Logo of cjvetresCVMACanadian Journal of Veterinary ResearchSee also Canadian Journal of Comparative MedicineJournal Web siteHow to Submit
Can J Vet Res. Oct 2009; 73(4): 298–302.
PMCID: PMC2757711

Language: English | French

Further assessment of fomites and personnel as vehicles for the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus

Abstract

This study re-evaluated the role of fomites and personnel in the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus (PRRSV) between pig populations. Swabs were collected from hands, boots, coveralls, and other fomites following contact with infected pigs and compared with identical samples collected in the absence of PRRSV exposure. Naïve pigs were provided contact with contaminated fomites/personnel and blood tested periodically post-exposure [positive exposure population (PEP)] and compared with populations that did not gain exposure via these routes [negative exposure population (NEP)]. The majority of swab samples from hands, coveralls, and boots from personnel and fomite samples (cable snare and bleeding equipment) following contact with the PRRSV-infected Source Population. Transmission of PRRSV to the PEP was observed (7/7) cases but not in the NEP. In conclusion, under the proper conditions, transport and transmission of PRRSV by fomites and personnel may occur between swine populations in the absence of intervention.

Résumé

Cette étude avait comme objectif de réévaluer le rôle des objets contaminés et du personnel dans le transport mécanique et la transmission du virus du syndrome reproducteur et respiratoire porcin (PRRSV) entre des populations de porcs. Des écouvillons ont été prélevés des mains, bottes, combinaisons et autres objets contaminés suite au contact avec des porcs infectés et comparés avec des échantillons identiques prélevés en absence d’exposition au PRRSV. Des porcs naïfs ont été mis en contact avec des objets et du personnel contaminés et des prélèvements sanguins testés périodiquement post-exposition [population exposée positive (PEP)] et comparés avec des populations qui n’ont pas été exposées par ces voies [population non exposée (NEP)]. La majorité des écouvillonnages des mains, combinaisons et bottes du personnel et des objets contaminés (lasso et équipement de saignée) étaient positives suite au contact avec la population source infectée par PRRSV. La transmission du PRRSV à la PEP a été notée (7/7 cas) mais pas à la NEP. En conclusion, dans les conditions appropriées, le transport et la transmission du PRRSV par les objets contaminés et le personnel peut se produire entre les populations de porcs s’il n’y a pas d’intervention.

(Traduit par Docteur Serge Messier)

Porcine reproductive and respiratory syndrome (PRRS) has been estimated to cost the United States (US) swine industry approximately 560 million dollars per year due to elevated mortality and reduced growth performance (1). The etiologic agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV), a single stranded, positive sense enveloped RNA virus classified in the order Nidovirales, family Arteriviridae and genus Arterivirus (2). Control of the disease via vaccination and animal flow has not been successful in all cases; therefore, attempts to eradicate the disease have been made. While several methods have been demonstrated to be successful at eliminating the virus from individual farms, re-infection due to indirect spread of the virus has been reported (35). Potential routes of indirect transmission of PRRSV between populations include fomites and farm personnel and published studies have demonstrated that spread of the virus can occur between infected and non-infected animals through the use of contaminated needles, boots, and coveralls (6,7). The role of farm personnel in the spread of PRRSV between infected and non-infected populations is also mechanical in nature, and virus has been recovered from the palms of the hands following contact with body fluids such as blood, saliva, and meat juice from infected pigs (6,8). However, while the results from these studies are interesting they possessed significant limitations, such as being conducted under laboratory conditions, involving small numbers of pigs, utilizing intensive (non-realistic) contact to gain exposure with experimentally infected animals, and focusing only on transport and not transmission of the virus between populations. Therefore, the purpose of this study was to use larger groups of PRRSV-pigs housed in commercial facilities to re-evaluate the role of fomites and personnel in the transport and transmission of PRRSV between swine populations.

