Evaluation of 4 intervention strategies to prevent the mechanical transmission of porcine reproductive and respiratory syndrome virus

Transcription

1 Article Evaluation of 4 intervention strategies to prevent the mechanical transmission of porcine reproductive and respiratory syndrome virus Scott Dee, John Deen, Carlos Pijoan A b s t r a c t Four intervention strategies were tested for their ability to prevent the mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV): the use of disposable plastic boots to prevent contamination of personal footwear, the use of boot baths to disinfect PRRSV-contaminated plastic boots, the use of plastic slatted (Polygrate) flooring in the anteroom to prevent PRRSV contamination of incoming personal footwear, and the use of bag-in-a-box shipping methods to prevent PRRSV contamination of the contents of a container destined for a swine farm. Ten PRRSV-positive replicates and 10 PRRSV-negative (sham-inoculated) replicates were used for each strategy. Swabs were collected from selected sites and tested by TaqMan polymerase chain reaction for PRRSV RNA and by swine bioassay to confirm the presence of infectious PRRSV. Results indicated that the use of disposable boots, bleach boot baths or bag-in-a-box shipping methods was highly efficacious in preventing mechanical transmission of PRRSV. In contrast, the use of Polygrate flooring in the anteroom did not prevent contamination of personal footwear. The numbers of PRRSV-positive samples from the Polygrate surface and the soles of incoming footwear placed directly on the Polygrate surface were not significantly different (P = 0.24) from those of footwear that directly contacted the floor of the contaminated anteroom. Although these results are promising, this study should be considered a pilot project and the intervention strategies not considered biosecurity protocols. The model used may or may not represent field conditions. Therefore, the information should be used to develop larger experimental studies, with sufficient statistical power, in combination with field-based epidemiologic studies to better assess the role of mechanical transmission of PRRSV under field conditions. R é s u m é Quatre stratégies d intervention ont été vérifiées quant à leur capacité à prévenir la transmission mécanique du virus du syndrome reproducteur et respiratoire porcin (PRRSV) : l utilisation de bottes en plastique jetables pour prévenir la contamination des chaussures du personnel, l utilisation de pédiluves afin de désinfecter des bottes de plastique contaminées à l aide de PRRSV, l utilisation d un plancher latté en plastique (Polygrate) dans l anti-chambre afin d empêcher une contamination par le PRRSV des souliers du personnel entrant, et l utilisation de méthodes d expédition «bag-in-a-box» pour empêcher la contamination du contenu d un contenant destiné à une ferme porcine. Pour chacune des stratégies, 10 réplications avec PRRSV et 10 réplications sans PRRSV ont été faites. Des écouvillonnages de sites sélectionnés ont été faits et la présence d ARN du PRRSV vérifiée par TaqMan PCR et la présence de PRRSV infectieux confirmée par bio-essai chez le porc. Les résultats indiquent que l utilisation de bottes jetables, de pédiluves contenant de l hypochlorite ou l expédition «bag-in-a-box» étaient très efficaces pour prévenir la transmission mécanique du PRRSV. Au contraire, l utilisation du plancher Polygrate dans l antichambre n a pas permis d empêcher la contamination des chaussures personnelles. Le nombre d échantillons positifs pour PRRSV à partir de la surface Polygrate et de la semelle des chaussures entrantes placées directement sur la surface de Polygrate n étaient pas significativement différentes (P = 0,24) du nombre obtenu en plaçant les chaussures directement sur le plancher de l antichambre contaminée. Bien que ces résultats soient prometteurs, cette étude n est qu un projet pilote et les stratégies d intervention étudiées ne devraient être considérées comme des protocoles de biosécurité. Le modèle utilisé pourrait ou non représenter des conditions de terrain. Ainsi, les informations devraient être utilisés pour développer une étude expérimentale élargie, avec suffisamment de puissance statistique, en combinaison avec des études épidémiologiques sur le terrain afin de mieux évaluer le rôle de la transmission mécanique du PRRSV dans des conditions de terrain. (Traduit par Docteur Serge Messier) Swine Disease Eradication Center, University of Minnesota College of Veterinary Medicine, Room 385C, 1988 Fitch Avenue, St. Paul, Minnesota 55108, USA. Address all correspondence and reprint requests to Dr. Scott Dee; telephone: (612) ; fax: (612) ; deexx004@umn.edu Received May 15, Accepted July 14, ;68:19 26 The Canadian Journal of Veterinary Research 19

