Swine Bioassay Research Articles

67 Decontamination of a feed manufacturing environment following inoculation of porcine epidemic diarrhea virus, porcine reproductive and respiratory virus, and Seneca Valley virus 1

Abstract Feed mill decontamination is difficult because equipment is not designed to be cleaned with water. Alternate strategies may improve the ability of a mill to decontaminate in the event of viral contamination. The objective was to evaluate different decontamination strategies within a mill following the inoculation of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca Valley virus 1 (SVV1) contaminated feed. A batch of feed was inoculated with PEDV, PRRSV, and SVV1 and ran through a mixer, bucket elevator, corn cleaner, screw conveyor, distributor head, and spout. Following mill inoculation, decontamination strategies were implemented with environmental samples collected after each decontamination step. Decontamination strategies included: 1) complete facility decontamination including organic matter removal with hot water power washing, disinfection with 1% peroxygen (Virkon S, Lanxess, Germany) followed by a rinse, and a second disinfection with 5% sodium hypochlorite (Clorox, Oakland, CA) followed by a heating period for 48 h once the facility reached 60°C; 2) chlorine dioxide application (ProOxine AH, Bio-Cide International, Inc., Norman, OK); 3) organic matter removal using vacuums (Ridge Tool Company, Elyria, OH) and chlorine dioxide application; 4) heat up with portable electric heaters for exactly 48 h; and 5) organic matter removal and heat up with portable heaters for exactly 48 h. Samples were analyzed via triplex PCR. Cycle threshold and proportion PCR positive were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). A swine bioassay was completed to determine the infectivity of samples following the final decontamination step of each treatment. A treatment × decontamination step × location interaction was observed (P < 0.05) for SVV1, where less RNA was detected post-treatment compared with pre-treatment following complete facility decontamination on surfaces including the mixer, corn cleaner, screw conveyor, and floor around the feed discharge (P < 0.05). There was no statistical difference between pre- and post-treatment samples for other combinations of decontamination treatment and location (P > 0.05), although numerical increases in Ct were generally observed following treatment. Across all treatments, the act of decontamination reduced detectable PEDV (P < 0.05) and PRRSV (P < 0.05) RNA when compared with samples immediately following inoculation, but complete facility decontamination was the only treatment where PEDV and PRRSV was non-detectable in all locations. Pigs given samples post-treatment showed no evidence of SVV1 or PEDV infection. Although PRRSV RNA was rarely found via PCR from samples collected within the mill, PRRSV infection was observed in pigs given the chlorine dioxide with and without organic matter removal treatments and the organic matter removal plus heat treatment. Overall, all treatments reduced detectable RNA for all viruses between the inoculation step and the final decontamination step; however, PRRSV particles remained infectious following decontamination regardless of PCR results in a swine bioassay.

Evaluation of Extended Storage of Swine Complete Feed for Inactivation of Viral Contamination and Effect on Nutritional, Microbiological, and Toxicological Profiles

The extended storage of feed ingredients has been suggested as a method to mitigate the risk of pathogen transmission through contaminated ingredients. To validate the approach of extended storage of complete swine feed for the inactivation of swine viruses, an experiment was conducted wherein swine feed was inoculated with 10 mL of 1 × 105 TCID50/mL of porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and Senecavirus A (SVA) and stored for 58 d at 23.9 °C. Measures of feed quality were also evaluated at the initiation and conclusion of the storage period including screening for mycotoxins, characterization of select microbiological measures, and stability of phytase and dietary vitamins. Storing feed for 58 d under either ambient or anaerobic and temperature-controlled storage conditions did not result in substantial concerns related to microbiological profiles. Upon exposure to the feed following 58 d of storage in a swine bioassay, previously confirmed naïve pigs showed no signs of PEDV or SVA replication as detected by the PCR screening of oral fluids and serum antibody screening. Infection with SVA was documented in the positive control room through diagnostic testing through the State of Minnesota. For PRRSV, the positive control room demonstrated infection. For rooms consuming inoculated feed stored for 58 d, there was no evidence of PRRSV infection with the exception of unintentional aerosol transmission via a documented biocontainment breach. In summary, storing feed for 58 d at anaerobic and temperature-controlled environmental conditions of 23.9 °C validates that the extended storage of complete swine feed can be a method to reduce risks associated with pathogen transmission through feed while having minimal effects on measures of nutritional quality.

