Considerations in the use of processing fluids for the detection of PRRSV RNA and antibody

Abstract

Surveillance is mandatory for tracking the progress of porcine reproductive and respiratory syndrome virus (PRRSV) control and elimination efforts in breeding herds. Processing fluids, the fluid recovered from tissues collected at castration and/or tail docking, are used for breeding herd surveillance by large segments of the industry, but the basic diagnostic characteristics of processing fluids are largely undescribed. We undertook 3 studies to address this information gap. In study 1, we found no differences among the PRRSV RT-rtPCR results obtained with 4 commercial RNA extraction kits. In study 2, we found that PRRSV RNA was highly stable in processing fluid samples at −20°C or 4°C, but detrimental effects were observed at ≥22°C within 24 h. In study 3, using a modified PRRSV ELISA at a sample:positive cutoff of ≥0.5, we found excellent discrimination in the detection of PRRSV antibody (IgM, IgA, IgG) in processing fluids from herds of known PRRSV status. Judicious handling of processing fluid samples from sow herds, and the use of methods available in veterinary diagnostic laboratories, can provide a foundation for reliable PRRSV surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV; Arteriviridae, Betaarterivirus, Betaarterivirus suid 1) continues to be a significant health and welfare problem for the swine industry. ² Irrespective of the specific PRRSV control or elimination measures implemented in a herd, surveillance is needed to track the effectiveness of these measures. In breeding herds, surveillance using processing fluid samples, the fluid recovered from tissues collected at castration and/or tail docking, was first reported in 2010 and has been adopted widely by the industry.^1,4,10

Processing fluid samples have been demonstrated to provide a practical, sensitive, and cost-effective approach for PRRSV detection in commercial herds.^{3,4,6,8,11,12,14} In addition to its utility for PRRSV detection, early reports suggested the use of processing fluid samples for the surveillance of other pathogens of swine (e.g., porcine circovirus 2, ⁵ porcine deltacoronavirus, ⁹ Mycoplasma hyopneumoniae, ¹³ and senecavirus A ⁷ ). However, the diagnostic characteristics of processing fluids are sparsely described. Therefore, we undertook 3 studies. In studies 1 and 2, we evaluated the impact of RNA purification procedures and sample storage conditions on PRRSV PCR results. In study 3, we evaluated detection of PRRSV antibody (IgM, IgA, IgG) in processing fluids from herds of known PRRSV status using a commercial PRRSV ELISA (PRRS X3 Ab test; Idexx).

The processing fluids used in our studies were collected in commercial herds by placing a disposable plastic bag inside a clean container, covering the top of the container with disposable gauze held in place with a rubber band, and then placing the testicle and tails on the gauze as piglets were processed. ⁶ At the end of processing, the fluid that collected in the plastic bag was transferred to a sterile tube and submitted for testing. Processing fluids in studies 1 and 2 were samples collected on PRRSV-naïve farms and submitted to the Health Assurance Testing Services (HATS) laboratory at the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL; Ames, IA, USA) for routine PRRSV RNA testing. Samples were first confirmed negative for PRRSV RNA (MagMAX viral RNA isolation kit; Applied Biosystems) and then stored at −20°C in their original tubes. Processing fluids in study 3 consisted of samples submitted to the HATS laboratory from PRRSV-naïve herds and presumed antibody-positive processing fluid samples collected from 2 PRRSV-unstable breed-to-wean herds (herds with endemic PRRSV circulation). For all studies, each processing fluid sample was the product of 20–30 litters (i.e., 230–345 piglets; Table 1).

Table 1.

Source and porcine reproductive and respiratory syndrome virus (PRRSV) status of processing fluid samples.

	Studies 1, 2	Study 3
Herd-of-origin PRRSV status	Naïve*	Naïve*	Positive†
Total samples	70	89	224
Litters per sample	20–30	20–30	20–30

^*Samples submitted from PRRSV-naïve herds for testing at the Health Assurance Testing Services (HATS) laboratory, Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.

^†Field processing fluid samples collected from PRRSV-positive commercial herds.