2 I n t r o d u c t i o n A thorough understanding of routes of transmission of porcine reproductive and respiratory syndrome virus (PRRSV) is critical for successful prevention, control, and eradication of the syndrome. Known routes of PRRSV transmission between swine farms include infected pigs and semen, contaminated fomites (boots, needles, and coveralls), mosquitoes, and houseflies (1 6). Recently, mechanical transmission of the virus throughout a coordinated sequence of events that replicated the common behaviour of farm workers during warm weather (10 C to 16 C) and cold weather ( 9 C to 2 C) was assessed with the use of field-based models (7,8). Both models involved fomites (boots and shipping containers), vehicle sanitation, transport, and personnel movement. Mechanical transmission of PRRSV was detected in 8 out of 10 replicates during cold weather and in 2 out of 10 replicates during warm weather. In both experiments, infectious PRRSV (identified by virus isolation or swine bioassay) was recovered from the concrete floor of livestock truck washes, from the floor mats in the transport vehicle, and from the soles of personal footwear. It was also shown that infectious PRRSV could be transported via contaminated fomites into a simulated farm setting, resulting in contamination of the floor of the farm anteroom and the ventral surfaces of containers after contact with the contaminated anteroom floor. From these results, risk factors for the mechanical transmission of PRRSV in commercial swine production systems were identified; these included the interior of vehicles, truck wash floors, personal footwear, the anteroom floor, and shipping containers. The purpose of this study was to measure the efficacy of intervention strategies to prevent mechanical transmission of PRRSV by minimizing the risk factors identified with the previous model. The specific aims of the study were as follows. 1. Assess the ability of disposable plastic boots to prevent contamination of personal footwear. 2. Evaluate the efficacy of boot baths for disinfecting PRRSVcontaminated plastic boots in the presence or absence of fecal material. 3. Determine whether the use of Polygrate flooring (Faroex; Gimli, Manitoba) in the anteroom could prevent PRRSV contamination of incoming footwear. 4. Test the efficacy of bag-in-a-box shipping methods for preventing PRRSV contamination of the contents of a container destined for a swine farm. Definitions M a t e r i a l s a n d m e t h o d s The specific components of the model were defined as follows: A carrier was defined as a medium that enhances the survivability of PRRSV outside of the pig. From data in the previous cold-weather study, snow and water were again selected as carrier materials and were inoculated with PRRSV as before (7). The components of the interior of a transport vehicle that were specific for this study were the floor mats, the steering wheel, and the accelerator and brake pedals. The contamination point was defined as the site in which Figure 1. Anteroom at the research farm of the University of Minnesota Swine Disease Eradication Center. study personnel contacted contaminated carriers during vehicle cleaning. The concrete floor of the garage at the research farm of the University of Minnesota Swine Disease Eradication Center (SDEC) was used to mimic the concrete floor of a commercial truck wash. The anteroom was defined as the area encountered immediately upon entering the front doorway of a swine farm using a shower-in, shower-out procedure (Figure 1). The anteroom at the SDEC research farm was used for this purpose. In most commercial swine facilities, the anteroom is separated from the farm office by a wall and a passthrough window. To enter the office, personnel must take a shower and don boots and coveralls designated by the farm. Because of this physical separation, the anteroom is considered contaminated and the office considered free of pathogens. Two pair of boots (men s 25.4-cm outdoor pull-up boots, IC ; Cabela s, Sydney, Nebraska, USA), the same as used in the previous studies (7,8), were used throughout this study by study personnel and referred to as personal footwear. A box of disposable tie boots made of plastic 6 mm thick was purchased for the study (Professional Veterinary Products, Omaha, Nebraska, USA); this type of disposable footwear is commonly used by commercial swine producers and livestock vehicle operators. Four sections of Polygrate flooring were used in the study. Each section was made of plastic and was cm. The dorsal surface contained 78 perforations that were cm and 24 that were cm. Each section contained interlocking edges, which allowed the 4 sections to be joined. Each side of a section contained a raised edge, which elevated the Polygrate 6 cm off the anteroom floor. Finally, to replicate a method used to introduce animal health products to commercial farms, we used the Sno Ball Pak Biosecure system (Newport Laboratories, Worthington, Minnesota, USA). The bag-in-a-box container consisted of a cardboard box lined with Styrofoam (36 cm long, 31 cm high, and 3.8 cm wide) that contained a plastic bag holding 12 plastic 250-mL bottles full of sterile water, simulating animal health products. 20 The Canadian Journal of Veterinary Research 2000;64:0 00