Evaluation of Truck Cab Decontamination Procedures following Inoculation with Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus.

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest.

Characterization of Senecavirus A Isolates Collected From the Environment of U.S. Sow Slaughter Plants.

Vesicular disease caused by Senecavirus A (SVA) is clinically indistinguishable from foot-and-mouth disease (FMD) and other vesicular diseases of swine. When a vesicle is observed in FMD-free countries, a costly and time-consuming foreign animal disease investigation (FADI) is performed to rule out FMD. Recently, there has been an increase in the number of FADIs and SVA positive samples at slaughter plants in the U.S. The objectives of this investigation were to: (1) describe the environmental burden of SVA in sow slaughter plants; (2) determine whether there was a correlation between PCR diagnostics, virus isolation (VI), and swine bioassay results; and (3) phylogenetically characterize the genetic diversity of contemporary SVA isolates. Environmental swabs were collected from three sow slaughter plants (Plants 1-3) and one market-weight slaughter plant (Plant 4) between June to December 2020. Of the 426 samples taken from Plants 1-3, 304 samples were PCR positive and 107 were VI positive. There was no detection of SVA by PCR or VI at Plant 4. SVA positive samples were most frequently found in the summer (78.3% June-September, vs. 59.4% October-December), with a peak at 85% in August. Eighteen PCR positive environmental samples with a range of Ct values were selected for a swine bioassay: a single sample infected piglets (n = 2). A random subset of the PCR positive samples was sequenced; and phylogenetic analysis demonstrated co-circulation and divergence of two genetically distinct groups of SVA. These data demonstrate that SVA was frequently found in the environment of sow slaughter plants, but environmental persistence and diagnostic detection was not indicative of whether a sampled was infectious to swine. Consequently, a more detailed understanding of the epidemiology of SVA and its environmental persistence in the marketing chain is necessary to reduce the number of FADIs and aide in the development of control measures to reduce the spread of SVA.

Evidence of viral survival in representative volumes of feed and feed ingredients during long-distance commercial transport across the continental United States.

The hypothesis that feed ingredients could serve as vehicles for the transport and transmission of viral pathogens was first validated under laboratory conditions. To bridge the gap from the laboratory to the field, this current project tested whether three significant viruses of swine could survive in feed ingredients during long‐distance commercial transport across the continental US. One‐metric tonne totes of soybean meal (organic and conventional) and complete feed were spiked with a 10 ml mixture of PRRSV 174, PEDV and SVA and transported for 23 days in a commercial semi‐trailer truck, crossing 29 states, and 10,183 km. Samples were tested for the presence of viral RNA by PCR, and for viable virus in soy‐based samples by swine bioassay and in complete feed samples by natural feeding. Viable PRRSV, PEDV and SVA were detected in both soy products and viable PEDV and SVA in complete feed. These results provide the first evidence that viral pathogens of pigs can survive in representative volumes of feed and feed ingredients during long‐distance commercial transport across the continental United States.

Survival of swine pathogens in compost formed from preprocessed carcasses.

