Ethical Statement for experimental procedures. All animal procedures were conducted following the recommendations in the Guild for the Care and Use of Laboratory Animal of the National Research Council of Thailand according to protocols reviewed and approved by the Chulalongkorn University Animal Care and Use Committee under protocol number 2031015, animal use license number UI-00058-2558. All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org).
PRRSV vaccines, vaccination, and viruses. PRRSV vaccines used for vaccination were 2 PRRSV-2 MLVs including Ingelvac PRRS MLV (Boehringer Ingelheim, Rhein, Germany) and Prime Pac PRRS (MSD Animal Health, Boxmeer, The Netherlands), respectively. Prime Pac PRRS is available in 2 different preparations, intramuscular (IM) and intradermal (ID) vaccination. Dosage and administration routes were following the manufacturer’s instructions. In brief, a 2 ml and 1 ml dose of Ingelvac PRRS MLV (batch no2451218A) and Prime Pac PRRS (batch no. A065CE04) was used for IM vaccination, respectively. A 0.2 ml dose of Prime Pac PRRS (batch no. A065CE04) was used for ID vaccination. ID vaccination and injection were performed using IDAL 3G needle-free device (MSD Animal Health, Boxmeer, The Netherlands).
Homologous vaccine viruses and highly pathogenic (HP)-PRRSV-2 were used in the present study. Homologous vaccine viruses refer to vaccine strains that were used as recall antigens for in vitro stimulation assay in the measurement of IFN-γ-SC and IL-10 production, as previously described methods17. To challenge pigs, the FDT10US23 isolate was used as a virus inoculum at the fifth passage in MARC-145 cells. Thai PRRSV-2 (HP-PRRSV-2,) was used. The FDT10US23 isolate is a HP-PRRSV-2 variant genetically classified in sublineage 8.7/HP-PRRSV-2 based on international systematic classification according to the previously described method7. The ORF5 genome sequence is available in GenBank under accession number JN255836. This isolate was isolated from swine herds experiencing PRRS outbreaks in the western region of Thailand during 2010-201118. Pathogenesis and challenge studies of the challenged isolate were demonstrated according to previous studies18 − 22.
Experimental design. One hundred fifty-four, castrated-male, PRRSV-free pigs at three weeks of age were procured from a PRRSV-free herd. Upon arrival, sera were collected individually and assayed for the presence of viral RNA and PRRSV-specific antibodies using PCR and ELISA to confirm their negative status. In the present study, two separate experiments were conducted. In experiment A (Exp A), pigs were vaccinated once with PRRSV-2 MLV via intramuscular (IM) or intradermal (ID) routes. PRRSV-specific antibodies and cell-mediated were evaluated. The presence of the vaccine virus in tissues and organs and the shedding pattern to sentinel pigs were determined. In experiment B (Exp B), pigs were intranasally challenged with HP-PRRSV-2. At 7 days post-challenge (DPC), the highest level of viremia, challenged pigs were injected with Diluvac Forte, either IM or ID route, using a conventional needle or needle-free device, respectively. The same needle or needle-free device was used to inject the Diluvac Forte into sentinel pigs. Seroconversion of the sentinel pigs was determined. Pigs in each group were kept in separate rooms with separated air spaces and monitored daily for physical condition and clinical respiratory disease throughout the experiment.
Experiment A. One hundred and twelve pigs were randomly allocated based on weight stratification into 4 groups of 21 pigs each including IM/Ingelvac MLV (G1), IM/Prime Pac (G2), ID/Prime Pac (G3), and NoVac (G4) as showed in Table 1. The IM/Ingelvac MLV (G1) group was IM vaccinated once with a 2 ml dose of Ingelvac PRRS MLV (Boehringer Ingelheim, Rhein, Germany). The IM/Prime Pac (G2) and ID/Prime Pac (G3) groups were vaccinated once via IM and ID routes with a 1 ml and 0.2 ml dose of Prime Pac PRRS (MSD Animal Health, Boxmeer, The Netherlands), respectively. ID vaccination was performed using IDAL 3G needle-free vaccinator. The NoVac (G4) group was left non-vaccination and the remaining 38 pigs were served as age-matched sentinel pigs.
