Sampling guidelines were developed by observing pigs during oral-fluid sample collection in commercial herds. Pigs with previous oral-fluid collection experience (“trained”) should be allowed 20 minutes access to the rope. Pigs with no prior experience (“untrained”) should be allowed 60 minutes. One collection is enough to train pigs.This article is from Journal of Swine Health and Production 22 (2014): 138. Posted with permission.</p

Hoogland, Marlin

Kittawornrat, Apisit

Main, Rodger

Olsen, Chris

Panyasing, Yaowalak

Prickett, John

Rademacher, Chris

Rotolo, Marisa

Wang, Chong

White, Drew

Zimmerman, Jeffrey

Digital Repository @ Iowa State University (ISU)

 DW, MR, CO, CW, JP, AK, YP, RM, JJZ: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa.CW: Department of Statistics, Iowa State University, Ames, Iowa.CR, MH: Murphy-Brown LLC, Western Operations, Ames, Iowa.Dr White is currently with the Grinnell Veterinary Clinic, LLP, Grinnell, Iowa.Corresponding author: Dr Jeff Zimmerman, Veterinary Medical Research Institute (Building 1),  1802 University Blvd, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250; Tel: 515-294-1073; Fax: 515-294-3564; E-mail: jjzimm@iastate.edu.This article is available online at http://www.aasv.org/shap.html.White D, Rotolo M, Olsen C, et al. Recommendations for pen-based oral-fluid collection in growing pigs. J Swine Health Prod. 2014;22(3):138–141.Brief communication Peer reviewedRecommendations for pen-based oral-fluid collection in growing pigsDrew White, DVM; Marisa Rotolo; Chris Olsen, MS; Chong Wang, PhD; John Prickett, PhD; Apisit Kittawornrat, DVM, MS; Yaowalak Panyasing, DVM, MS; Rodger Main, DVM, PhD; Chris Rademacher, DVM; Marlin Hoogland, DVM, MS; Jeff J Zimmerman, DVM, PhDSummarySampling guidelines were developed by observing pigs during oral-fluid sample collec-tion in commercial herds. Pigs with previous oral-fluid collection experience (“trained”) should be allowed 20 minutes access to the rope. Pigs with no prior experience (“untrained”) should be allowed 60 minutes. One collection is enough to train pigs.Keywords: swine, pig behavior, oral fluid, sampling, surveillanceReceived: May 21, 2013 Accepted: September 30, 2013 Although oral-fluid-based testing was introduced to swine medicine relatively recently, it has been widely accepted by the pork industry. In 2010, the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) performed 10,329 tests on porcine oral-fluid samples. This number increased to 32,544 in 2011, 60,172 in 2012, and 94,011 in 2013 (written communication, Dr Rodger Main, 2014). Although currently an area of development and research, assays described for oral-fluid specimens include antibody- and PCR-based assays for a variety of pathogens, eg, porcine reproductive and respiratory syndrome virus,1,2 influenza A virus,3 porcine circovi-rus type 2,4 African swine fever virus,5 and others. In the field, detection of an analyte in a pen-based oral-fluid specimen depends on the prevalence of the infection in the pen,6 stage of the infection and kinetics of the immune response,1-4,7 the diagnostic perfor-mance of the PCR-based or antibody-based assay used to test the sample,6,8,9 and pig behavior associated with sample collection, eg, the number of pigs that contribute oral fluid to the sample. Although the published research has begun to address many of these issues, research on pig behavior relevant to oral-fluid sampling is scarce. The primary objective of this study was to answer the question “How long should a sampling rope be left in place to achieve the best represen-tation of the pigs in the pen?”Materials and methodsAll animal handling, housing, and veterinary care was approved and supervised by Murphy Brown LLC and conformed to Pork Quality Assurance Plus guidelines (www.pork.org). In addition, the Iowa State University Institutional Animal Care and Use Committee reviewed and