Experimental design
Individual pig samples (rectal swabs and/or serum) and pen samples (fecal and/or oral fluid specimens) were collected longitudinally from one PEDV-positive commercial wean-to-finish (WTF) barn in Missouri USA (Site One) and one PEDV-negative commercial WTF barn in Iowa USA (Site Two). Fecal samples and oral fluids were tested by PEDV real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Serum and oral fluid specimens were tested by two PEDV antibody WV ELISAs (IgA, IgG). Testing results were used to describe PEDV shedding, establish the performance parameters of two PEDV WV ELISAs (IgA, IgG), and characterize antibody kinetics in a commercial pig production system. This project was approved in writing both by an agent representing the livestock producer and the Iowa State University Office for Responsible Research.
Site descriptions
Site One was a 52-pen WTF barn stocked with ~800 pigs. Pens were separated by metal gates, with 26 pens on each side of the walk way. Pens were equipped with automatic feeders, bowl drinkers, and fully slatted floors. The facility was designed with negative pressure tunnel ventilation and a deep pit (2.4 m) manure handling system. Pigs were placed in the facility at the time of weaning (~3 weeks of age). Pen samples (feces and oral fluids) and pig samples (rectal swabs and serum) were collected from the same 6 pens and a convenience sample of 5 pigs in each of the 6 pens at each sampling point. Sampling began when the pigs were ~3 weeks of age and continued at ~2-week intervals for 27 weeks. At 10 weeks post-placement, i.e., when pigs were approximately 13 weeks of age, the producer exposed the pigs (replacement gilts) to PEDV by mixing PEDV-positive fecal material with water and spraying feed and the pigs’ oral-nasal area with the mixture using a hand-held sprayer.
Site Two consisted of 3 identical 40-pen WTF barns, each stocked with ~900 pigs. Pens were separated by solid walls, with 20 pens on each side of the walk way. Pens were equipped with automatic feeders, bowl drinkers, and half-slatted floors. The barns were constructed with natural ventilation and deep pit (2.4 m) manure handling systems. Pigs were placed in the facility at the time of weaning (~3 weeks of age). Pen oral fluid samples were collected from 36 pens (4 pens were not stocked) in each of the 3 barns and serum samples were collected from a convenience sample of 20 pigs in 2 pens (10 pigs per pen) in each barn. Sampling began at two weeks post placement (pigs were ~5 weeks of age) and continued weekly for a total of 9 samplings. Individual pig rectal swabs and pen fecal specimens were not collected on Site Two.
Sample collection
Individual pigs were restrained and bled using 12.5 mL vacutainer tubes (Covidien, Minneapolis, MN USA) and 20 gauge x 3.81 cm (1 ½ in.) needles (Smiths Medical, Dublin, OH USA). Blood samples were centrifuged at the laboratory, aliquoted, and stored at −20 °C.
Fecal swabs were collected from individual pigs using a commercial collection and transport system (StarswabII®, Starplex® Scientific Inc., Cleveland, TN USA) and stored at −20 °C. Prior to testing, swabs were suspended in 1 mL of PBS (1X pH 7.4, Invitrogen Corporation, Carlsbad, CA USA), vortexed, and the liquid submitted for testing by PEDV rRT-PCR.
Each pen-level fecal sample consisted of a convenience sample of 3-to-5 fresh semi-solid feces from throughout the pen. Approximately equal portions of pen feces were placed in one 50 mL tube (Thermo Fisher Scientific, Waltham, MA USA) and stored at −20 °C. Prior to testing, samples were homogenized (stirred), ~1.0 g placed in 1 mL of PBS (1X pH 7.4, Invitrogen Corporation) and submitted for PEDV rRT-PCR testing.
Pen-based oral fluids were collected as described elsewhere [6]. In brief, 3-strand, 100 % cotton rope was cut with the free end at shoulder height to the animals and suspended in the pen for 20 to 30 min. Pigs actively sought out and chewed the rope, leaving the strands moistened with oral fluids. The rope was then removed from the pen and the wet portion placed in a single-use plastic bag. Oral fluids were extracted by either manual or mechanical compression (wringer) of the wet rope, after which the fluid was decanted into 50 mL centrifuge tubes (Fisher Scientific) and stored at −20 °C.
