Animals and housing
Eight female 8-week-old Belgian Landrace piglets of approximately 20 kg, purchased from a commercial swine herd known to be free of PRRS virus (TaqMan NA and EU PRRSV Reagents, Life Technologies) and negative for PRRSV-specific antibodies (PRRS X3 Ab Test, Idexx), were housed individually on slatted floors at air-filtered level-2 biosecurity facilities (CODA-CERVA, Machelen). Each pig was randomly assigned to control or vaccination or infection groups. Water and food were provided ad libitum. Animal experiments were performed in accordance with EU and Belgian regulations on animal welfare in experimentation. The protocol was approved by the joined ethical committee of CODA-CERVA and the Scientific Institute of Public health Belgium (procedure agreement no. 120112–01).
Vaccination and infection
After one week of acclimatization, two experiments were conducted: a vaccination experiment and an infection experiment.
For the vaccination experiment, an attenuated PRRSV vaccine (Porcilis PRRS, Intervet) was used. The vaccine virus was diluted in Diluvac Forte according to the manufacturer’s instructions and each vaccine dose contained at least 104 50% tissue culture infectious doses (TCID50) of PRRS vaccine virus strain DV. Three pigs were vaccinated intramuscularly with 2 mL vaccine/pig at 9 and 11 weeks of age. One pig was not vaccinated and served as a control animal.
For the infection experiment, a 5th passage on MARC-145 cells of the PRRSV prototype strain Lelystad virus was used. Lelystad virus was cultivated and titrated on MARC-145 cells as described before [19]. A virus stock containing 1 × 105 TCID50/mL was aliquotted and stored at -80°C until use. Three nine-week-old pigs were inoculated with PRRSV Lelystad virus via aerosol inoculation (105 TCID50/nostril). One pig was not inoculated and served as a control animal.
Sample collection
During the week before the start of the experiments, the animals were trained to chew on six different rope materials – two natural fibred ropes (cotton and hemp) and four synthetic ropes (polyester and polyamide (water absorbing); polypropylene and polyethylene (water repellent)) – by positive reinforcement.
In the vaccination experiment, “stimulated” oral fluids (oral fluids collected by masticatory or gustatory stimulation such as chewing [20]) were collected 1 day before the first vaccination and 14 days after each vaccination with four different rope materials according to procedures described by Prickett et al. [17]. In brief, one rope (length 1 m; diameter 14 mm) was suspended in each pen and was left in place for 30 min, during which the animal could chew on it and moisten it with oral fluid. At each time-point, animals were presented with the cotton ropes first, followed by hemp, polyamide and polyester. To recover the oral fluid samples, ropes were manually wrung and the oral fluids were collected in 50 mL conical centrifuge tubes (BD Falcon). “Unstimulated” oral fluid (oral fluid collected without exogenous gustatory, masticatory, or mechanical stimulation [20]) was collected at the same time points by means of suction with a small suction catheter. Prior to the oral fluid collection, animals were deprived of food for 8 h to prevent cross-reaction of food products with antibodies. Blood samples were collected from each pig at -7 and 28 days post vaccination using venepuncture.
In the infection experiment, stimulated oral fluids were collected with two different rope types (cotton first, followed by polyester) at -1, 14 and 28 days post infection as described above. Blood samples were obtained at the same time points. The time points for sample collection were chosen based on literature describing that PRRSV specific IgM and IgA antibodies could be detected at 14 days p.i. and IgG antibodies at 28 days p.i. in BAL fluids of PRRSV inoculated pigs [21]. All oral fluid samples were immediately chilled on ice, centrifuged at 1147 × g (Jouan CR312) for 10 minutes to remove insoluble materials, and stored at -80°C until use. From the blood samples, serum was collected, incubated for 30 min at 56°C and kept at -80°C until use.
All samples were first collected from the negative control animals, followed by the vaccinated animals and then the infected animals, in order to avoid contamination of the samples as much as possible.
