Rearing pigs with play opportunities: viral load and clinical, behavioural, performance, and immune data

Positive emotions can reduce disease susceptibility during infectious challenges in humans, and emerging evidence suggests similar effects in farm animals. Because play behaviour may support a positive emotional state in pigs, this study investigates whether rearing pigs with regular intermittent play opportunities enhances disease resilience when challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Litters were assigned to either play (PLY; n = 5 L) or control (CON; n = 4 L) treatments at birth. In PLY, play was promoted with extra space and enrichment items for three hours daily from five days of age (doa). At weaning (25 ± 2 doa; mean ± SD), 28 pigs (14/treatment) were selected for a disease challenge, based on weight, sex, and sow. The pigs were transported to a disease containment facility and at 43 ± 2 doa (day 0 post-inoculation, DPI) inoculated with PRRSV. Skin lesions, blood, rectal temperature, clinical signs, body weight, and behaviour were collected pre- and post-inoculation. Play opportunities for PLY continued every other day until euthanasia of all pigs at 65 ± 2 doa (22 DPI). PLY pigs exhibited fewer skin lesions following transport and throughout the infection compared to CON. Although the viral load did not differ between treatments, PLY pigs had a lower probability of experiencing moderate and severe respiratory distress, with a shorter duration. PLY also performed better throughout the infection, showing higher ADG and greater feed efficiency. The immune response differed as well. PLY pigs had fewer monocytes on 8 DPI than CON, with levels returning to baseline by 21 DPI, whereas CON levels exceeded baseline. Regardless of day of infection, lymphocyte counts tended to be lower in PLY than in CON, and white blood cells and neutrophils were also lower, but only in slow-growing pigs. PLY pigs continued to play during the infection, demonstrating less sickness behaviour and emphasizing the rewarding properties of play. Results suggest that PLY pigs were less affected by PRRSV and developed increased resilience to PRRSV compared to CON. This study demonstrates that rearing pigs in an environment supporting positive experiences through provision of play opportunities can enhance resilience against common modern production challenges, underscoring the value of positive welfare in intensive pig farming.

Methods
Animal measures in detail, including tables, are described in the published paper.

Citation: Steinerová K, Harding JC, Parker SE, Wilson HL, Nery Finatto A and Seddon YM (2024) Rearing pigs with play opportunities: the effects on disease resilience in pigs experimentally inoculated with PRRSV. Front. Vet. Sci. 11:1460993. doi: 10.3389/fvets.2024.1460993

Behaviour and skin lesions

The duration of play (locomotor, social and object) and exploratory behaviour (Table 2) were scored with continuous sampling within the initial 10 min of the play sessions only in the PLY treatment at -2 (baseline), 3, 7, 11, 16 and 20 DPI. The scoring commenced after an experimenter exited the playpen and closed the gate. On the same days, to assess pig activity during the challenge, the frequency of active, inactive, and feeding behaviours (Table 2) in the PLY and CON treatments were assessed through instantaneous sampling within the first half of the play sessions at 5-min intervals (90 min, 18 scans/pig/DPI). Additionally, scans were collected at 10-min intervals when no play sessions were occurring for two 90-min periods without human presence in the pen, in the morning (between 7:30 – 9:30 AM) and evening (between 5:00 – 7:00 PM), totalling 180 min per day (18 scans/pig/DPI). In the chosen periods, human presence in the BSL2 was recorded only in the AM, and when it happened, an experimenter continued scanning but noted a person in the room. The pigs were individually marked with spray paint (Raidex GmbH, Dettingen/Erms, Germany) at least two hours before video recording.

All behaviours were videotaped with Lorex cameras (4K Ultra HD IP Security Camera, Lorex Technology, Markham, ON, Canada) in the farrowing room (one camera/two neighbouring home pens), the nursery room (one camera/home pen, one camera/two playpens) and the BSL2 room (one camera/pen; Figure 1). The behaviours were scored from the video recordings by one experimenter using the Observer software XT14 (version 14.2.1127, Noldus, Leesburg, VA, USA). The experimenter could not be blinded to the treatments due to clear distinctions between treatments in the experimental set up (playpens in PLY) and restricted number of trained personnel allowed to access to the disease containment facility.