For each site, all samples were completely randomized (random.org) within specimen type and submitted for testing at the end of the collection period.
Diagnostic testing
PEDV RNA extraction and real-time reverse transcriptase PCR (rRT-PCR)
In brief, 90 μl of viral RNA was eluted from rectal swabs, fecal samples and oral fluid specimens using the Ambion® MagMAX™ viral RNA isolation kit (Life Technologies, Carlsbad CA USA) and a KingFisher® 96 magnetic particle processor (Thermo-Fisher Scientific) following the procedures provided by the manufacturers. Samples were tested for PEDV using a PEDV N gene-based rRT-PCR described in Madson et al. [7] and performed routinely at the Iowa State University-Veterinary Diagnostic Laboratory (ISU-VDL SOP 9.5263). The forward primer sequence was 5′-CGCAAAGACTGAACCCACTAACCT-3′, the reverse primer sequence was 5′-TTGCCTCTGTTGTTACTTGGAGAT-3′, and probe sequence was 5′-FAM-TGTTGCCAT/ZEN/TACCACGACTCCTGC-Iowa Black-3′. The eluted RNA, primers, and probe were mixed with commercial reagents TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) and the rRT-PCR reactions were conducted on an ABI 7500 Fast instrument (Life Technologies) in fast mode as follows: 1 cycle at 50 °C for 5 min, 1 cycle at 95 °C for 20 s, 40 cycles at 95 °C for 3 s, and 60 °C for 30 s. The results were analyzed using an automatic baseline setting with a threshold at 0.1. Quantification cycle (Cq) values < 35 were considered positive for the corresponding coronavirus. Data were reported as ‘adjusted Cqs’:
$$ \mathrm{Adjusted}\ \mathrm{C}\mathrm{q} = \left(35\ \hbox{--}\ \mathrm{sample}\ \mathrm{C}\mathrm{q}\right) $$
(1)
PEDV whole virus (WV) antibody ELISA
A U.S. prototype PEDV isolate (USA/NC35140/2013, [8]) was used in the PEDV WV antibody ELISA. Each batch of approximately 1000 mL of PEDV was propagated on Vero cells (ATCC CCL-81). Briefly, one 75 cm2 flask (Thermo Fisher Scientific) of confluent Vero cells was inoculated with 3 mL of PEDV stock (1x105 TCID50 per mL) followed by the addition of 50 mL of cell culture medium composed of MEM 1X (Minimum Essential Medium, Life Technologies) supplemented with 0.3 % tryptose phosphate broth, 0.02 % yeast extract, 5 ug per mL Trypsin 250 (Sigma-Aldrich, St. Louis, MO USA), plus penicillin/streptomycin (10 U per mL), gentamicin (0.05 mg per mL) and amphotericin (0.25 μg per mL) as antibiotics. After 3 to 4 days at 37 °C in a 5 % CO2 incubator and when cytopathic effects were apparent, the contents of the flask (53 mL) were used to further expand the virus by inoculating each of 4 875 cm2 flasks (BD Falcon, San Jose, CA) containing confluent Vero cell monolayers with 13 mL of the harvested PEDV plus 240 mL of culture medium. After 3 to 4 days of incubation and when cytopathic effects were apparent, the fluid was frozen (−80 °C), thawed, poured off, and then centrifuged at 4,000 x g for 15 min to remove cell debris. The virus was pelleted by ultracentrifugation at 140,992 x g for 3 h, after which the pellet was washed twice with sterile PBS (1X pH 7.4) to remove culture medium components. The purified virus was re-suspended in 100µl PBS (1X pH 7.4) at a 1:100 dilution of the original supernatant volume and stored at −80 °C. Following titration and optimal dilution (PBS pH 7.4), polystyrene 96-well microtitration plates (Nalge Nunc, Rochester, NY USA) were manually coated (100 μl per well) with the viral antigen solution and incubated at 4 °C overnight. After incubation, plates were washed 5 times, blocked with 300 μl per well of a solution containing 1 % bovine serum albumin (Jackson ImmunoResearch Inc., West Grove, PA USA), and incubated at 25 °C for 2 h. Plates were then dried at 37 °C for 4 h and stored at 4 °C in a sealed bag with desiccant packs. The performance of each lot of plates was standardized using a panel of reference PEDV negatives and positives. Plate lots with a coefficient of variation ≥10 % were rejected.