Detection of total IgA, IgG and IgM in oral fluid samples
Total amounts of IgA, IgG and IgM antibodies present in oral fluid samples from vaccinated animals were measured using a commercial direct sandwich ELISA (IgA, IgG and IgM (pig) – ELISA, Celltrend GmbH), following the manufacturer’s recommendations. Oral fluid samples were diluted 1:500 (IgA), 1:250 (IgG) and 1:50 (IgM) in phosphate-buffered saline (PBS), respectively. A standard curve was generated by assaying 2-fold dilutions of a standard concentrate containing 1000 ng/mL IgA (x-axis: log, Ig concentration; y-axis: linear, absorbance) and oral fluid results that fell within the linear part of the reference curve were extrapolated to determine concentrations, taking the dilution factor of the oral fluid samples into account. When the absorbance was outside the standard curve a subsequent determination with changed sample dilutions was carried out. Western Blotting was used to confirm ELISA results. Briefly, oral fluid samples were diluted 1:2 in PBS (pH 7.2) and mixed with NuPAGE Sample Reducing Agent (Life Technologies). The samples were separated by electrophoresis in 12% Tris–HCl gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) via Western Blotting. Membranes were blocked overnight in 5% non-fat dry milk in PBS containing 0.05% Tween-20 at 4°C. Membranes were consecutively incubated with a 1:50,000 dilution of biotinylated goat anti-swine IgA, IgG or IgM antibodies (AbD Serotec; 1.5 h, room temperature) and a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated streptavidin (GE Healthcare; 1.5 h, room temperature). Subsequently, labeled proteins were visualised using the SuperSignal West Dura Chemiluminescent Substrate ECL kit (Thermo Scientific). The emitted chemiluminescent signal was detected with a VersaDoc MP 4000 image analyser (Bio-Rad). ImageJ software (NIH Image) was used to estimate the relative amounts of protein (relative to antibody concentrations in unstimulated oral fluid).
Detection of PRRSV-specific antibodies with IgA and IgG isotype in serum samples and oral fluid samples
PRRSV-specific IgG antibodies in sera were detected using the PRRS X3 Ab Test (Idexx) according to the manufacturer’s instructions. S/P ratios were calculated, using the following formula: S/P = (OD of sample – OD of negative control)/(OD of positive control – OD of negative control). Samples with an S/P ratio above 0.4 were considered positive for PRRSV-specific IgG antibodies. In addition, PRRSV-specific IgG antibody titres in sera were determined using the immunoperoxidase monolayer assay (IPMA) technique described by Labarque et al. [21]. Briefly, Marc-145 cells were seeded in 96-well cell culture plates, inoculated with PRRSV Lelystad virus and incubated for 24 h at 37°C and 5% CO2. Subsequently, culture medium was removed, cells were washed with PBS and dried at 37°C for 1 h. The plates were kept at -80°C until use. Plates were thawed and cells were fixed in 4% paraformaldehyde for 10 min and then washed twice with PBS. A solution of 1% H2O2 in methanol was added to the cells for 5 min, and cells were subsequently washed twice with PBS. 4-fold dilution series (starting dilution 1:10) of the sera in phosphate-buffered saline supplemented with 1% Tween 80 (PBS-T) and 10% negative goat serum were then added to the infected Marc-145 cells and cells were incubated for 1 h at 37°C. Subsequently, serum dilutions were removed and cells were washed with PBS-T. Cells were then incubated with peroxidase-labeled goat anti-swine IgG antibodies (Dako; dilution 1:500 in PBS-T + 10% negative goat serum) at 37°C for 1 h. Finally, the plates were washed with PBS-T and 50 μL of a substrate solution of 5% 3-amino-9-ethyl-carbazole in 0.05 M Na-acetate buffer (pH 5) supplemented with 0.05% H2O2 was added to each well and incubated at room temperature for 20 min. The reaction was stopped by replacing the substrate solution with Na-acetate buffer and the staining was analyzed via light microscopy (Axiovert 25, Zeiss). Positive and negative control samples were included on each plate.