3 Experimental design Study personnel and inoculation of carriers Two people (persons A and B) were involved in the field phases of the study. All laboratory phases were conducted at the Minnesota Veterinary Diagnostic Laboratory. For construction of carriers, 15 ml of nonchlorinated well water was added to 150 g of snow. The materials were manually compressed into the shape of a sphere averaging 25.4 cm in circumference; each sphere weighed approximately 115 g and had a core temperature of 0 C. A field strain of PRRSV (MN ) was used throughout the study to inoculate carriers. This isolate had been recovered from a chronically infected sow after an acute outbreak of PRRS within a commercial swine system (9) and had been used in the previous studies (7,8). For inoculation of carriers, 1 ml ( TCID 50 /ml) of the isolate (third passage) was injected into the centre of each carrier with a 3-mL syringe (Monoject; Becton- Dickinson, St. Louis, Missouri, USA). The same concentration and passage had been used in the previous studies (7,8). Along with PRRSV-inoculated carriers (virus-positive carriers), sham-inoculated carriers (virus-negative carriers) were used for each replicate, each receiving 1 ml of sterile minimum essential medium (MEM). Intervention 1: Use of disposable plastic boots to prevent PRRSV contamination of personal footwear This study was conducted on the concrete garage floor of the SDEC research farm at an environmental temperature of 2 C. To initiate the study, a PRRSV-positive carrier was constructed as described and placed on the concrete floor approximately 1 m from the driver s door of the vehicle. Person A donned disposable plastic boots over personal footwear and stepped on the carrier, crushing it with both feet. Person A then entered the vehicle, posterior first, and sat on the driver s seat with feet and legs extending from the cab. The disposable boots were then removed by grasping the heel of each boot and pulling it off the personal footwear. The soles of the disposable boots were swabbed with sterile Dacron swabs (Fisher Scientific, Hanover Park, Illinois, USA) that had been moistened with MEM; the swabs were drawn from the toe region down to the heel, in a left-to-right zigzag pattern, such that the swab contacted the entire ventral surface. The disposable boots were then placed in a plastic garbage bag. The feet and legs of person A were drawn into the cab, and person A assumed a driving position. The right foot contacted the accelerator pedal, the left foot contacted the brake pedal, and both hands grasped the steering wheel. The right hand was placed in the 2 o clock position and the left hand in the 10 o clock position. The positions were held for 10 s, and then swabs were collected from the soles of the personal footwear, both pedals, and the steering wheel, as described for the soles of the disposable boots. All swabs were placed in plastic tubes (Falcon, Franklin Lakes, New Jersey, USA) containing 3 ml of MEM, stored on ice, and delivered to the Minnesota Veterinary Diagnostic Laboratory for testing. Between each of the 10 PRRSV-positive replicates, a sham-inoculated (negative-control) replicate was conducted with the use of carriers inoculated with MEM that was void of PRRSV. For positive controls, the procedure was repeated 10 times without the use of disposable plastic boots. Between all replicates, tested surfaces and hands were disinfected with ammonia spray (Lysol; Reckitt Benckiser, Wayne, New Jersey, USA), wiped with paper towels, and allowed to dry for 10 min. Lysol spray was selected because of its commercial availability and widespread use on farms and in veterinary practices. Intervention 2: Use of boot baths to disinfect PRRSV-contaminated plastic boots in the presence or absence of fecal material This study was conducted in the anteroom of the SDEC research farm and consisted of 2 phases. Phase 1 evaluated boot-bath efficacy in the absence of fecal contamination; phase 2 evaluated boot-bath efficacy after exposure to feces. During both phases, the anteroom temperature was set at 20ºC and a PRRSV carrier was allowed to melt on the linoleum anteroom floor, forming an aqueous pool. For phase 1, a boot bath consisting of 6% sodium hypochlorite (undiluted Clorox bleach; Clorox Company, Oakland, California, USA) in a round plastic pan was placed on the floor directly behind the PRRSVpositive carrier pool. This product was selected primarily for its low cost, its commercial availability, and its frequency of use on farms. Person A donned disposable plastic boots and stepped into the carrier pool for 5 s, then swabbed the soles of the plastic boots as previously described. Person A then immersed the boots in the