An introduction of a Foreign Animal Disease (FAD) like African Swine Fever Virus (ASF) would be financially devastating. For example, ASF, a highly contagious pathogen with high mortality rates, is a World Health Organization reportable disease that has recently been spreading across Asia and Europe. Control of ASF would likely require mass euthanasia of infected and exposed animals similar to the United Kingdom’s elimination of Foot and Mouth Disease (FMD). Subsequent disposal of infectious carcasses must adequately eliminate the virus and prevent further transmission of the disease. Although composting swine carcasses is widely used throughout the industry, limited data is available describing pathogen survival or elimination during this process. While current methods have evaluated the composting of swine carcasses under temperature‐controlled settings, they have not considered the effects of adverse weather conditions (e.g., cold winter conditions) where composting is routinely performed. This study utilized preprocessing (grinding) of swine carcasses prior to composting, which decreases the amount of required carbon material and land space. The ability of composting to reduce the level of viral nucleic acid during cold weather conditions and the risk of environmental contamination that may occur during preprocessing was evaluated. In this study, pigs challenged with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Epidemic Diarrhea Virus (PEDV), common domestic diseases, before euthanasia provided infectious carcasses containing pathogen surrogates. Composting of preprocessed carcasses achieved adequate temperatures necessary to eliminate FAD and common swine pathogens during cold weather conditions (monitored by compost temperature over time, virus diagnostic testing, and swine bioassay for PRRSV and PEDV). Under the conditions of this study, composting preprocessed carcasses presents minimal risk to air and groundwater contamination. In conclusion, composting preprocessed euthanized swine under adverse weather conditions is a safe and feasible option for mass disposal of infected carcasses.

UV-C irradiation is able to inactivate pathogens found in commercially collected porcine plasma as demonstrated by swine bioassay

Liquid porcine plasma is an animal origin raw material for the manufacturing process of spray-dried porcine plasma that is used in pig nutrition worldwide. In previous studies we found that the application of ultraviolet light C (UV-C) in liquid plasma that was inoculated with a variety of bacteria or viruses of importance in the swine industry can be considered as redundant safety steps because in general achieve around 4 logs reduction for most of these pathogens. However, the final validation of the UV-C light as safety feature should be conducted with commercial liquid plasma and using the pig bioassay model. As a first objective, the potential infectivity of a raw liquid plasma product collected from an abattoir was tested by means of a swine bioassay. We used Porcine circovirus 2 (PCV-2), a ubiquitous virus that has been systematically detected by PCR in porcine plasma at abattoirs as selection criteria for commercial liquid plasma lot. As a second aim of the study, the effects of different doses of UV-C irradiation on the selected raw liquid plasma were assayed in the animal bioassay. Moreover, other swine infecting agents, including Porcine reproductive and respiratory syndrome virus (PRRSV), were also determined in the original plasma and monitored in the inoculated animals. Pigs negative for PCV-2 and PRRSV genome and antibodies were allotted to one of five groups (6 to 8 pigs/ group) and injected intra-peritoneally with 10 mL of their assigned inoculum at 50 d of age. Negative control pigs (group 1) were injected with PBS. Positive control pigs (group 5) were injected with a PCV-2 inoculum. Groups 2, 3 and 4 were injected with liquid porcine plasma that had been subjected to 0 (raw plasma), 3000 or 9000 J/L UV-C irradiation, respectively. Group 2 pigs (0 J/L UV-C) got infection by PRRSV but no PCV-2 infection or seroconversion. However, one pig from group 2 seroconverted to Rotavirus A (RVA) and Hepatitis E virus (HEV) and three group 2 pigs seroconverted to Porcine parvovirus (PPV). Groups 1, 3 and 4 pigs showed no evidence of infection or seroconversion associated with the tested viruses or any other pathogens found in the liquid plasma before UV-C irradiation. Group 5 pigs developed PCV-2 infectivity as expected. UV-C irradiation of liquid plasma at 3000 and 9000 J/L was effective in preventing PRRSV and other pathogens transmission. Moreover, raw liquid plasma was non-infectious for PCV-2 in naïve pigs.

Survival of viral pathogens in animal feed ingredients under transboundary shipping models.

The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients (“high-risk combinations”) under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels.

Alkaline stabilization of manure slurry inactivates porcine epidemic diarrhea virus

Hydrated lime manure treatment was evaluated to determine porcine epidemic diarrhea virus (PEDV) susceptibility to alkaline stabilization. At pH 10, PEDV decreased (quantitative polymerase chain reaction) and lost infectivity (swine bioassay). Although ammonium decreased above pH 9 (up to 25%), alkaline stabilization managed to control potential infection from manure sources.

An evaluation of porcine epidemic diarrhea virus survival in individual feed ingredients in the presence or absence of a liquid antimicrobial.