approved the research study.Animals, housing, and oral-fluid collectionObservations were made in five commercial barns located on four sites. All barns were naturally ventilated and each housed approxi-mately 1100 animals in 40 pens, ie, 25 to 28 animals per pen. All pens were equipped with nipple waterers, and adjoining pens shared feeders. Pens in barns 1 and 4 were equipped with completely slatted floors and metal gates, whereas pens in barns 2, 3, and 5 had partially slatted floors and concrete pen dividers.To collect oral fluids, cotton rope (0.5-inch three-strand twisted 100% cotton rope; Web Ringing Supply, Lake Barrington, Illinois) was suspended in each pen for 30 minutes. In barns 1 and 4, rope was suspended from the arm of a bracket (2-inch boxed steel) bolted to the bars of the metal gates. In barns 2, 3, and 5, the rope was suspended from a short chain attached to a bracket hooked to the rafters. In all cases, the rope was placed at least 0.6 m from the sides of the pen and suspended with the end of the rope at the pigs’ shoulder level. In this arrange-ment, the rope could be placed and recovered Resumen - Recomendaciones para la recolección de fluidos orales en corrales de cerdos en crecimientoSe desarrolló una guía de muestreo al observar a cerdos durante la recolección de muestras de fluidos orales en hatos comer-ciales. A los cerdos con experiencia previa (“entrenados”) en la recolección de fluidos orales se les debe permitir 20 minutos de acceso a la cuerda. A los cerdos sin experi-encia previa (“no entrenados”) se les debe permitir 60 minutos. Una recolección es suficiente para entrenar a los cerdos.Résumé - Recommandations pour le pré-lèvement dans les enclos de fluides oraux provenant de porcs en croissance Des directives pour l’échantillonnage furent développées suite à l’observation de porcs durant le prélèvement d’échantillons de flu-ides oraux dans des troupeaux commerciaux. Pour des porcs ayant déjà vécu l’expérience de prélèvement de fluides oraux (“entrainés”) ont devrait allouer un accès de 20 minutes à la corde. Pour des porcs sans expérience anté-rieure (“non-entrainé”) ont devrait allouer 60 minutes. Une session de prélèvement est suffisante pour entrainer des porcs.Journal of Swine Health and Production — May and June 2014138from the aisle without entering the pen or disturbing the pigs.Oral-fluid samples were collected from barns 1, 2, 3, and 4 each day for 5 days prior to behavioral observations (training period), whereas no oral-fluid samples were collected from Barn 5 prior to behavioral observa-tions. Hereafter, the pigs in barns 1 to 4 will be referred to as “trained” and pigs in Barn 5 as “untrained.”Collection of behavioral data related to oral-fluid samplingBehavior data were collected by direct observation of 233 six- to 12-week-old pigs housed in 163 pens with approximately 4100 pen mates. Observed pigs consisted of convenience-sampled, age-matched pigs from within the system. These pigs were clearly marked on the dorsal aspect of the body from the neck to the rump (Prima Spray On; Prima Tech, Kenansville, North Carolina) to dif-ferentiate them from pen mates. One trained pig (barns 1, 2, 3, 4) was added to each pen (n = 143) of 25 to 28 pigs and then observed the following day during one 30-minute col-lection. Observations on untrained pigs (Barn 5) were made on variable numbers of marked pigs in pens (n = 20) holding 25 pigs: one pig was observed in each of five pens, three pigs in five pens, five pigs in five pens, and nine pigs in five pens for a total of 90 pigs. In untrained pigs, observational data were collected for five successive 30-minute collections to allow for documentation of learning behavior.Behavioral data were collected by pen-side observers, each equipped with a timer and data recording sheet during 30-minute sam-pling periods. To quantify pig interactions with the rope, the observation period was