boot bath for 5 s, removed the boots, and swabbed the soles of the disposable plastic boots and personal footwear; a 5-mL sample of the boot-bath liquid was also collected. The process was repeated 10 times, each time with new boots. Between replicates, the boot bath was emptied and then refilled with new bleach solution. To test the effect of the bleach in the boot bath, 10 replicates of the procedure were conducted with the use of a disinfectant-free (water-only) boot bath. The same types of samples were collected during each replicate. Negative controls consisted of 10 replicates with bleach boot baths, disposable plastic boots, and sham-inoculated carriers. Shaminoculated replicates were conducted with a different boot-bath pan, between PRRSV-positive replicates. Swabs from all replicates were collected and processed as previously described. Phase 2 was similar to phase 1 except that person A stepped into a pile (908 g) of swine feces before coming in contact with the PRRSV-positive pool. The feces had been obtained from a PRRSVnaïve farm and were placed directly in front of the area of the anteroom that was contaminated with a melted PRRSV-positive carrier pool. After contact with first feces and then PRRSV, person A swabbed the sole of each boot, went through the boot-bath disinfection process, and once again sampled the plastic boots and collected a 5-mL liquid sample, all as previously described. Samples were immediately placed on ice and submitted to the Minnesota Veterinary Diagnostic Laboratory for testing. After testing, the samples were stored at 70 C. No attempts were made to neutralize the disinfectant after sampling. The disposable plastic boots and the boot-bath liquid were changed between replicates. After 10 replicates, the entire area was disinfected with undiluted bleach, all boot baths were emptied and cleaned, and the process was repeated with a water-only boot bath. Once again, each replicate was conducted after a change of boots and boot-bath liquid. Between PRRSVpositive replicates, sham-inoculated negative-control replicates were again conducted with the use of fecal exposure and boot baths with bleach. All samples were processed as previously described. Intervention 3: Use of Polygrate flooring in the anteroom to prevent PRRSV contamination of incoming personal footwear This study utilized the SDEC concrete garage floor and the linoleum floor of the anteroom. Four interlocking sections of the previously described 2000;64:0 00 The Canadian Journal of Veterinary Research 21

4 Polygrate flooring were placed on the anteroom floor, directly adjacent to the entry door. Prior to anteroom entry, the soles of the personal footwear of person A contacted a PRRSV-positive carrier placed on the concrete garage floor immediately outside the entry door. Carriers were crushed underfoot in an effort to force residual snow and ice into the boot treads. Person A then opened the door and removed the footwear, placing both boots on the Polygrate floor. The footwear was allowed to sit at 20 C for 15 min to promote melting of ice and snow in the treads, then was removed from the Polygrate surface. Person A then placed an identical pair of unused boots on the Polygrate surface for 15 min. The soles of both pairs of boots were swabbed, as was any area of moisture observed on the Polygrate surface. Swabs were also inserted through the perforations of the Polygrate floor to sample areas of moisture on the underlying linoleum floor secondary to dripping from the PRRSV-contaminated footwear. This procedure was repeated 10 times, and shaminoculated replicates were conducted between each PRRSV-positive replicate. Positive controls consisted of replication of the procedure with the use of PRRSV-positive carriers but not Polygrate flooring, all personal footwear being placed directly on the anteroom floor. After each PRRSV-positive replicate, the personal footwear, Polygrate floor, and linoleum floor were disinfected with Lysol spray, wiped with paper towels, and allowed to dry. Samples were collected and processed as previously described. Intervention 4: Use of bag-in-a-box shipping methods to prevent PRRSV contamination of the contents of a container This study was conducted with the use of the SDEC research anteroom and an adjacent closet area modified to mimic the design of an office in a commercial swine facility. A sheet of plywood 1.5 m high was nailed to a doorframe of the closet. This physical barrier allowed for separation of the clean office (closet) from the dirty anteroom. Person A was stationed on the anteroom side to act as a delivery person. Person B was stationed in the office area to simulate a farm employee who had entered the farm via the shower and was now on the clean side of the farm. The open area above the plywood wall served as the pass-though window. For the first phase, a bag-in-a-box shipping container with 12 bottles of sterile water (250 ml