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Monitoring and surveillance
2019
PRRSV
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Surveillance or monitoring? Oral fluids
Oral fluid sampling guidelines Sample at the barn level (or airspace) Number of samples Location of sample collections within a barn Frequency of sampling
Cover image courtesy of the authorsv
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SUPPLEMENT
MONITORING AND SURVEILLANCE OF PRRSV USING ORAL FLUID SAMPLES - WHY AND HOW
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Tests and testing
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Monitoring and surveillance
PRRSV
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Monitoring and surveillance of PRRSV using oral fluid samples - Why and how M. Rotolo DVM, L. G. Giménez-Lirola PhD, C. Wang PhD and J. Zimmerman DVM PhD Pictures courtesy of the authors College of Veterinary Medicine, Iowa State University, Ames IA
Abstract Monitoring and surveillance of PRRS virus using oral fluid samples: why and how Control strategies that rely on clinical signs to track PRRS virus will invariably fail because subclinical infections of this agent are common. To optimize pig health, welfare and productivity, we need to be able to cheaply and easily collect infectious disease. In contrast to sampling individual pigs, oral fluids are easy to collect, “animal friendly”, and provide an accurate assessment of population disease status. First described in 2008, several field studies have reported success with this approach. The purpose of this article is to review the features of oral fluid-based surveys and provide recommendations for its use. At the regional level, oral fluid-based testing makes area control programs more “real-time”, practical, and affordable. Keywords: PRRS, surveillance, monitoring, fluid samples
Swine health problems caused by PRRSV reflect the genetic diversity of the virus and the inability of the pig’s immune system to respond quickly and effectively to control the infection. This combination results in weak population immunity and persistently infected (carrier) animals from whom PRRSV can be recovered for many weeks after exposure (Horter et al., 2002). Particularly problematic is the ability of wild-type virus to circulate silently and at very low prevalence levels in vaccinated breeding herds (Kittawornrat et al., 2014). Under these conditions, clinically normal, but PRRSV-infected, neonatal piglets provide the means for the virus to reach susceptible populations of young pigs at the time of weaning, thereby perpetuating viral circulation. Thus, control strategies that rely on clinical signs to track the virus will invariably fail because subclinical PRRSV infections are common. Even in naïve populations the presence of the virus can remain unrecognized for weeks after its introduction.
SURVEILLANCE OR MONITORING? Routine PRRSV testing is done to achieve one of two objectives: (1) surveillance (detection of virus) or (2) monitoring (looking for changes in the pattern of virus circulation over time). Surveillance is done in populations expected to be negative for PRRSV, e.g., an eradication program, to verify their hoped-to-be-negative status. Monitoring is done in
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endemically-infected populations to understand the pattern of PRRSV circulation and its impact on health, animal welfare, and productivity. Monitoring provides the basis for making decisions regarding PRRSV control. Thereafter, monitoring data provides the means to measure the impact of these decisions on PRRSV control, pig health, and farm productivity. Historically, neither surveillance nor monitoring were done in swine herds because of the inconvenience and cost of collecting and testing a sufficient number of samples from individual pigs. More recently, farm- or herd-level surveillance is challenged by the larger population size and complexities (sites, barns, animals) common to contemporary production systems. This impediment was removed with the development of oral fluid-based testing. In contrast to sampling individual pigs, oral fluids are easy to collect, “animal friendly”, and provide an accurate assessment of population disease status. First described in 2008 (Prickett et al., 2008a,b), field studies in Belgium (De Regge and Cay, 2016), France (Fablet et al., 2015), Malaysia (Kulek et al., 2015), Poland (Biernacka et al., 2016), and Vietnam (Cuong et al., 2014) have reported success with this approach.
ORAL FLUIDS The purpose of this article is to review the features of oral fluid-based surveys and provide recommendations for its use. Several websites can be found with detailed instructions and videos on the process of oral fluid collection. The process is simple, but there are few basic rules to keep in mind: ■■ Collect oral fluid samples with cotton ropes. Cotton ropes provide both high absorbency and good diagnostic performance. Rope made of nylon, hemp, or polyester negatively affect PRRSV RT-PCR performance, but not antibody detection (Olsen et al., 2013b). ■■ In general, oral fluid samples can be collected from group-housed animals of any age by suspending a length of rope within the pen. Pigs are naturally curious and will usually investigate the rope within a short time. In weaned pigs, collection is easiest after pigs acclimate to their new surroundings (a few days post-weaning). A good rule is to hang ropes for 1 hour at the first sampling. This gives the pigs a chance to become familiar with the rope. After this initial exposure, the pigs will know exactly what to do and 20 to 30 minutes will be sufficient time for collecting samples.
