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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 a co-inoculants of PRRSV (1 × 105 TCID50 per mL) and PEDV (1 × 105 TCID50 per 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 minute, 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 were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon was 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 > 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.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5′-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5′-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5′-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID50/ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1.

The overall objective of this study was to compare the efficacy of medium-chain fatty acids (MCFA) to other common fat sources to minimize the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination in a pig bioassay. Treatments were feed with mitigants inoculated with PEDV after application and were: 1) positive control with no chemical treatment; 2) 0.325% commercially available formaldehyde-based product; 3) 1% blend of 1:1:1 caproic (C6), caprylic (C8), and capric acids (C10) and applied with an aerosolizing nozzle; 4) treatment 3 applied directly into the mixer without an aerosolizing nozzle; 5) 0.66% caproic acid; 6) 0.66% caprylic acid; 7) 0.66% capric acid; 8) 0.66% lauric acid; 9) 1% blend of 1:1 capric and lauric acids; 10) 0.3% commercially available dry C12 product; 11) 1% canola oil; 12) 1% choice white grease; 13) 2% coconut oil; 14) 1% coconut oil; 15) 2% palm kernel oil; 16) 1% palm kernel oil; 17) 1% soy oil and four analysis days (0, 1, 3, and 7 post inoculation) as well as 1 treatment of PEDV-negative feed without chemical treatment. There was a treatment × day interaction (P < 0.002) for detectable PEDV RNA. The magnitude of the increase in Ct value from d 0 to 7 was dependent upon the individual treatments. Feed treated with individual MCFA, 1% MCFA blend, or commercial-based formaldehyde had fewer (P < 0.05) detectable viral particles than all other treatments. Commercial-based formaldehyde, 1% MCFA, 0.66% caproic, 0.66% caprylic, and 0.66% capric acids had no evidence of infectivity 10-d old pig bioassay, while there was no evidence the C12 commercial product or longer chain fat sources inhibited PEDV infectivity. Interestingly, pigs given the coconut oil source with the highest composition of caprylic and capric only showed signs of infectivity on the last day of bioassay. These data suggest some MCFA have potential for reducing post feed manufacture PEDV contamination.

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

New regulatory and consumer demands highlight the importance of animal feed as a part of our national food safety system. Porcine epidemic diarrhea virus (PEDV) is the first viral pathogen confirmed to be widely transmissible in animal food. Because the potential for viral contamination in animal food is not well characterized, the objectives of this study were to 1) observe the magnitude of virus contamination in an animal food manufacturing facility, and 2) investigate a proposed method, feed sequencing, to decrease virus decontamination on animal food-contact surfaces. A U.S. virulent PEDV isolate was used to inoculate 50 kg swine feed, which was mixed, conveyed, and discharged into bags using pilot-scale feed manufacturing equipment. Surfaces were swabbed and analyzed for the presence of PEDV RNA by quantitative real-time polymerase chain reaction (qPCR). Environmental swabs indicated complete contamination of animal food-contact surfaces (0/40 vs. 48/48, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05) and near complete contamination of non-animal food-contact surfaces (0/24 vs. 16/18, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05). Flushing animal food-contact surfaces with low-risk feed is commonly used to reduce cross-contamination in animal feed manufacturing. Thus, four subsequent 50 kg batches of virus-free swine feed were manufactured using the same system to test its impact on decontaminating animal food-contact surfaces. Even after 4 subsequent sequences, animal food-contact surfaces retained viral RNA (28/33 positive samples/total samples), with conveying system being more contaminated than the mixer. A bioassay to test infectivity of dust from animal food-contact surfaces failed to produce infectivity. This study demonstrates the potential widespread viral contamination of surfaces in an animal food manufacturing facility and the difficulty of removing contamination using conventional feed sequencing, which underscores the importance for preventing viruses from entering and contaminating such facilities.

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi.

As the product of interspecific hybridization between its two ancestral octoploid (2n = 8x = 56) species (Fragaria chiloensis and F. virginiana), the cultivated strawberry (F. ×ananassa) is among the most genomically complex of crop plants, harboring subgenomic components derived from as many as four different diploid ancestors. To physically visualize the octoploids’ subgenome composition(s), we launched molecular cytogenetic studies using genomic in situ hybridization (GISH), comparative GISH (cGISH), and rDNA-FISH techniques. First, GISH resolution in Fragaria was tested by using diploid and triploid hybrids with predetermined genome compositions. Then, observation of an octoploid genome was implemented by hybridizing chromosomes of pentaploid (2n = 5x = 35) hybrids from F. vesca × F. virginiana with genomic DNA probes derived from diploids (2n = 2x = 14) F. vesca and F. iinumae, which have been proposed by phylogenetic studies to be closely related to the octoploids yet highly divergent from each other. GISH and cGISH results indicated that octoploid-derived gametes (n = 4x = 28) carried seven chromosomes with hybridization affinities to F. vesca, while the remaining 21 chromosomes displayed varying affinities to F. iinumae, indicating differing degrees of subgenomic contribution to the octoploids by these two putatively ancestral diploids. Combined rDNA-FISH revealed severe 25S rDNA loss in both the F. vesca- and F. iinumae-like chromosome groups, while only the prior group retained its 5S loci.

Citation: Huss, A. R., Schumacher, L. L., Cochrane, R. A., Poulsen, E., Bai, J. F., Woodworth, J. C., . . . Jones, C. K. (2016). Proof-of-concept method to sanitize a feed mill contaminated with Porcine Epidemic Diarrhea Virus. Journal of Animal Science, 94, 102-103. doi:10.2527/msasas2016-217

Porcine Epidemic Diarrhea Virus (PEDV) was the first virus of wide scale concern to be linked to possible transmission by livestock feed or ingredients. Measures to exclude pathogens, prevent cross-contamination, and actively reduce the pathogenic load of feed and ingredients are being developed. However, research thus far has focused on the role of chemicals or thermal treatment to reduce the RNA in the actual feedstuffs, and has not addressed potential residual contamination within the manufacturing facility that may lead to continuous contamination of finished feeds. The purpose of this experiment was to evaluate the use of a standardized protocol to sanitize an animal feed manufacturing facility contaminated with PEDV. Environmental swabs were collected throughout the facility during the manufacturing of a swine diet inoculated with PEDV. To monitor facility contamination of the virus, swabs were collected at: 1) baseline prior to inoculation, 2) after production of the inoculated feed, 3) after application of a quaternary ammonium-glutaraldehyde blend cleaner, 4) after application of a sodium hypochlorite sanitizing solution, and 5) after facility heat-up to 60°C for 48 hours. Decontamination step, surface, type, zone and their interactions were all found to impact the quantity of detectable PEDV RNA (P < 0.05). As expected, all samples collected from equipment surfaces contained PEDV RNA after production of the contaminated feed. Additionally, the majority of samples collected from non-direct feed contact surfaces were also positive for PEDV RNA after the production of the contaminated feed, emphasizing the potential role dust plays in cross-contamination of pathogen throughout a manufacturing facility. Application of the cleaner, sanitizer, and heat were effective at reducing PEDV genomic material (P < 0.05), but did not completely eliminate it.