ELISA conditions for the detection of anti-PEDV IgA and IgG antibodies in serum and oral fluid specimens, including coating and blocking conditions, reagent concentrations, incubation times, and buffers, were identical. Serum samples were diluted 1:50 and oral fluid samples were diluted 1:2, after which plates were loaded with 100 μl of the diluted sample per well. Plates were incubated at 25 °C (serum) or 37 °C (oral fluid) for 1 h and then washed 5 times with PBS (1X pH 7.4). Positive and negative plate controls, i.e., antibody-positive and -negative experimental serum samples, were run in duplicate on each ELISA plate.
To perform the assay, 100 μl of peroxidase-conjugated goat anti-pig IgG (Fc) antibody (Bethyl Laboratories Inc., Montgomery, TX USA) diluted 1:20,000 for serum and 1:3,000 for oral fluid samples or goat anti-pig IgA (Bethyl Laboratories Inc.) diluted 1:7,000 for serum and 1:3,000 for oral fluid samples was added to each well and the plates incubated at 25 °C (serum) or 37 °C (oral fluid) for 1 h. After a washing step, the reaction was visualized by adding 100 μl of tetramethylbenzidine-hydrogen peroxide (Dako North America, Inc., Carpinteria, CA USA) substrate solution to each well. After 5 min incubation at room temperature, the reaction was stopped by the addition of 50 μl of stop solution (1 M sulfuric acid) to each well. Reactions were measured as optical density (OD) at 450 nm using an ELISA plate reader (Biotek® Instruments Inc., Winooski, VT USA) operated with commercial software (GEN5™, Biotek® Instruments Inc.). The antibody response in serum and oral fluid samples was represented as sample-to-positive (S/P) ratios calculated as:
$$ \mathrm{S}/\mathrm{P}\ \mathrm{ratio} = \frac{\left(\mathrm{sample}\ \mathrm{O}\mathrm{D}\ \hbox{--}\ \mathrm{negative}\ \mathrm{control}\ \mathrm{mean}\ \mathrm{O}\mathrm{D}\right)}{\left(\mathrm{positive}\ \mathrm{control}\ \mathrm{mean}\ \mathrm{O}\mathrm{D}\ \hbox{--}\ \mathrm{negative}\ \mathrm{control}\ \mathrm{mean}\ \mathrm{O}\mathrm{D}\right)} $$
(2)
Data analysis
Statistical analyses were performed using commercial statistical software (SAS® Version 9.4, SAS® Institute, Inc., Cary, NC) using test results on serum (Site One, n = 330; Site Two, n = 540), oral fluid (Site One, n = 66; Site 2, n = 972), rectal swabs (Site One, n = 330), and pen feces (Site One, n = 66). A mixed-effects repeated measures model (Proc GLIMMIX) was used to analyze the association between the detection of PEDV by rRT-PCR and the variables of interest, i.e., sample specimen (oral fluids, rectal swab, pen feces, serum) and day post exposure (DPE) using pen as a random effect. Fixed effects were considered significant at α = 0.05. Differences in the proportion of PEDV rRT-PCR positive oral fluid, rectal swab, and pen feces was compared using the Fisher Exact Test. Point and interval estimates of the sensitivity and specificity of the PEDV WV IgG and IgA ELISAs for serum and oral fluid samples were calculated using the exact Binomial formula and confidence intervals.