BackgroundContaminated complete feed and porcine plasma are risk factors for PEDV introduction to farms and a liquid antimicrobial has been proven useful for reducing risk. This study provides information on the survivability of PEDV across common swine feed ingredients in the presence or absence of the liquid antimicrobial.ResultsEighteen ingredients commonly included in commercial swine diets were selected, including 3 grain sources (corn, soybean meal (SBM), dried distillers grains with solubles (DDGS)), 5 porcine by-products (spray-dried plasma, purified plasma, intestinal mucosa, meat and bone meal and red blood cells (RBCs)), 3 vitamin/trace mineral (VTM) mixes (sow, nursery, finishing), 2 fat sources (choice white grease and soy oil), 3 synthetic amino acids (lysine HCL, D/L methionine, threonine), as well as limestone and dry choline chloride. Complete feed and stock PEDV served as controls. Thirty grams of each ingredient were inoculated with 2 mL PEDV. A matched set of samples were treated with the formaldehyde-based liquid antimicrobial SalCURB® (LA). All samples (n = 320) were stored outdoors under winter time ambient conditions for 30 days. Samples were submitted on 1, 7, 14 and 30 days post-inoculation (DPI) and tested by PCR and virus isolation (VI). All VI-negative samples were tested by swine bioassay. Viable PEDV was detected by VI or swine bioassay at 1, 7, 14 and 30 DPI from SBM, DDGS, meat & bone meal, RBCs, lysine HCL, D/L methionine, choice white grease, choline chloride, complete feed and stock virus control and at 7 DPI in limestone and at 14 DPI in threonine. Supplementary testing of complete feed and SBM indicated viable virus out to 45 and 180 DPI, respectively. All other samples were negative by VI and bioassay. In contrast, treatment with LA inactivated PEDV across all ingredients on 1 DPI and induced RNA reduction over time.ConclusionsUnder the conditions of this study, PEDV viability in feed was influenced by ingredient with extended survival in SBM. Furthermore, LA treatment rendered virus inactive, independent of ingredient type.

An evaluation of ultraviolet light (UV 254) as a means to inactivate porcine reproductive and respiratory syndrome virus on common farm surfaces and materials

A study was conducted to assess the effect of UV 254 on the concentration and viability of PRRSV on surfaces and materials commonly encountered on swine farms. A standard quantity (5 × 10 6 TCID 50, total dose) of a PRRSV modified live vaccine virus was inoculated onto 2 matched sets of surfaces/materials including wood, plastic, latex, rubber, styrofoam, metal, leather, cloth, concrete, cardboard, glass and paper. One set was exposed to UV 254 radiation (treatments) and the other to incandescent light (controls) for a 24 h period. During this time, treatments and controls were swabbed at 10 min intervals from 0 to 60 min post-inoculation (PI) and again at 24 h PI. The quantity of PRRSV RNA on each item at each sampling time was calculated by RT-PCR and the presence of viable PRRSV in each sample was determined by swine bioassay. A significant reduction ( p < 0.0001) in the quantity of PRRSV RNA was demonstrated at 24 h PI independent of treatment. In addition, a significant reduction ( p = 0.012) in the number of UV 254-treated surfaces which harbored viable virus was observed at 60 min (0/12 positive) when compared to control surfaces (5/12 positive). In addition, all UV 254 treated samples collected between 10 and 50 min PI were bioassay negative. These results suggest that UV 254 is an effective means to inactivate PRRSV on commonly encountered farm surfaces and materials and inactivation can be accomplished following 10 min of exposure.