each) was used to simulate a shipment of animal health products destined for entry into the farm. The container consisted of a cardboard box with an interior plastic bag that contained the bottles. Person A melted a PRRSVpositive carrier on the anteroom floor and set the container in the middle of the pool for 10 s to contaminate the exterior of the box. Person A then opened the container, raising it to the level of the pass-through window. At that point, person B grasped the bag and pulled it from the box and into the office area, avoiding contact with the box. Person B then opened the bag and removed the contents. Swabs were collected from the box, the bag, the contents of the bag, and the hands of the personnel and processed as previously described. This procedure was repeated 10 times, each replicate using a new bag-in-a-box container. The study was repeated without the use of an interior bag (phase 2). Person A contaminated the ventral surface of the box as before, then handed the box to person B, who took it into the office and placed it on a table (site of box placement). Person B then opened the box and removed the contents. Swabs were collected from the site of box placement, the box, the box s contents, and the hands of the personnel and processed as previously described. Ten replicates were conducted, each with the use of a new box. Samples were placed on ice and immediately delivered to the laboratory for testing. Ten sham-inoculated negative-control replicates were conducted between each PRRSV-positive replicate as before. Between each replicate, the office area was disinfected with undiluted bleach solution and Lysol spray. Both persons washed and disinfected their hands between replicates. Diagnostic analysis Upon arrival at the laboratory, samples were centrifuged at 1500 g for 15 min, and supernatants were tested for PRRSV nucleic acid by polymerase chain reaction (PCR), with the TaqMan PCR assay (Perkin-Elmer Applied Biosystems, Foster City, California, USA) (10). Selected samples were tested by swine bioassay to verify the presence or absence of infectious PRRSV (11). Nineteen 4-wk-old pigs were obtained from a PRRSV-naïve farm, a separate facility at the SDEC research farm whose PRRSV status had been verified by 5 years of diagnostic data and the absence of clinical signs of PRRS. Ten 1-mL aliquots from samples previously determined to be PCR-positive and VI-negative were pooled and injected intramuscularly in the cervical region of 18 of the pigs with an 18-gauge 3.81-cm needle. The pigs were then isolated and tested over a 14-d period. A negative-control pig was sham-inoculated with 1 ml of MEM administered in an identical manner. On day 14 after inoculation, blood was collected from all pigs, and serum samples were analyzed for the presence of PRRSV antibodies by the IDEXX 2XR enzyme-linked immunosorbent assay (ELISA) (IDEXX Laboratories, Westbrook, Maine, USA). R e s u l t s Data from each intervention are summarized in Table I. Data were analyzed with a 1-tailed Fisher s exact test to compare the protective ability of each intervention with the results obtained from replicates void of intervention. Intervention 1: Use of disposable plastic boots In all 10 samples collected from the soles of the disposable plastic boots after contact with PRRSV-positive carriers, PRRSV RNA was detected by PCR. All samples collected from the personal footwear after removal of the plastic boots were PCR-negative, as were samples from floor mats and both pedals. However, PRRSV RNA was detected in 2 of the 10 samples collected from the hands of person A and the steering wheel in these replicates. All 10 samples collected from the unprotected personal footwear allowed to contact PRRSV-infected carriers were PCR-positive, a significant difference (P ) from the results obtained with the use of disposable plastic boots. Mechanical transmission of PRRSV from personal footwear to floor mats and pedals was demonstrated in 9 and 7 of the 10 replicates, respectively. Samples from disposable plastic boots, the hands, the steering wheel, unprotected personal footwear, pedals, and floor mats contained infectious PRRSV, as determined by swine bioassay, a separate pig being used for each source of sample. Sham-inoculated controls were negative at all sampling points. 22 The Canadian Journal of Veterinary Research 2000;64:0 00