Skin lesions were scored as a proxy measure of aggression (Turner et al., 2006) two days pre-weaning (age: D23 ± 2 (days); mean ± S.D.), one-day post-weaning (age: D26 ± 2), one day before transport (age: D33 ± 2), one day after transport (age: D35 ± 2), pre-inoculation (-2 DPI, age: D40 ± 2) and at the end of the trial (21 DPI, age: D64 ± 2). The body was divided into six regions: ears, face, front (neck, shoulders, and front legs), middle (the body after the shoulders up to the frontal tip of the hind legs), rear (the hind legs), and tail[1] (modified from Turner et al., 2006). Each body region was scored individually and was assigned a score from 0 to 3: score 0 (none) = no lesions; score 1 (mild) = less than five superficial scratches; score 2 (moderate) = 5-10 superficial scratches and/or less than three deep wounds; score 3 (severe) = more than 10 superficial scratches and/or more than three deep wounds. A total body skin lesion score was calculated by summing all body region scores per pig and day (maximum score of 18/pig/day). One experimenter, who could not be blinded to the treatments, directly scored the skin lesions while standing outside the pen.

Rectal temperature and body weight

Rectal temperature (RT) was taken on 0 (baseline), 2, 4, 8, 13, 17, and 21 DPI using a digital thermometer with a flexible tip and a resolution of 0.1ºC. On 0 DPI, a baseline RT was measured pre-inoculation. However, due to technical difficulties with the thermometer, the initial baseline data were discarded, and the baseline RT was recorded three hours post-inoculation before the onset of detectable viremia (6-48 hours post-exposure; Zimmerman et al., 2019a) using a new thermometer that was used thereafter. The pigs were weighted on 0, 8, 13, 17, and 21 DPI on a digital scale with a resolution of 0.1 kg. The ADG post-inoculation was calculated between each subsequent weigh period per pig.

Blood collection and clinical signs

Blood (serum, EDTA) was collected from the jugular vein with the pig restrained in a supine position on -1, 2, 4, 8, 13, 17, and 21 DPI. Tubes with EDTA were gently inverted 8-10 times to ensure thorough mixing with the anti-coagulant and stored on ice. Rectal temperature, body weight, and blood were collected between 8 to 10 am in the aforementioned order.

To prevent cross-contamination, the negative control pigs in the BSL1 were blood sampled (serum, EDTA), and weighted and their RT was collected on -1 (blood) or 0 (weight, RT), 13, and 21 DPI before the pigs in the BSL2.

The pigs in the BSL2 were monitored for PRRSV clinical signs with scores assigned based on severity (0: not present, 4: severe) in the AM and PM. The negative control pigs in the BSL1 were monitored in the AM only by a separate team, from the first day in the acclimation period until 21 DPI. Monitored clinical signs included: respiratory distress (RD), coughing, responsiveness, appetite, colour of the skin, consistency of the faeces, body condition, and additionally lameness as a clinical sign not specific to PRRSV (see description in Table 2 in the suppl. mat.).

Pen feed intake

Pen feed intake was recorded in the PM pre-inoculation on -8, -5, and -1 DPI, and post-inoculation on 2, 6, 9, 13, 16, 20, and 21 DPI. Feed intake was divided into periods: pre-inoculation (DPI -8 to -1; 8 days), one-week post-inoculation (DPI 0 to 6; 7 days), second-week post-inoculation (DPI 7 to 13; 7 days), third-week post-inoculation (DPI 14 to 21; 8 days), from which the average feed intake per pig per day in a given period was calculated. Feed-to-gain ratio (F:G) was calculated per pen (total (from 0 to 21 DPI) feed intake per pen / total gain per pen) and averaged per treatment.

Gross lung lesions

At necropsy, lungs were rinsed with water and carefully placed on a tray, and their ventral and dorsal surface showing left and right cranial (CR), middle (M), caudal (CA), and accessory (A) lobes were photographed for later examination of pathomorphological changes. A consistent observer utilized a lung drawing from Halbur et al. (1995) to shade areas on the lobes exhibiting the colour change observed in the photographs. Lung lesions typical of interstitial pneumonia and differing in severity with colour ranging from tan to dark red and purple (Zimmerman et al., 2019b) were identified. A 9 mm by 9 mm grid was placed on the shaded lung drawing to calculate the proportion of the affected lobes (number of shaded grid squares (with a precision of ¾ of a square) / total number of grid squares). This proportion was then multiplied with a pre-defined score assigned to each lobe (ventral left and right – CR: 10, M: 10, CA: 25, A: 5; dorsal left and right – CR: 10, M: 10, CA: 30; Halbur et al. 1995), resulting in an estimate of the percentage of the affected lobe, and thereafter summed to determine the total affected area of the lungs. Other characteristics of the gross lung lesions, such as the consistency of the lungs (slightly firm to rubbery) (Zimmerman et al., 2019b) were not possible to record from the photographs.