Efficacy of experimentally produced spray-dried plasma on infectivity of porcine circovirus type 21

The value of incorporating spray-dried plasma (SDP) into the diet of weanling pigs to improve feed intake and growth performance has been well documented. However, limited work has been done to confirm that the spray-drying process eliminates all viral contaminates including porcine circovirus type 2 (PCV2). To determine the effect of spray-drying on PCV2 infectivity, colostrum-fed, crossbred, specific-pathogen-free (SPF) pigs were inoculated with PCV2-contaminated SDP intraperitoneally (SDP-IP) or by oral gavage (SDP-OG), inoculated intraperitoneally with PCV2-positive plasma (POS), or left uninoculated (NEG). The plasma used for the experimentally produced SDP was collected from a SPF pig experimentally infected with a PCV2b isolate. Pigs in the NEG group remained seronegative, and PCV2 viremia was not detected. All pigs in the POS group became PCV2 viremic by 14 d postinoculation (DPI) and seroconverted by 28 DPI. In the SDP-IP group, all pigs became viremic by 35 DPI and seroconverted by 49 DPI. In the SDP-OG group, all animals became viremic by 35 DPI and 2/3 pigs seroconverted by 35 DPI. There were no significant (P > 0.05) differences between anti-PCV2-IgG antibody sample-to-positive ratios among pigs in the POS, SDP-OG, or SDP-IP groups. This work provides direct evidence that the experimental spray-drying process used in this study was not effective in inactivating PCV2b in the plasma of a PCV2-infected pig based on a swine bioassay using PCV2-naïve pigs. This work suggests that SDP sourced from pigs could represent a biosecurity risk for the industry.

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.

Cross‐species infection of specific‐pathogen‐free pigs by a genotype 4 strain of human hepatitis E virus

Hepatitis E virus (HEV) is an important pathogen. The animal strain of HEV, swine HEV, is related to human HEV. The genotype 3 swine HEV can infect humans and genotype 3 human HEV can infect pigs. The genotype 4 swine and human HEV strains are genetically related, but it is unknown whether genotype 4 human HEV can infect pigs. A swine bioassay was utilized in this study to determine whether genotype 4 human HEV can infect pigs. Fifteen, 4-week-old, specific-pathogen-free pigs were divided into three groups of five each. Group 1 pigs were each inoculated intravenously with PBS buffer as negative controls, group 2 pigs similarly with genotype 3 human HEV (strain US-2), and group 3 pigs similarly with genotype 4 human HEV (strain TW6196E). Serum and fecal samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days postinoculation (dpi) and tested for evidence of HEV infection. All pigs were necropsied at 56 dpi. As expected, the negative control pigs remained negative. The positive control pigs inoculated with genotype 3 human HEV all became infected as evidenced by detection of HEV antibodies, viremia and fecal virus shedding. All five pigs in group 3 inoculated with genotype 4 human HEV also became infected: fecal virus shedding and viremia were detected variably from 7 to 56 dpi, and seroconversion occurred by 28 dpi. The data indicated that genotype 4 human HEV has an expanded host range, and the results have important implications for understanding the natural history and zoonosis of HEV.

Inactivation of infectious hepatitis E virus present in commercial pig livers sold in local grocery stores in the United States

Hepatitis E virus (HEV) is a zoonotic pathogen and pigs are a known reservoir. Recently we showed that approximately 11% of commercial pig livers sold in local U.S. grocery stores for food consumptions are contaminated by infectious HEV. In this study, a swine bioassay was used to determine if the infectious HEV in contaminated commercial pig livers could be inactivated by traditional cooking methods. Group 1 pigs ( n = 5) were each inoculated intravenously (I.V.) with a HEV-negative liver homogenate as negative controls, group 2 pigs ( n = 5) were each inoculated I.V. with a pool of two HEV-positive pig liver homogenates as positive controls, groups 3, 4 and 5 pigs ( n = 5, each group) were each inoculated I.V. with a pool of homogenates of two HEV-positive livers incubated at 56 °C for 1 h, stir-fried at 191 °C (internal temperature of 71 °C) for 5 min or boiled in water for 5 min, respectively. As expected, the group 2 positive control pigs all became infected whereas the group 1 negative control pigs remained negative. Four of the five pigs inoculated with HEV-positive liver homogenates incubated at 56 °C for 1 h also became infected. However, pigs in groups 4 and 5 did not become infected. The results indicated that HEV in contaminated commercial pig livers can be effectively inactivated if cooked properly, although incubation at 56 °C for 1 h cannot inactivate the virus. Thus, to reduce the risk of food-borne HEV transmission, pig livers must be thoroughly cooked.