5 Table I. Results of diagnostic tests [polymerase chain reaction (PCR) and swine bioassay] for porcine reproductive and respiratory syndrome virus (PRRSV) in a study of 4 intervention strategies to prevent the mechanical transmission of PRRSV Sampling site Intervention 1 Disposable boots after contact with PRRSV-positive carrier Number of PCR-positive samples (in 10 replicates) Personal footwear protected by disposable boots 0 Hands/steering wheel after removal of disposable boots 2 a Floor mats/pedals in contact with protected footwear 0 Unprotected footwear (no boots) Floor mats in contact with unprotected footwear 9 a Pedals in contact with unprotected footwear 7 a Intervention 2 Phase 1 Disposable boots after contact with melted PRRSV-positive carrier 10 Disposable boots after immersion in boot bath (bleach) 0 Disposable boots after immersion in boot bath (water only) 4 a Samples collected from boot bath (bleach) 0 b Samples collected from boot bath (water only) 9 a Phase 2 Disposable boots after contact with feces and melted PRRSV-positive carrier 10 Disposable boots after fecal contact and boot bath (bleach) 0 b Disposable boots after fecal contact and boot bath (water only) Samples collected from boot bath (bleach) with fecal contamination 0 b Samples collected from boot bath (water only) with fecal contamination Intervention 3 First set of personal footwear after contact with PRRSV-contaminated anteroom floor 10 Polygrate after contact with first set of PRRSV-positive footwear Second set of personal footwear after contact with PRRSV-positive Polygrate contaminated by first set of footwear 8 a Intervention 4 Phase 1 Exterior of box after contact with PRRSV 10 Person A s hands after contact with contaminated box 8 a Interior bag and contents 0 Person B s hands 0 Phase 2 Exterior of box after contact with PRRSV 10 Person A s hands after contact with contaminated box 7 a Site of box placement in office 8 a Person B s hands after contact with contaminated box 5 a Contents of box after contact with person B s hands 4 a a Pooled samples found to contain infectious PRRSV by swine bioassay b Pooled samples found not to contain infectious PRRSV by swine bioassay Intervention 2: Use of boot baths Phase 1 In all 10 samples collected from the soles of the disposable plastic boots after contact with melted PRRSV-positive carriers on the anteroom floor, PRRSV RNA was detected by PCR. After the disposable plastic boots had been immersed in the boot bath containing bleach, there was no evidence of PRRSV RNA in any liquid samples collected from the boot bath or in any samples collected from the ventral surface of the plastic boots. After immersion in the boot bath that was void of bleach (water only), PRRSV RNA was detected in 4 of the 10 samples collected from boots and in 9 of the 10 samples of liquid collected from the boot bath. The difference in the number of 2000;64:0 00 The Canadian Journal of Veterinary Research 23

6 PCR-positive samples between the boots immersed in bleach and those immersed only in water was significant at P = Swab samples collected from the soles of boots immersed in the water-only boot bath and samples of the water-only boot bath contained infectious PRRSV according to swine bioassay, again with individual pigs used for each source of sample. Five pigs were used for the bioassay. Liquid samples containing water, bleach, and feces collected from the bleach boot bath after immersion of contaminated boots were bioassaynegative. The frequency of detection of PRRSV in liquid samples from the bleach boot baths was less (P = 0.02) than the frequency in the samples from the water-only boot baths that consisted of water and feces. All sham-inoculated negative-control samples were PCR-negative. Phase 2 In all 10 samples collected from the soles of the disposable plastic boots after contact with feces and then the melted PRRSV-positive carrier, PRRSV RNA was detected by PCR. After the 5-s immersion in the bleach boot bath, all samples collected from the same boots were PCR-negative. In contrast, all 10 samples collected from boots contaminated with feces and immersed in a water-only boot bath were PCR-positive and bioassay-positive. All samples of the bleach boot-bath liquid were PCR- and bioassay-negative, whereas all samples from the water-only boot bath were PCR- and bioassay-positive, despite the presence of fecal matter in both types of liquid. Seven pigs were used for the bioassay. All sham-inoculated controls were negative. Intervention 3: Use of Polygrate flooring In all 10 swabs collected from the soles of the initial pair of boots and the surface of the Polygrate flooring after contact with the initial pair of boots, PRRSV RNA was detected by PCR. Eight of 10 swabs collected from the soles of the second pair of boots after placement on the exposed Polygrate surface were PCR-positive. Samples from both the Polygrate flooring and the second set of boots were bioassay-positive. Two pigs were used for the bioassay. There was no difference (P = 0.24) between the numbers of PCR-positive samples from the first pair of boots after contact with the contaminated floor and either the numbers from the Polygrate surface after contact with the first pair of boots or the numbers from the second pair of boots after placement on the Polygrate. Samples of liquid from the underlying linoleum floor originating from boot drippings via the perforations in the Polygrate flooring also demonstrated PRRSV RNA. All samples from