In oral fluid specimens, PRRSV-specific IgA and IgG antibodies were detected using IgA- and IgG-based IPMAs, immunofluorescence assays (IFA), a commercial IgG-based ELISA (Idexx PRRS OF) and a commercial PRRSV ELISA modified for the detection of PRRSV-specific IgA in swine oral fluid [12]. The IPMA used for detection of PRRSV-specific IgA and IgG in oral fluid samples was a modified version of the IPMA for serum samples described above. Two-fold serial dilutions (starting dilution 1:2) of oral fluid samples were made in PBS and added to the infected Marc-145 cells. After 1 h of incubation at 37°C, oral fluid dilutions were removed and cells were washed with PBS-T. Subsequently, cells were incubated with biotinylated goat- anti-swine IgA (AbD Serotec; 1:10 in PBS) or biotinylated goat- anti-swine IgG (AbD Serotec; 1:10 in PBS) for 1 h at 37°C. Following a washing step with PBS-T, cells were incubated with Streptavidine-HRP (GE Healthcare; 1:20 in PBS) for 1 h at 37°C. Visualization of antibody-labeled, PRRSV-infected cells was identical as described above. For IFA, Marc-145 cells were seeded on glass inserts in 24-well plates, infected with PRRSV Lelystad virus and incubated for 24 h at 37°C and 5% CO2. Cells were then fixed in 100% methanol for 10 min at -20°C, air dried, and stored at -80°C until use. Cells were thawed and incubated with 1:2 dilutions of the oral fluids in PBS for 2 h at 37°C. Subsequently, cells were washed and incubated with either goat anti-swine IgA (dilution 1:10) (AbD Serotec) or goat anti-swine IgG (dilution 1:20) (AbD Serotec) for 1 h at 37°C. Finally, cells were washed and incubated with anti-goat FITC (dilution 1:50 or 1:100 when previously incubated with goat anti-swine IgA or IgG respectively) for 1 h at 37°C. Cells were washed, embedded in a glycerine-PBS solution (0.9/0.1 v/v) containing 2.5% 1, 4-diazabicyclo-(2,2,2)-octane, mounted and analysed using a fluorescence microscope (Dialux 20, Leitz Wetzlar) for FITC-stained PRRSV positive cells.
Oral fluid samples were also tested for the presence of PRRSV-specific IgG antibodies using the PRRS OF kit (Idexx), according to the manufacturer’s instructions. The OD values were converted into S/P ratios, using the formula as described above. Samples with an S/P ratio above 0.4 were considered positive for PRRSV-specific IgG antibodies. PRRSV-specific IgA in oral fluid samples was measured with a modified PRRS X3 Ab Test (Idexx) as described by Kittawornrat et al. [12]. Reference standard samples (=oral fluid samples collected from 37 pens at 0, 10, 15, 20, 28, 35, 41, 49, 56, 75, and 91 days after intramuscularly vaccination of 5-week-old pigs (n = ~1,100) with 2 ml of a type 2 PRRS MLV vaccine, kindly donated by prof. Zimmerman, College of Veterinary Medicine,
Iowa State University) were included in this modified test to verify its correct performance. The raw plate data were converted into S/P ratios, using the formula as described above with the OD values of reference samples at day 0 and 35 as negative and positive controls, respectively.
Statistical analyses
Differences in oral fluid volumes collected with different rope materials; differences between IgA, IgG and IgM concentrations collected with a specific rope; and differences between ropes for IgA, IgG and IgM collection were compared by analysis of variance (ANOVA) and a Bonferroni post hoc test was applied for multiple comparisons. Statistical analyses were performed using SPSS Statistics V21.0 (IBM) software and p values ≤ 0.05 were considered to be significant.