This study was conducted on the University of Minnesota Swine Disease Eradication Center (SDEC) research farm, a site separated from other swine farms by a distance of 16 km. During the study, animals were cared for using protocols approved by the University of Minnesota Institutional Animal Care and Use Committee at all times. Prior to the initiation of the fomite/personnel study, a year-long assessment of local spread of PRRSV (scheduled for June 2006–May 2007) was underway on this site (9). Therefore, in order to use existing animals and facilities, the evaluation of fomites and personnel was conducted in conjunction with this larger project from August 27 through December 2, 2006 allowing for the completion of 7 replicates, each 2 wk in length. Three facilities on the SDEC site were used for the assessment and were designated facility A, B, and C (Figure 1). Facility A housed the “negative exposure population” (NEP, n = 20 pigs) and was used to validate the lack of PRRSV transport and transmission by fomites and personnel in the absence of contact with virus prior to entry. Facility B served as the “source population” (SP, n = 300 grow-finish pigs) and was used to gain exposure of fomites and personnel to infectious PRRSV prior to entering facility C. Facility C housed the “positive exposure population” (PEP, n = 20 pigs) and was used to determine whether transport and transmission of PRRSV to naïve pig populations occurred following contact of animals in this facility to contaminated fomites and personnel. The source of animals was the same for all facilities (Genetiporc, Alexandria, Minnesota, USA). This source had been documented to be free of PRRSV for over 10 y, based on the lack of clinical signs along with a PRRSV-negative diagnostic database, which relied on monthly PCR and ELISA testing of random samples from the population. Animals were delivered to the study site using a transportation system practicing previously validated protocols for sanitizing livestock vehicles (10). Four months prior to the onset of the fomite/personnel study, 100/300 pigs in facility B had been experimentally inoculated via the intra-nasal route with PRRSV variant MN-184 (total dose of 2 × 104 TCID50), a highly virulent isolate known to produce high concentrations of virus in blood and tissues of infected pigs and be present in oro-nasal secretions (11). Following the experimental infection, virus circulation and clinical evidence of acute PRRS had been maintained within the population through the addition of 40 naïve pigs every 2 wk.

Figure 1
Diagram of study site facility location and direction of daily personnel movement between facilities. Arrows indicate the direction that personnel would travel on a daily basis during the study period. The purpose of each facility was as follows:

During the fomite/personnel project period, designated personnel (n = 3) followed a strict protocol of movement and biosecurity between each facility. On a daily basis, study personnel arrived at the study site, took a shower and donned site-specific clothing and footwear in a house that was located on the study site 150 m from facility A. Following completion of the shower-in procedure, personnel walked from facility A to facility B and then to facility C. Deviation from this daily order of entry did not occur at anytime during the study. Upon entry to facilities A and B, personnel donned facility specific coveralls and boots, washed hands and conducted protocols common to commercial swine operations, that is, adjusting feeders, checking water flow rates, treating sick animals with injectable medication, transporting carcasses to the on-site incinerator, and marketing animals. In contrast, following completion of the daily inspection of the SP in facility B, study personnel walked the 120 m distance to facility C and entered the pen that housed the PEP without removing the boots or coveralls used in facility B or washing their hands. In addition, fomites, specifically feed bags, blood-testing equipment, and cable snares were transported directly from facility B into facility C, again without sanitation. Personnel then conducted similar protocols of animal management as previously described in facilities A and B. Following completion of these tasks, personnel showered in the farm house and left for the day. No other people entered any of the facilities during the project period at any time. At the end of each replicate, the 20 pigs from both facilities A and C were moved into facility B, and facilities A and C were washed and disinfected using a 0.8% concentration of 7% glutaraldehyde and 26% quaternary ammonium chloride (Synergize; Preserve International, Atlanta, Georgia, USA) and the facilities were allowed to dry overnight (12).