Wild-type PRRSV2 was initially isolated on ZMAC cells (derived from alveolar macrophages in pig lungs; ATCC PTA-8764) from serum samples collected at the time of a PRRSV outbreak in a commercial herd in the Midwest United States. The virus was propagated on ZMAC cells (ATCC PTA-8764) cultured in RPMI-1640 medium containing L-glutamine and HEPES (Corning) supplemented with MEM non-essential amino acids (Corning), sodium pyruvate (Corning), L-glutamine (Corning), glucose (Corning), fetal bovine serum (MilliporeSigma), mouse macrophage colony stimulating factor (Shenandoah Biotechnology), and antibiotics (gentamicin, penicillin, streptomycin, amphotericin; Invitrogen/Thermo Fisher) at 37°C under 5% CO₂ following established procedures. ¹⁵ Propagation was done in cell culture flasks with ZMAC cells and then stored in 20-mL aliquots at −80°C.

To perform studies 1 and 2, 70 PRRSV RNA-negative processing fluid samples were thawed (4°C for 14 h), aggregated, stirred, and re-tested by PRRSV reverse-transcription real-time PCR (RT-rtPCR) to re-confirm PRRSV RNA-negative status. To create PRRSV RNA-positive processing fluid samples, 10 mL of a ZMAC-propagated PRRSV clarified culture supernatant sample was thawed (4°C for 24 h), added to PRRSV-free processing fluid at a ratio of 1:10, and aliquoted (1 mL). For study 1, 70 sample aliquots were stored at −80°C; for study 2, 24 sample aliquots were used immediately.

Study 1. In study 1, we compared the effect of 4 PRRSV RNA extraction protocols on PRRSV RT-rtPCR cycle threshold (Ct) results: extraction I (RealPCR DNA/RNA magnetic bead kit; Idexx); extraction II (MagMAX viral RNA isolation kit; Applied Biosystems); extraction III (MagMAX total nucleic acid isolation kit; Applied Biosystems); and extraction IV (QIAamp RNA blood mini kit; Qiagen).

For extraction I, we used the RealPCR DNA/RNA magnetic bead kit and automated instrumentation (Kingfisher Flex magnetic particle processor; Thermo Fisher). In brief, a 200-µL sample and 200 µL of lysis solution were incubated for 15 min followed by a 5-min incubation with the bead solution (600 µL binding buffer + 20 µL magnetic beads). Magnetic beads were collected and washed with solutions I, II, and 80% ethanol (600 µL for 3 min each) followed by 10 min of drying after the final wash. Thereafter, nucleic acids were eluted from the magnetic beads (100 µL of elution buffer for 5 min). All of the above-mentioned procedures were performed at room temperature.

For extraction II, we used the MagMAX viral RNA isolation kit and automated instrumentation (Kingfisher Flex magnetic particle processor). In brief, a 100-µL sample, 240 µL of lysis/binding solution, and 20 µL of the magnetic bead solution were combined in extraction plates and mixed for 5 min. Magnetic beads were collected for 15 s (3 s × 5 times), washed 2 times with wash solution I (300 µL for 1 min each time), 2 times with wash solution II (450 µL for 15 s each time), and then dried for 1 min after the final wash. Thereafter, nucleic acids were eluted at 65°C from the magnetic beads (90 µL of elution buffer for 3 min).

For extraction III, we used the MagMAX total nucleic acid isolation kit. In brief, a 175-µL sample and 235 µL of lysis solution were combined with zirconia beads in bead tubes and agitated (Mini-Beadbeater; BioSpec) for 5 min. The beads were then pelleted by centrifugation (16,000 × g for 3 min) after which 115 µL of lysate was transferred to a 96-well plate containing 65 µL of 100% isopropanol and 20 µL of the magnetic bead solution (20 µL of bead mix) in each well. The plate was then placed on a purification instrument (Kingfisher Flex magnetic particle processor). Magnetic beads were collected for 15 s (3 s × 5 times) and then washed 2 times with wash solution I (300 µL for 1 min each time), 2 times with wash solution II (450 µL for 15 s each time), and dried for 1 min after the final wash. Thereafter, nucleic acids were eluted at 65°C from the magnetic beads (90 µL of elution buffer for 3 min).