Lab analyses

Immediately after the blood collection, whole blood (-1, 2, 4, 8, 13, 17, and 21 DPI) was submitted to Prairie Diagnostic Services (PDS) for a total count of white blood cells (WBC) and differential counts of lymphocytes, neutrophils, and monocytes counted in a haematology analyser (Advia 2120i, Siemens Healthcare Diagnostics, Erlangen, Germany). Serum was extracted from serum-separating tubes (Vacutainer® SST™) by centrifuging 1500 g for 10 min at 4°C, aliquoted to vials and stored at -80°C until further analysis. Serum samples (-1, 2, 4, 8, 13, 17, and 21 DPI) were analysed for total circulating triiodothyronine (T3), and PRRSV RNA. Triiodothyronine was quantified in PDS in the IMMULITE® 2000 Systems Analyser (Siemens Healthcare Diagnostics, Erlangen, Germany). The samples were assayed in duplicate with a calibration range of 0.61 to 9.2 nmol/L and analytical sensitivity of 0.29 nmol/L, with the protocol followed without any modifications.

Quantification of PRRSV RNA

The concentration of PRRSV strain NVSL 97-7895 RNA was determined using an in-house quantitative reverse transcription PCR assay (qRT-PCR). Each sample was individually assessed for the presence of target PRRSV RNA copies/mL. RNA was extracted from 140 µL of serum using the QIAamp Viral RNA mini kit (Qiagen Inc., Toronto, ON) according to the manufacturer’s instructions. The concentration (A260) and purity (A260/A280) of the extracted viral RNA were determined using spectrophotometry (NanoDrop 2000c, Thermo Fisher Scientific).

To quantify PRRSV RNA levels in the serum of pigs, a probe-based qRT-PCR assay previously described by Ladinig et al., (2014) was employed. The primers and probe targeted the highly conserved region at the C-terminal end of ORF7 of NVSL 97-7895. The primer sequences were as follows: PRRS-2F primer 5’-TAA TGG GCT GGC ATT CCT-3’, PRRS-1R primer 5’-ACA CGG TCG CCC TAA TTG-3’, and the probe 5’-HEX-TGT GGT GAA TGG CAC TGA TTG RCA-BHQ2-3’. A dilution series (1.8 x 108 to 1.8 x 102 copies/µL) of HindIII linearized plasmid, pCR2.1TOPO-NVSL, containing a 446 bp sequence of ORF7, was used as a standard curve. The standards were run in triplicate on each PCR plate, while the tested samples were run in duplicate. All qRT-PCR reactions were performed on a 96-well plate (Hard-Shell 96-Well PCR Plate, Applied Biosystems), sealed with a Microseal ‘B’ PCR Plate Sealing Film (Applied Biosystems), and analysed on a Step-One Plus Real-Time PCR System (Bio-Rad Laboratories). Each qRT-PCR reaction consisted of 2 µL of sample or standard, 6 µL of RNAse-free water, 10 µL of iTaq Universal Probes 1-step kit (Bio-Rad Laboratories), 10 µM of PRRS-2F primer, 10 µM of PRRS-1R primer, 10 µM of PRRS-P1 probe, and 0.5 µL of iScript reverse transcriptase (Bio-Rad Laboratories). The thermocycling protocol included a reverse transcription step at 50°C for 30 min, followed by an initial activation step at 95°C for 10 min, and 40 cycles of denaturation (30 sec at 95°C) and annealing/extension (30 sec at 59°C). Individual samples were re-run if the cycle quantification (Cq) standard deviation between duplicates was >1.0 or if one of the duplicates had no cycle threshold (Ct) value. The results were reported as PRRSV RNA concentration per mL of serum. The limits of quantification were determined based on the least and most concentrated standards. Samples were considered negative if the target RNA was not detected or DNQ (detected, but not quantifiable).