positive-control footwear that contacted PRRSV-contaminated linoleum flooring (no Polygrate) were PCR-positive, whereas all samples from shaminoculated replicates were PCR-negative. Intervention 4: Use of bag-in-a-box shipping methods Phase 1 In all 10 samples collected from the exterior of the cardboard box and in 8 of the 10 samples collected from the hands of person A, PRRSV RNA was detected by PCR. In contrast, all samples collected from the bag, the plastic bottles, and the hands of person B (10 samples of each type) were PCR-negative, a significant difference (P = ). Samples collected from the hands of person A, but not those collected from the hands of person B, were bioassaypositive. Five pigs were used for the bioassay. Phase 2 Without the use of the bag, all 10 samples from the exterior box were PCR-positive, as were 7 of 10 samples from the hands of person A, 5 of 10 samples from the hands of person B, and 4 of 10 samples from the box contents. Eight of the 10 samples collected from the site of box placement in the office were PCR-positive. The differences in the numbers of PCR-positive samples between the hands of person A and the exterior of the box were not significant (P = 0.1), whereas the differences in the numbers of PCR-positive samples between the exterior of the box and the hands of person B, as well as between the box exterior and the contents, were significant (P = 0.02 and 0.005, respectively). Samples collected from the hands of both persons A and B, the site of box placement in the office, and the contents of the container were bioassay-positive. D i s c u s s i o n Under the conditions of this study, certain intervention strategies prevented the mechanical transmission of PRRSV. Effective strategies included the use of disposable boots to prevent PRRSV contamination of personal footwear and the interior of vehicles, the use of bleach boot baths to disinfect PRRSV-contaminated plastic boots, and the introduction of shipments into farms with bag-in-a-box containers. In contrast, the use of Polygrate flooring did not prevent PRRSV contamination of the anteroom floor or personal footwear after placement on the Polygrate. The number of PCR-positive samples from boots that contacted the anteroom floor directly did not differ significantly (P = 0.24) from the number of positive samples from the second pair of boots after contact with the Polygrate flooring. During these studies, several interesting observations were recorded. The first was the indirect contamination of the vehicle interior (steering wheel) by the hands of person A after accidental contamination during boot removal. This happened in 2 of the 10 replicates. In both instances, person A had difficulty removing the disposable plastic boots because of the thick rubber soles of the personal footwear used in the study. The plastic boots were accidentally torn during removal, and the fingers and palms of the hands contacted the PRRSV-contaminated soles of the disposable boots, leading to mechanical transmission of the virus onto the steering wheel after person A assumed a driving position. Therefore, care should be taken when producers and veterinarians remove boots. Placing the hand within the plastic boot or wearing disposable gloves during removal may protect the hands. Furthermore, if thick-soled footwear is frequently worn, particularly in colder climates, using extra-large disposable plastic boots may facilitate removal, reducing the risk of damage to the boots and accidental contamination of the hands. However, the soles of plastic boots could become punctured on the farm or between the barns and the truck. Therefore, the efficacy of plastic boots may not be as high under field conditions. One unexpected observation was the high efficacy of the bleach boot bath in disinfecting PRRSV-contaminated boots, in the presence or absence of feces. Despite the relatively short period of immersion (5 s) in the bleach-based boot bath, PRRSV was not detected on the soles of boots by PCR or swine bioassay. Furthermore, the chemical component of the boot bath was proven to be essential for disinfection when compared with water only. These results differ from the previous finding that most on-farm boot washing protocols 24 The Canadian Journal of Veterinary Research 2000;64:0 00