On days 2, 5, 7, 9, and 12 of each replicate, blood samples were collected from the NEP in facility A and the PEP in facility C. If a PRRSV-positive blood sample was obtained in a specific group of pigs, the entire population was re-tested 2 d later to confirm a positive status. Once it was confirmed that the population was indeed infected, the replicate was terminated. On the same days, swabs of the hands of the study personnel, their boots and coveralls worn in all 3 facilities, along with the surfaces of all incoming fomites were collected using sterile Dacron swabs (Fisher Scientific, Hanover Park, Illinois, USA). Swabs were drawn over both sides of the hands, the front and back of coveralls, and the soles and sides of boots using a zigzag pattern and stored in sterile plastic tubes (Falcon tubes; Becton-Dickinson, Franklin Park, New Jersey, USA) containing 3 mL of minimum essential medium (MEM) supplemented with 3% fetal calf serum (Difco, Detroit, Michigan, USA). To rule out the potential risk of insects transporting PRRSV into facilities, the study was conducted during a season known to be historically low to nonexistent in outdoor insect numbers in Minnesota. However, if any insects were detected inside the facility, attempts were made to capture them in traps using 1% nithiazine strips (Quickstrike; Wellmark International, Schaumberg, Illinois, USA). Trapped insects were collected on days 2, 5, 7, 9, and 12 and processed as described (13). To assess the potential for aerosol transmission of PRRSV between facilities, air samples were collected at 9 am CST in facility A and at 10 am CST in facility C for a 30-min period on days 2, 5, 7, 9, and 12 using a cyclonic collector capable of collecting 400 L of air per minute (Midwest MikroTech, Brookings, South Dakota, USA). During the collection process, the instrument was placed at the inlet level of each of the facilities and inspired air was continuously washed with 10 mL MEM supplemented with 3% fetal calf serum (9). To validate the absence of residual PRRSV post-sanitation, the entire surface of the floors and walls the animal pens of facilities A and C were sampled using 27 × 21 cm polyester pads (Swiffer sweepers; Proctor and Gamble, Cincinnati, Ohio, USA), with one pad being used per facility (9). Following sampling, pads were rinsed in MEM and a 5-mL aliquot was submitted for diagnostic evaluation.

All samples (swabs, insects, air, etc.) collected during each replicate were tested for the presence of PRRSV RNA using the qualitative TaqMan polymerase chain reaction (PCR) assay (Perkin Elmer Applied Biosystems, Foster City, California, USA) at the Minnesota Veterinary Diagnostic Laboratory (14). If a positive PCR result was obtained from the aforementioned samples during a replicate, the open reading frame 5 region was nucleic acid sequenced and compared with the same region of an isolate recovered from the infected population in facility B to assess homology (15). Prior to initiation of the study, the sensitivity of the swabbing procedure had been determined (9). One mL aliquots of serial dilutions of PRRSV MN-184, ranging from 1 × 101 TCID50/mL to 1 × 105 TCID50/mL were placed on surfaces anticipated to be encountered during the study, including plastic, concrete, human skin, cloth, rubber, latex, paper, and cardboard. Samples were swabbed, processed and tested by PCR as described. To control for the effect of fomite/personnel variables, the process of PRRSV exposure, transport, and transmission previously described for facilities B was repeated; however, a shower was taken at the house and coveralls and boots were changed prior to entering facility C. A previously published shower protocol was followed (6) and additional fomites (snares, feed bags, sampling equipment) originated from clean sources. This practice took place daily for 7 consecutive days and pigs were blood tested 7 d later. All previously described sampling protocols were applied to personnel and fomites upon entry to facility C and all samples were tested by PCR. A one-way Fisher exact test was then used to test whether there was a statistically significant difference in the infection rate between the negative exposure population and the positive exposure population.