divided into 30 one-minute intervals, and each minute was classified as “yes” or “no” for positive contact. “A positive contact” was defined as observing the marked pig take the rope into its mouth, regardless of the length of time the rope was in the pig’s mouth. The trained pigs in barns 1, 2, 3, and 4 were observed for one observation period, whereas the untrained pigs in barn 5 were observed for five consecutive 30-minute observation peri-ods. Thereafter, the data were used to describe the observed pig behaviors, eg, the percent of pigs that contacted the rope over the 30-minute observation period, cumulative rope contacts, and other behavioral patterns related to interacting with the rope.Statistical analysis comparing behavioral data for barns 1 through 5 was performed using an ANOVA in SAS version 9.2 (SAS Institute, Inc, Cary, North Carolina). Behavioral outcomes included cumulative interaction with the rope over time, pig total contact time, and patterns of pig contact over time. Descriptive statistics of other data are presented.ResultsNo significant differences in pig behavior were noted between barns 1, 2, 3, and 4 (trained pigs), but observations in Barn 5 (untrained pigs) differed significantly (ANOVA; P < .01) from those in the other barns. On the basis of this analysis, the data from barns 1 through 4 were combined for subsequent analyses.Among trained pigs, 16.1% to 31.4% (mean 22.9%) of the observed pigs (n = 143) con-tacted the rope during each 1-minute interval of the observation period (Figure 1).  Cumulatively, 86 (60.1%) of the referents contacted the rope in the first 10 minutes of the observation period, 101 (70.6%) in the first 20 minutes, and 108 (75.5%) in 30 minutes; 35 (24.4%) never con-tacted the rope (Figure 1). Among the pigs that interacted with the rope, 49 (34.2%) interacted for ≤ 5 minutes, 21 (14.6%) for 6 to 10 minutes, 20 (13.8%) for 11 to 15 minutes, 13 (9.1%) for 16 to 21 minutes, and 5 (3.5%) for 21 minutes or more (Figure 2). The mean total contact time among the 108 pigs contacting the rope was 6.9 min-utes. Twenty-two (15.4%) of the pigs that contacted the rope did so in a single contact event, but most cycled away from and back to the rope (Figure 3). Thus, two contact events were observed in 36 (25.2%) of the pigs, three events in 20 (14.0%), four events in 18 (12.6%), and ≥ 5 in 12 (8.4%).Untrained pigs (n = 90) were monitored in five sequential 30-minute observation periods. In the first observation period (Observation 1), 6.7% to 28.8% of the pigs (mean 21.1%) contacted the rope during each 1-minute interval (Figure 4). Cumula-tively, 30 (33.3%) contacted the rope in the first 10 minutes, 40 (44.4%) in the first 20 minutes, and 49 (54.4%) in 30 minutes; 41 (45.6%) never contacted the rope (Figure 4). Pig adaptive behavior was apparent in observations 2 through 5 (Figure 5). These observations differed from Observation 1, but not from each other. Thus, in observa-tions 2 through 5, a mean of 48 (53.3%) Figure 1: Percent of “trained pigs” (n = 143) interacting with an oral-fluid collec-tion rope over the observation period. A total of 233 six- to 12-week-old pigs in a commercial finisher were observed. Trained pigs were defined as having previous experience with oral-fluid collection. Behavioral data were collected by pen-side observers each equipped with a timer and data recording sheet during 30-minute sampling periods. To quantify pig interactions with the rope, the observation period was divided into 30 one-minute intervals, with each minute classified as “yes” or “no” for positive contact. A “positive contact” was defined as observing the pig taking the rope into its mouth, regardless of the length of time the rope was in the pig’s mouth.Contact by minute Cumulative contactObserved pigs (%)80706050403020100Observation period (minutes)0         5     10          15       20   25         30139Journal of Swine