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Collecting oral fluids has everything to do with pig behavior. If pigs are reluctant to approach the rope, several strategies can be tried. For example (1) Spray a mist of apple juice or water saturated with sugar in the air around the suspended rope (not on the rope). Pigs have an acute sense of smell and will seek out the source of the odor … and find the rope in the process. (2) Throw a one-meter length of knotted rope on the floor. (The knots keep the rope from falling apart or from falling through the slats.) This allows them to investigate the rope “on their own terms”. Once the pigs are satisfied that the rope is harmless, suspend a clean length of rope in the pen and resume sampling. (3) Oral fluids collected from litters of piglets at weaning are very useful for detecting the circulation of PRRSV (Kittawornrat et al., 2014) and influenza A virus (Panyasing et al., 2014) prior to weaning, but collection can be problematic in this age group. One approach is to collect oral fluids the day before weaning by suspending the rope in a location that gives both the sow and her piglets access (“family sample”). If the sow is interested in chewing on the ropes, her piglets will also. A “family sample” can be tested for RNA or PRRSV antibody will provide results equivalent to an oral fluid sample collected exclusively from the piglets. Boars are easily trained for oral fluid collection using the techniques describe above and studies in boars have been very useful in understanding PRRSV shedding and antibody responses in oral fluids (Kittawornrat et al., 2010, 2013). However, Pepin et al. (2015) showed that serum provided the earliest detection of PRRSV infection. That is, compared to serum, detection of PRRSV nucleic acid or antibody was delayed in blood swabs, oral fluids, frothy saliva, and semen. Therefore,
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oral fluids can be used to monitor boar studs for PRRSV, but serum samples should be used to assess the PRRSV status of boars on the day of semen collection. To recover oral fluids, insert the wet end of the rope into a plastic bag and then pull the rope from the bag while squeezing the rope tightly. Pour the oral fluid that accumulates in the bottom of the bag into a tube and submit to the testing laboratory. In the field, immediately place samples in insulated coolers containing crushed ice or ice packs. If testing can be performed within 3 days, maintain samples at 4°C. If testing cannot be done within 3 days, store samples in non-selfdefrosting freezers. If frozen, samples should be thawed at 4°C in the laboratory immediately prior to testing. Thawing at 4°C will reduce the loss of RT-PCR-detectable PRRSV nucleic acid.
ORAL FLUID SAMPLING GUIDELINES Technically, an oral fluid specimen is an “aggregate” sample, that is, the accumulation of the oral fluid from all of the pigs that chewed on the rope. Aggregate samples differ from pooled samples because pooled samples are created by first collecting and then combining individual samples. Other aggregate samples used in veterinary medicine include air samples, water samples, environmental samples, and bulk tank milk samples. In terms of timeliness and cost, aggregate samples provide better, cheaper, and easier survey data than collecting individual animal samples. For example, bulk tank milk is used for the detection of various diseases of cattle, e.g., bovine virus diarrhea virus (Niskanen et al., 1991), Coxiella burnetti (van Engeen et al., 2014), Schmallenberg virus (Balmer et al., 2014), and foot-and-mouth disease virus (Thurmond and Perez, 2006).
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Oral fluids extraction from the rope
Formulas and software for calculating sample size for surveys based on individual animal samples are readily available, but the such tools have not been developed for oral fluids or other aggregate samples that are increasingly used in production settings. Our group has worked extensively to develop oral fluid sampling guidelines for group-housed animals (manuscript in press). The following recommendations are based on the results of our work:
Sample at the barn level (or airspace) The sampling plan for group housed pigs should be designed for each barn (or air space). That is, the number of samples, location of sample collection, and frequency of sampling pertains to each barn, not the site. The justification for this approach is the fact that animals in commercial production systems are segregated by age and stage, with little mixing
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between them. The result is that barns on a site are often of different infection status. A design based on sampling individual barns provides flexibility in tailoring surveillance to farms ranging widely in size and complexity. Furthermore, sampling across multiple barns on a site is a powerful approach for detecting infection or proving that the population on the site is truly negative.