Evaluation of an industry-based sanitation protocol for full-size transport vehicles contaminated with porcine reproductive and respiratory syndrome virus

Objective: To test a protocol for sanitation of full-size commercial transport vehicles contaminated with porcine reproductive and respiratory syndrome virus (PRRSV), utilizing conditions found on commercial swine production units. Conditions included use of cold water for washing (21°C), application of a commercial disinfectant via a low-pressure foamer, and rapid completion of ≤ 2 hours. Materials and methods: Fifteen sites in a trailer were experimentally contaminated with IngelVac PRRS MLV vaccine (Boehringer Ingelheim Vetmedica Inc, St Joseph, Missouri; total of 5 × 105 median tissue culture infectious doses per site). Ten replicates were conducted. The presence or absence of PRRSV RNA was evaluated by polymerase chain reaction (PCR) testing of swabs taken from the trailer’s interior before treatment and 120 minutes post treatment. Swabs that were PCR-positive were then evaluated for viable PRRSV by swine bioassay. Treatment consisted of washing with cold water then disinfecting with a 1% solution of modified potassium monopersulfate applied via low-pressure foaming. The trailer was not dried. Results: In 10 of 150 samples collected across the 10 replicates, PRRSV RNA was detected 120 minutes post treatment. Differences in the percentages of PCR-positive swabs collected at 0 and 120 minutes post treatment in treatment and control replicates were significant (P &lt; .001; Fisher’s exact test). Viable virus was not detected by swine bioassay. Implication: High-pressure washing of transport trailers, followed by 120 minutes exposure to 1% modified potassium monopersulfate applied with a hydrofoamer, will most likely eliminate residual infectious PRRSV

Evaluation of an industry-based sanitation protocol for transport vehicles contaminated with porcine reproductive and respiratory syndrome virus

Objective: To test a protocol, using conditions found on commercial swine production units, for sanitation of 1:150 scale models of commercial transport vehicles contaminated with porcine reproductive and respiratory syndrome virus (PRRSV). Methods: Model trailers were experimentally contaminated with PRRSV MN 30-100 and either pressure-washed with cold water alone (Treatment 1) or washed and then disinfected with modified potassium monopersulfate (Treatment 2), quaternary ammonium chloride (Treatment 3), or a phenolic product (Treatment 4), each applied using a hydrofoamer. In Phase One, the presence of PRRSV RNA was evaluated by polymerase chain reaction (PCR) testing of swabs collected from the trailers’ interiors immediately after washing and 30, 60, 90, and 120 minutes post treatment. In Phase Two, the presence of viable PRRSV was evaluated by swine bioassay (injection of supernatants from PCR-positive swabs) and housing pairs of sentinel pigs in treated trailers for 2 hours, beginning 90 minutes post treatment. Results: Swabs were PCR-positive 120 minutes post treatment in 18 of 20 trailers receiving Treatment 1, none of 20 trailers receiving Treatments 2 and 3, and two of 20 trailers (suspect reactions) receiving Treatment 4. Viable virus was detected both by swine bioassay and sentinel pig exposure protocols in trailers receiving Treatment 1, and by swine bioassay alone in trailers receiving Treatment 4. Implications: High-pressure washing of transport trailers, followed by 90 to 120 minutes exposure to either modified potassium monopersulfate or quaternary ammonium chloride disinfectants applied with a hydrofoamer is likely to eliminate residual infectious PRRSV.