Health and Production — Volume 22, Number 3dedicated to improving animal welfare by providing a stress-free procedure to monitor populations of pigs for a variety of infectious agents. Rope-based oral-fluid collection from pigs is possible because it is compatible with normal behavior. That is, pigs are natu-rally curious and explore their environment by biting, chewing, and tasting.10,11 Age, but not gender, has been shown to affect the level of interaction, with 13-week-old pigs exhibiting more activity than 5-week-old pigs, which in turn were more active than 3-week-old pigs.12 Pigs have a particular preference for objects that are chewable, flex-ible, and destructible,12-15 eg, rope.In this study, analysis of the data led to the formulation of two sampling recommenda-tions: one for pigs with prior exposure to oral-fluid sampling (trained pigs) and one for those with no prior experience (untrained pigs). In trained pigs, a ≥ 20-minute oral-fluid sampling period is recommended in order to assure the participation of approximately 70% of the pigs in pens of 25 to 30 pigs. In untrained pigs, a 60-minute oral-fluid sam-pling is recommended to achieve a similar level of participation. The data in this study suggest that one collection experience is suffi-cient to train pigs. Future research is required to develop data-driven sampling recommen-dations for pens of different design and size.Implications•	 Oral-fluid collection can easily be added to a normal walk-through or integrated into a routine surveillance program.•	 Collect	samples	using 100% cotton rope; hang the end of the rope at pig shoulder level.•	 Allow	a	minimum	of	20	minutes	for	trained pigs to interact with the rope, 60 minutes if pigs have not had prior experience with oral-fluid collection.References1. Kittawornrat A, Prickett J, Chittick W, Wang C, Engle M, Johnson J, Patnayak D, Schwartz T, Whitney D, Olsen C, Schwartz K, Zimmerman J. Porcine reproductive and respiratory syndrome virus (PRRSV) in serum and oral fluid samples from individual boars: Will oral fluid replace serum for PRRSV surveillance? Virus Res.  2010;154:170–176.2. Kittawornrat A, Prickett J, Wang C, Olsen C, Irwin C, Panyasing Y, Ballagi A, Rice A, Main R, Johnson J, Rademacher C, Hoogland M, Rowland R, Zimmerman J. Detection of Por-cine reproductive and respiratory syndrome virus (PRRSV) antibodies in oral fluid specimens using a commercial PRRSV serum antibody enzyme-linked immunosorbent assay. J Vet Diagn Invest.  2012;24:262–269.Figure 2: Distribution of total contact time with the rope among trained pigs as described in Figure 1 (n = 143).Observed pigs (%)Total contact time (minutes)25201510500         5  10            15                 20           25      30Figure 3: Number of positive-contact events among trained pigs (n =143) as described in Figure 1. While a few pigs contacted the rope in a single contact event, the majority cycled away and back to the rope over the observation period.0 1 32 4 5 6 7(n = 35) (n = 22) (n = 20)(n = 36) (n = 18) (n = 9) (n = 2) (n = 1)90th percentile75th percentile50th percentile25th percentile10th percentileMeanTotal contact time (minutes)302520151050Number of “pig-on-rope” contact events(n = number of pigs)observed pigs contacted the rope in the first 10 minutes, 59 pigs (65.6%) in 20 minutes, and 63 pigs (70.0%) in 30 minutes; 27 pigs (30.0%) never contacted the rope. Cumula-tively, it took 64 minutes for the untrained pigs to reach a 70% participation level.DiscussionThe use of oral-fluid-based surveillance facilitates health monitoring while providing a positive image for pork producers. That is, the use of oral fluids shows that we are Journal of Swine Health and Production — May and June 20141403. Panyasing Y, Goodell CK, Wang C, Kittaworn-rat A, Prickett JR, Schwartz KJ, Ballagi A, Lizano S, Zimmerman JJ. Detection of influenza A virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking ELISA. Transbound Emerg Dis. 2014;61:171–184.  