For extraction IV, we used the QIAamp RNA blood mini kit. In brief, a 140-µL sample and 560 µL of lysis solution were pipetted into a 1.5-mL microcentrifuge tube, vortexed for 15 s, incubated at room temperature for 10 min, and then 560 µL of 100% ethanol was added and the sample vortexed for 15 s. Thereafter, 630 µL of the solution was transferred to a QIAamp mini column nested in a 2-mL collection tube and centrifuged (6,000 × g for 1 min). The QIAamp mini column was then placed into a new 2-mL collection tube and the previous tube containing the filtrate was discarded (this step was done twice). Buffer AW1 was added (500 µL) and then the QIAamp mini column was centrifuged (6,000 × g for 1 min); as described above, the QIAamp mini column was then placed into a new clean 2-mL collection tube and the previous tube containing the filtrate was discarded. Then 500 µL of buffer AW2 was added and the column was centrifuged (20,000 × g for 3 min). The QIAamp mini column was then placed in a new 2-mL collection tube and centrifuged (20,000 × g for 1 min) to eliminate possible buffer AW2 carryover. The QIAamp mini column was then placed in a clean 1.5-mL microcentrifuge tube, 60 µL of buffer AVE was added, and the sample was incubated at room temperature for 1 min. Finally, the sample was centrifuged (6,000 × g for 1 min) to elute viral RNA.

We assayed nucleic acid extracts using a commercial PRRSV RT-rtPCR kit (RealPCR PRRS type 1 and type 2 multiplex RNA mix and master mixes; Idexx). Plates containing purified RNA, multiplex RNA mix, and master mixes were loaded onto a thermocycler (7500 fast real-time PCR system; Applied Biosystems), and the following cycling conditions with standard ramp rate were used: 1 cycle at 50°C for 15 min, 1 cycle at 95°C for 1 min, 45 cycles at 95°C for 15 s, and 60°C for 30 s. Amplification data were analyzed using the “auto baseline” and “auto Cq” functions, with Ct values <37 considered PRRSV positive. Assay controls in each run included a negative extraction control, positive and negative amplification controls, and an internal sample control (ISC; i.e., endogenous, pig-specific genetic material to monitor sample quality, extraction, and amplification). ISC primers and probes targeting host nucleic acid in the sample produced an amplification curve that validated the reaction.

Regardless of the extraction method, all 24 PRRSV-inoculated processing fluid samples tested by RT-rtPCR in study 1 were positive and met quality control criteria. The Ct results were normally distributed based on quantile-quantile plot (Q-Q plot) analysis (v.9.4; SAS Institute). For samples processed with extraction I, mean Ct values (95% CIs) were calculated as 18.2 (17.6, 18.7), extraction II as 18.2 (17.7, 18.8), extraction III as 17.9 (17.4, 18.5), and extraction IV as 18.0 (17.4, 18.5). Using Ct values as the response variable and treatment as the independent variable, one-way ANOVA found no differences among extraction methods (p = 0.84).

Study 2. In study 2, we evaluated the stability of PRRSV RNA in processing fluid samples by time (0, 24, 48, 72, 120, 336 h) and temperature (−20°C, 4°C, 22°C, 34°C). Each temperature included 6 replicates, with time 0 serving as the baseline for comparison. Samples were tested using extraction I and the PRRSV RT-rtPCR kit used in study 1. Samples held at 4°C were highly stable through 336 h (Table 2). In contrast, samples held at 22°C or 34°C had higher Ct relative to baseline values (time 0) at both 24 and 48 h (Tukey test, p < 0.005).

Table 2.

Effect of temperature and time on processing fluid porcine reproductive and respiratory syndrome virus RT-rtPCR Ct values (95% CIs) based on 6 replicates per treatment.

Time, h	−20°C	4°C	22°C	34°C
0		18.6 (18.4, 18.9)	18.6 (18.4, 18.9)
24		18.9 (18.6, 19.1)	19.4 (19.1, 19.7)*	25.3 (25.0, 25.6)*
48		18.4 (18.1, 18.6)	20.0 (19.8, 20.3)*	27.4 (27.2, 27.7)*
72		18.9 (18.6, 19.1)
120	18.8 (18.5, 19.1)	18.8 (18.5, 19.0)
336	18.0 (17.8, 18.3)	18.9 (18.6, 19.2)

Blank cells indicate condition not assessed.

^*Different from time 0 (p < 0.005).