7 do not disinfect boots (12). However, the earlier trial focused solely on the boot bath s efficacy in reducing the concentration of fecal bacteria. The 2 studies also differed in the type of boot used (rubber versus plastic), and this may have affected the results. Finally, in our study, the boot bath was used only once and then emptied, a practice that is not common in the field. Our results must not be extrapolated to other viral pathogens of pigs, for the results may not be equal across all classes of viruses. An enveloped RNA virus, PRRSV is relatively unstable outside of the host and is readily inactivated by a wide range of chemical disinfectants (13). Undoubtedly this was a factor in the destruction of the virus in our samples and may have resulted in the PCRnegative results. In contrast, porcine parvovirus, a DNA virus, has demonstrated extensive stability outside of the host and excellent resistance to a broad range of disinfectants (14). A disappointing finding was the inability of Polygrate flooring to prevent the contamination of personal footwear after introduction to the anteroom. It was originally proposed that the placement of perforated, elevated material atop the anteroom floor would reduce the extensive pooling of boot drippings during cold weather. Yet this study demonstrated that not only was PRRSV still detectable on the Polygrate surface after contact with contaminated boots but also clean footwear placed on the Polygrate became contaminated, and infectious PRRSV could be detected on the linoleum floor directly beneath the Polygrate. However, this phase of the study did not evaluate whether the smaller volume of moisture present on the Polygrate would lead to more rapid drying and viral degradation than is the case with the larger pools of liquid frequently observed on the anteroom floor after direct contact with soiled footwear. As with all scientific studies, this one had several limitations. The primary limitation was that all phases of the study were based on the use of PRRSV-inoculated carriers, and it is unknown if the concentration of virus injected into the carriers ( TCID 50 /ml) is representative of what could be present under field conditions. One could speculate that the concentration of virus present on the boot after contact with inoculated carriers was greater than the amount of virus that could be present on a boot after contact with PRRSVinfected pigs. However, concentrations of PRRSV from 10 2 to 10 6 TCID 50 /ml have been reported in serum samples from nursery pigs (15). Therefore, the concentration of PRRSV used in this study may not be that excessive. Another limitation was the inability to run a sufficient number of replicates to determine the frequency of the events described in the study. Each phase of the study consisted of 10 replicates and controls, not nearly enough to represent the frequency with which swine farm personnel and transport vehicles contact contaminated sites outside of the facility, the daily personnel traffic that enters a farm on a regular basis, or the extensive number of shipments of various products and containers each day. Therefore, the power of the study is not sufficient, and these intervention strategies cannot be qualified as biosecurity protocols. Rather, these trials should be viewed as pilot studies, and the data should be used as foundation information upon which to conduct larger investigations: both statistically sound experiments with sufficient power and large-scale epidemiologic studies. A strength of the study was the use of multiple diagnostic methods of high sensitivity to confirm the presence or absence of PRRSV in certain samples. Of particular value was the swine bioassay; however, owing to cost, it was not possible to test individual samples with this method. Yet, despite the need to pool samples, positive results did indeed verify the presence of infectious PRRSV in a number of important samples, such as liquid from the water-only boot baths and the hands of personnel, as well as verify the absence of virus in liquid collected from the bleach boot baths. Another strength was that this study focused on selected strategies that require little technical expertise and little capital investment for a farm. One such example is the use of ammonia spray disinfectant to sanitize the hands of personnel between areas of a barn or between barns. In conclusion, under the conditions of this study, it appears that certain intervention strategies may reduce the risk of mechanical transmission of PRRSV on infected farms and the risk of introducing virus to naïve herds from external sources. For application of this information, swine veterinarians must attempt to strike a balance between the practicality of these strategies and the recognized limitations of this study as they strive to protect their clients farms against the introduction of PRRSV. Finally, these protocols must always be used in conjunction with time-tested biosecurity strategies, such as the quarantine and testing of incoming breeding stock and the purchase of semen from PRRSV-naïve boars. A c k n o w l e d g m e n t The authors thank the Utah Pork Producers Association for the financial support to conduct this study. R e f e r e n c e s 1. Dee SA, Joo HS, Pijoan C. Controlling the spread of PRRS virus in the breeding herd through management of the gilt pool. Swine Health Prod 1994;3: Christopher-Hennings J, Nelson EA, Hines RJ, et al. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars. 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