Lack of evidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection in domestic swine in Brazil

This report describes the first prevalence of antibodies and experimental inoculation of suspected samples of porcine reproductive and respiratory syndrome virus (PRRSV) from ELISA positive pigs from swine herds in Brazil. Based on the hypothesis that this agent is present in swine herds worldwide, the objective of this work was to establish a diagnostic methodology and to investigate the occurrence of PRRSV in Brazilian swine herds. Fifty-four swine herds, the total number which imported genetic material (live pigs or swine semen) from countries where PRRS was endemic from 1990 to December 2000, from eight Brazilian States all included in this study. The sampling used was such as to detect a prevalence of infection of 5%, with a confidence level of 95%. A total of 3785 serum samples were tested for PRRSV antibodies by ELISA. Following the ELISA test, which was performed with two different commercial kits, all serum positive pigs were retested, examined and additional materials were collected. Viral isolation in permissive tissue culture cells and swine bioassays were performed. Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR were also performed. We could not demonstrate the presence of PRRSV or RNA of PRRSV by viral isolation or RT-PCR (or nested RT-PCR), respectively in all of the analyzed samples. Furthermore, the pigs inoculated with PRRSV suspicion samples did not seroconvert nor produce characteristic PRRS lesions in the swine bioassay. Thus, our results indicate no evidence of PRRSV in the samples analyzed from swine herds in this study.

Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus.

Understanding the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) persistence in individual pigs is essential to the development of successful control programs. The objectives of this study were to investigate the proportion of inoculated pigs that become persistently infected with PRRSV and the duration of their infection. Additionally, different diagnostic techniques that detect persistent infections were compared. Twenty-eight 35-day-old pigs were inoculated with PRRSV. Serum and tonsil biopsy samples were collected on days 0, 7, 14, and 28 and then approximately monthly thereafter until day 251 postinoculation (p.i.). Tonsil, lymph node, and lung samples were collected following euthanasia on day 251 p.i. Virus was isolated from serum and tonsil biopsy samples that had been collected through days 28 and 56 p.i., respectively. Viral RNA was detected by reverse transcription (RT)-PCR in serum and tonsil biopsy samples that had been collected through day 251 p.i., although no serum samples collected from days 84 to 196 p.i. were positive and the presence of infectious PRRSV was not detected by swine bioassay of tissue samples collected at necropsy. The results confirmed that RT-PCR is more sensitive than virus isolation in identifying PRRSV-infected pigs. Six pigs that were persistently infected through days 225 or 251 p.i. remained seropositive throughout the study, although one pig had an enzyme-linked immunosorbent assay sample-to-positive ratio that was only slightly above the cutoff value of 0.40. Twenty of 28 tonsil biopsy samples collected on day 84 p.i. were positive by RT-PCR compared to only 1 positive biopsy sample out of 28 collected on day 119 p.i. The study's results indicate that most pigs clear PRRSV within 3 to 4 months, but that some may remain persistently infected for several months.

Characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS) virus infection results in clinically normal, but persistently infected animals. An understanding of the carrier state is necessary for prevention, control and/or elimination of PRRS virus. The objective of this experiment was to estimate the proportion of PRRS virus carriers over time and determine which combination of sample and diagnostic assay could most effectively identify persistently infected animals. Sixty 3-week-old pigs were inoculated with PRRS virus ATCC VR-2332 and followed for up to 105 days post-inoculation (PI). Sixty age-matched animals served as uninoculated controls. Samples (serum, peripheral blood leukocytes, oropharyngeal scrapings, tonsil, bronchoalveolar lavage, lung tissue and tracheobronchial lymph nodes) were collected periodically and tested for evidence of PRRS virus infection by virus isolation (VI), swine bioassay and reverse transcriptase-nested polymerase chain reaction (RT-nPCR). The PRRS virus-specific antibody response was monitored with a commercial enzyme-linked immunosorbent assay (ELISA). Overall, PRRS virus was found in 51 of the 59 (84%) necropsied animals by VI or swine bioassay between 63 and 105 days PI, including 10 of the 11 (91%) of animals at day 105 PI. RT-nPCR on oropharyngeal scrapings was the most effective combination of assay and sample for detecting carriers. There was no significant difference in the antibody response of carrier vs. non-carrier animals. Infectious PRRS virus is present in most pigs the first 105 days following infection. Antibody response, as measured by a commercial ELISA, cannot be used to determine carrier status. RT-nPCR is a useful tool for detection of carriers, but diagnostic sample selection is critical if false negative results are to be avoided.