Study 3. In study 3, we modified the protocol of a commercial PRRSV serum IgG ELISA (PRRS X3 Ab test, Idexx; i.e., sample dilution, assay conditions, and conjugate) to detect PRRSV isotype–specific antibody (IgG, IgA, IgM) in processing fluid samples from herds of known PRRSV status (i.e., 224 samples from PRRSV-positive breed-to-wean herds, and 89 samples from PRRSV-free breed-to-wean herds). We tested processing fluid samples with the optimized ELISA at a 1:10 dilution (serum is tested at 1:40) using kit sample diluent (100 µL final volume). The ELISA was performed as directed by the manufacturer with the following exceptions: the kit IgG conjugate was replaced with goat anti-pig IgG (Fc; Bethyl) diluted 1:10,000 in Idexx conjugate diluent; or goat anti-pig IgA (Bethyl) diluted 1:2,000 in Idexx conjugate diluent; or goat anti-pig IgM (Bethyl) diluted at 1:5,000 in Idexx conjugate diluent. The original incubation time and temperatures from the serum kit were changed to 2 h at 37°C for sample incubation, 1 h at 37°C for conjugate (IgG, IgA, or IgM) incubation, and 5 min at room temperature (20–24°C) for substrate incubation. All samples were run on plates with 2 positive and 2 negative internal controls for IgG, IgA, or IgM.

For the analysis of the IgM, IgA, and IgG ELISA results, sample:positive (S:P) ratios were transformed (log_ex) to normalize the data and then Welch t-test for groups with unequal variance was used to compare responses in processing fluids from PRRSV-free versus PRRSV-infected herds (R program v.3.5.0, https://www.r-project.org/). IgG (p < 0.0001) and IgA (p < 0.0001) S:P responses differed between the 2 groups (Table 3), but not IgM (p = 0.267; Fig. 1). Thereafter, receiver operating characteristic curve analyses were performed on non-transformed IgA and IgG S:P data, with breeding herd status assumed as true disease status (v.20.022; MedCalc Software).

Table 3.

Diagnostic performance (95% CIs) of a commercial porcine reproductive and respiratory syndrome virus (PRRSV) serum IgG ELISA (PRRS X3 Ab test; Idexx) modified for the detection of IgG or IgA in processing fluids.

S:P cutoff	PRRSV IgG ELISA		PRRSV IgA ELISA
	Diagnostic sensitivity	Diagnostic specificity	Diagnostic sensitivity	Diagnostic specificity
0.1	100.0 (98.4, 100.0)	40.4 (30.2, 51.4)	99.6 (97.5, 100.0)	54.0 (43.0, 64.6)
0.2	100.0 (98.4, 100.0)	82.0 (72.5, 89.4)	97.3 (94.3, 99.0)	80.9 (71.2, 88.5)
0.3	100.0 (98.4, 100.0)	91.6 (83.8, 96.4)	87.5 (82.4, 91.5)	89.9 (81.7, 95.3)
0.4	100.0 (98.4, 100.0)	96.6 (90.5, 99.3)	72.8 (66.3, 78.4)	97.8 (92.1, 99.7)
0.5	99.9 (98.2, 100.0)	100.0 (95.9, 100.0)	56.3 (49.5, 62.8)	97.8 (92.1, 99.7)

Figure 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) IgG, IgA, and IgM sample:positive (S:P) ratio responses (y-axis) in processing fluids from known PRRSV-negative (Neg) and PRRSV-positive (Pos) herds (x-axis). We used a commercial PRRSV serum IgG ELISA (PRRS X3 Ab test; Idexx) adapted to detect specific antibody isotypes in processing fluids. *Significant difference in mean S:P response when comparing samples from PRRSV-negative versus -positive herds (Welch t-test, p < 0.05).

Our results corroborate the performance of current RNA extraction procedures for PRRSV RT-rtPCR and provide guidelines for handling processing fluids on the farm. Notably, PRRSV RNA in processing fluids was stable at 4°C for up to 14 d. Thus, processing fluids collected in the field should be maintained at ≤4°C until arrival at the testing laboratory. Prior research showed that PRRSV IgG antibody could be detected in processing fluids ⁶ ; study 3 expanded on this report by demonstrating that a modified PRRSV IgG ELISA provided excellent discrimination at a cutoff of S:P ≥0.5. Thus, PRRSV IgG testing of processing fluids could be a highly cost-effective approach for the surveillance of breeding herds thought to be negative or naïve for PRRSV.