doi:1111/tbed.12019.4. Prickett JR, Johnson J, Murtaugh MP, Puvanendi-ran S, Wang C, Zimmerman JJ, Opriessnig T. Pro-longed detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation. Transbound Emerg Dis.  2011;58:121–127.5. Mur L, Gallardo C, Soler A, Zimmerman J, Pelayo V, Nieto R, Sánchez-Vizcaíno JM, Arias M. Potential use of oral fluid samples for serological diagnosis of African swine fever. Vet Microbiol.  2013;165:135–139.6. Olsen C, Wang C, Christopher-Hennings J, Doolittle K, Harmon KM, Abate S, Kittawornrat A, Lizano S, Main R, Nelson EA, Otterson T, Panyas-ing Y, Rademacher C, Rauh R, Shah R, Zimmer-man J. Probability of detecting Porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a func-tion of within-pen prevalence. J Vet Diagn Invest.  2013;25:328–335.7. Goodell CK, Prickett J, Kittawornrat A, Zhou F, Rauh R, Nelson W, O’Connell C, Burrell A, Wang C, Yoon K-J, Zimmerman JJ. Probability of detecting influenza A virus subtypes H1N1 and H3N2 in individual pig nasal swabs and pen-based oral fluid specimens over time. Vet Microbiol.  2013;166:450–460.8. Goodell CK, Prickett J, Kittawornrat A, John-son J, Zhang J, Wang C, Zimmerman JJ. Evaluation of screening assays for the detection of influenza A virus serum antibodies in swine. Transbound Emerg Dis. 2014. In press.  doi:10.1111/tbed.12214.9. Kittawornrat A, Wang C, Anderson G, Bal-lagi A, Broes A, Carman S, Doolittle K, Galeota J, Johnson J, Lizano S, Nelson E, Patnayak D, Pogranichniy R, Rice A, Scherba G, Zimmerman J. Ring test evaluation of the repeatability and repro-ducibility of a porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid enzyme-linked immunosorbent assay. J Vet Diagn Invest.  2012;24:1057–1063.10. Day JEL, Kyriazakis I, Lawrence AB. The effect of food deprivation on the expression of foraging and exploratory behaviour in the growing pig. Appl Anim Behav Sci.  1995;42:193–206.11. Van Putten G, Dammers J. A comparative study of the well-being of piglets reared conventionally and in cages. Appl Anim Ethol.  1976;2:339–356.12. Docking CM, Van de Weerd HA, Day JEL, Edwards SA. The influence of age on the use of potential enrichment objects and synchronisa-tion of behaviour of pigs. Appl Anim Behav Sci.  2008;110:244–257.13. Feddes R, Fraser D. Non-nutritive chewing by pigs: implications for tail-biting and behavioral enrichment. Trans ASAE.  1994;37:947–950.14. Gifford AK, Clotier S, Newberry RC. Objects as enrichment: Effects of object exposure time and delay interval on object recognition memory of the domestic pig. Appl Anim Behav Sci.  2007;107:206–217.15. Van de Weerd HA, Docking CM, Day JEL, Avery PJ, Edwards SA. A systematic approach towards developing environmental enrichment for pigs. Appl Anim Behav Sci.  2003;84:101–118. Figure 4: Percent of “untrained pigs” (n = 90) interacting with the rope over the first observation period. Observation periods described in Figure 1. Untrained pigs had no previous experience with oral-fluid collection, in contrast to the trained pigs (described in Figure 1).Observed pigs (%)80706050403020100Observation period (minutes)0         5     10          15       20   25         30Contact by minute Cumulative contactFigure 5. Percent of untrained pigs (n = 90; described in Figure 4) interacting with the rope (cumulative contact) over five sequential observation periods (described in Figure 1).Observed pigs (%)80706050403020100Observation period (minutes)0         5     10          15       20   25         30Observation 1Observation 4 Observation 5Observation 2 Observation 3141Journal of Swine Health and Production — Volume 22, Number 3

Recommendations for pen-based oral-fluid collection in growing pigs

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Recommendations for pen-based oral-fluid collection in growing pigs

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