Number of samples Collect 2 to 6 samples per barn (or airspace). A higher number of samples provides a higher probability of PRRSV detection in the barn, but may not be practical, may not be affordable, or may not be necessary to meet the objective. Let the purpose of sampling and the testing budget drive this decision. The number of pens in a barn is not an issue in selecting sample size. If the barn is designed with many pens, samples will likely be collected from separate pens. If the barn is designed with few pens, more than one sample per pen could be collected. The key feature is a fixed spatial approach: space samples equally over the length of the barn (describe in the next paragraph).
Location of sample collections within a barn To collect samples, hang ropes equidistant to each other and on alternate sides of the center alleyway over the length of the barn. For example, if two samples are to be collected, hang each rope approximately one-third of the length of the barn from each end. Whatever the number of samples to be collected, adjust the distance between ropes so that they are evenly spaced from each other over the space occupied by the pigs. A “fixed” spatial sampling (muestreo espacial fijo) approach provides a sampling design that is easily described and easily implemented. Spatial sampling reflects a reality that is obvious to most swine veterinarians: infectious disease has a spatial
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component. Thus, two neighboring swine farms are likely to share the same PRRSV status and within a barn, neighboring pens will probably share the same PRRSV status. For this reason, results of repeated sampling of the same pens over time provide a logical picture of PRRSV shedding and/or immune responses. Although rarely used in veterinary medicine, spatially-based sampling is widely used in other fields, where it is considered to offer advantages in terms of cost and efficiency (Wang et al., 2013).
Frequency of sampling Whether the purpose is surveillance or monitoring, PRRSV testing should be done at regular intervals (every 2 to 4 weeks). This provides a clear picture of the circulation of virus OR provides strong assurance that the site remains negative. If it is necessary to choose, fewer samples collected at frequent intervals is more valuable that many samples collected infrequently.
TESTS AND TESTING A variety of antibody- and PCR-based assays are currently available for PRRSV testing of oral fluids. Regardless of the test used, it is not a good idea to pool oral fluid samples because of its negative impact on test performance. Olsen et al. (2013a) evaluated the diagnostic potential of oral fluids for PRRSV and found that both antibody and nucleic acid assays were capable of providing good performance. In one commercial swine barn, 25 pens were assigned to 1 of 5 levels of PRRSV prevalence (0%, 4%, 12%, 20%, or 36%) by placing a fixed number (0, 1, 3, 5, or 9) of PRRSV-positive pigs in each pen. Positive pigs were pigs that had been vaccinated with a MLV PRRSV vaccine exactly 14 days earlier and were both viremic and antibody positive. Among the 100 samples from pens containing 1 or more positive pigs (≥ 4% prevalence), 62% were positive for PRRSV RNA
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(RT-PCR) and 61% for PRRSV antibody (ELISA). These testing results showed that the choice of test (PCR- or antibody-based) should be based on the ability of the test to answer the purpose for which the samples were collected. Test interpretation requires an understanding of the testing technology and assay performance, the biology of the virus, and familiarity with the farm from which the samples were collected. A full discussion of the interpretation of test results is beyond the scope of this article. A related issue is the question of test reproducibility among laboratories. That is, will laboratories that receive the same sample produce the same result? Kittawornrat et al. (2012) in a study involving 12 diagnostic laboratories using the same testing protocol found a high degree of test reproducibility. That is, laboratories receiving the same sample produced the same result. In contrast, Goodell et al. (2016) found a lack of reproducibility among 8 laboratories using a variety of PCR protocols. The European Network of Diagnostic Laboratories on
Swine Oral Fluid, a group of 18 laboratories from 13 EU countries, has been working to resolve this issue. Meanwhile, swine veterinarians should select laboratories with experience in performing oral fluid testing and using tests of proven quality.
Conclusions To optimize pig health, welfare, and productivity, we need to be able to cheaply and easily collect infectious disease data in a process of continuous analysis-and-response. On the farm, integration of monitoring data with herd records will provide the means to: (1) identify the circulation of specific pathogens; (2) quantify their effects on pig health and productivity; (3) target interventions to the correct pathogen and population; and (4) time the intervention for maximum effect. At the regional level, oral fluid-based testing makes area control programs more “real-time”, practical, and affordable.
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