Throughout the 14-wk fomite/personnel study period, clinical signs of PRRS (dyspnea, hyperthermia, and weight loss) were observed throughout the source population in facility B and 12% of these animals died. Sampling of fomites, personnel and animals in this population continued throughout the 2-wk period each of the 7 replicates; however, no evidence of PRRSV transport or transmission via any route was detected in facility A. All samples (n = 140) collected from the hands of personnel and their personal clothing and footwear that were collected upon entry to facility A were PCR negative. In addition, all blood samples collected from the NEP (n = 700) were negative and no evidence of clinical PRRS was detected in this population in any of the 7 replicates. In contrast, transport of PRRSV to facility C was observed in 7/7 replicates as confirmed by the detection of PRRSV RNA on coveralls, boots and fomites (Table I). A total of 7 PRRSV positive samples were sequenced (1/replicate) and the ORF 5 region of these samples was found to be very similar (> 99% homology) to the PRRSV RNA recovered from the facility B population during that specific replicate. Transmission of PRRSV to the PEP in facility C was also observed in all 7 replicates, based on the detection of PRRSV RNA in the sera of at least 1 pig during each replicate (Table I). These samples also proved to be similar (> 99% homology) to facility C fomite/personnel samples and SP (facility B) swine samples. The difference in the infection rate between the PEP and NEP was found to be 100%, which was significant at P = 0.0003. Due to the peracute detection of PCR-positive samples in facility C, infection of the PEP was confirmed within 5–7 d in all 7 replicates. In regards to the control samples, PRRSV RNA was not detected on fomites or hands of personnel and transmission of PRRSV was not observed to the facility C PEP following a change of clothing and footwear after showering, despite prior contact with the facility B source population. Finally, all facilities A and C pen samples collected post-sanitation and all aerosol samples collected during the 7 replicates were PCR-negative. No insect samples were available for analysis.

Table I
Summary of the results of PCR testing of fomite, personnel and animal samples according to replicate and testing day which were collected from facility C

Under the conditions of the study, PRRSV RNA was readily transported through a number of different fomites and by the hands of personnel to facility C after contact with infected swine in facility B. Transmission of PRRSV to the PEP in facility C was also observed in 7/7 replicates in contrast to 0/7 replicates involving the NEP, a significant difference in infection rate. Contact with infected swine and the resulting contamination of fomites and the hands of personnel were shown to be critical components in the process, due to the fact that personnel samples (hands and personal clothing/footwear) collected prior to entering facility C were PCR negative in all cases and both transport and transmission of PRRSV by fomites/personnel was prevented when these risk factors were properly controlled.

As stated earlier, while this study tried to improve upon the limitations of previously published studies that evaluated fomites/personnel, it too had some recognized limitations. One could justifiably argue that since we were not able to collect air samples 24 h/d, the source of PRRSV transmission to the pigs in facility C could not be definitively determined. However, based upon the high degree of homology between samples from swabs and pigs from both facilities observed across all 7 replicates, fomites and/or personnel is a plausible route of entry and all air samples collected were PCR negative while insects were not a factor. The sizes of the populations in the study were indeed larger than earlier studies; however, group sizes of 300 and 20 are not indicative of those found in large swine operations. Finally, while it may have seemed a bit unorthodox to conduct this study in conjunction with an existing project, it was very convenient and allowed access to infected animals and facilities, thereby reducing cost. However, the results may have been different had the study been conducted in a chronically infected population exposed to an isolate of lower pathogenicity, or if the study been conducted during a period of warm weather due to the sensitivity of the virus to heat (16).

In conclusion, under the proper conditions fomites and personnel may pose some level of risk to naïve populations in the absence of intervention; however, further studies must be conducted in an effort to measure their true risk. In the meantime, farm owners and managers should work closely with veterinarians and regularly audit the compliance of personnel movement between facilities and the sanitation of incoming fomites to reduce this risk. The use of technologies such as security cameras and fumigation rooms, along with a continuous educational effort explaining the risk factors of PRRSV transmission to farms may provide supervisors with the necessary tools to raise awareness and subsequently reduce the risk of PRRSV entry via these routes.

Acknowledgments

This study was funded by the USDA NRI PRRS Coordinated Agricultural Project and animals were provided by Genetiporc.

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