 did not undergo the procedure and two had missing data on anaesthetic strategy), 236 (30%) of 797 patients who had endovascular procedures were treated under GA. At baseline, patients receiving GA were younger and had a shorter delay between stroke onset and randomisation but they had similar pre-treatment clinical severity compared with patients who did not have GA. Endovascular thrombectomy improved functional outcome at 3 months both in patients who had GA (adjusted common odds ratio (cOR) 1·52, 95% CI 1·09–2·11, p=0·014) and in those who did not have GA (adjusted cOR 2·33, 95% CI 1·75–3·10, p<0·0001) versus standard care. However, outcomes were significantly better for patients who did not receive GA versus those who received GA (covariate-adjusted cOR 1·53, 95% CI 1·14–2·04, p=0·0044). The risk of bias and variability between studies was assessed to be low. Interpretation: Worse outcomes after endovascular thrombectomy were associated with GA, after adjustment for baseline prognostic variables. These data support avoidance of GA whenever possible. The procedure did, however, remain effective versus standard care in patients treated under GA, indicating that treatment should not be withheld in those who require anaesthesia for medical reasons