Table 1
Experimental design of experiment A (Exp A). Four treatment groups included 3 vaccinated groups and 1 non-vaccinated group, respectively. Routes of vaccine administration are included either intramuscular (IM) or intradermal (ID). ID vaccination was performed using IDAL 3G needle-free device.
Treatment groups
|
Number of pigs
|
Vaccination
|
Vaccines
|
Vaccine type
|
Dosage and route of administration
|
Manufacturers
|
IM/Inglevac MLV (G1)
|
21
|
Yes
|
Ingelvac PRRS MLV
|
PRRSV-2
|
2 ml, IM
|
Boehringer Ingelheim, Rhein, Germany
|
IM/Prime Pac (G2)
|
21
|
Yes
|
Prime Pac PRRS
|
PRRSV-2
|
1 ml, IM
|
MSD Animal Health, Boxmeer, The Netherlands
|
ID/Prime Pac (G3)
|
21
|
Yes
|
Prime Pac PRRS
|
PRRSV-2
|
0.2 ml, ID
|
MSD Animal Health, Boxmeer, The Netherlands
|
NoVac (G4)
|
21
|
No
|
-
|
-
|
-
|
-
|
Blood samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 days post-vaccination (DPV). Sera were assayed for PRRSV specific antibody and PRRSV RNA using ELISA and quantitative RT-qPCR (qPCR), respectively. Peripheral blood mononuclear cells (PBMC) were isolated and used for in vitro stimulation to measure IL-10 production using an ELISA kit and IFN-γ-SC using an ELISPOT assay. Three pigs from each group were necropsied on weekly basis starting from 7 to 42 DPV. Sera, nasal swabs, tonsils, lungs, bronchoalveolar lavages (BAL), and feces were collected and assayed for the presence of PRRSV using qPCR and virus isolation in cell culture. The shedding of vaccine virus to naïve animals was measured by placing age-matched sentinel pigs in contact with the vaccinated pigs at 1, 7, 14, 21, 28, 35, and 42 DPV. Sentinel pigs were comingled with vaccinated pigs for 3 consecutive weeks. Sera were collected at 0, and 21 days post commingle. Commingled pigs were weekly measured PRRSV specific antibody and PRRSV RNA using ELISA and qPCR, respectively.
Experiment B. Forty-two pigs were randomly allocated based on weight stratification into 5 groups with 3 pigs each including IM/High (T1), ID/High (T2), IM/Low (T3), ID/Low (T4), and NoChal (T5) as showed in Table 2. Two different dosages of HP-PRRSV-2 were used to inoculate pigs. The IM/High (T1) and ID/High (T2) groups were intranasally inoculated with 4 ml (2 ml/nostril) of HP-PRRSV-2 (FDT10US23 isolate, 106 TCID50/ml). The IM/Low (T3) and ID/Low (T4) groups were intranasally inoculated with 4 ml of HP-PRRSV-2 at a lower dose (FDT10US23 isolate, 103 TCID50/ml). The NoChal (T5) group was served as a control, and the remaining 17 pigs were used as age-matched sentinel pigs. Sera were collected at 0, 7, 14, 21, and 28 DPC and assayed for the presence of PRRSV-specific antibody and RNA using ELISA and qPCR, respectively.
Table 2
Experimental design of experiment B (Exp B). Five treatment groups included 4 challenged groups and 1 non-challenged group, respectively. Pigs were intranasally inoculated with HP-PRRSV-2 at 0 days post-challenge (DPC). Pigs in non-challenge (NoChal) served as the control. At 7 DPC, the challenged pigs were injected with Diluvac Forte via either IM or ID routes. ID injection was performed using IDAL 3G needle-free device.
Treatment groups
|
Number of pigs
|
Challenge
|
Detail
|
Dosage and challenge route
|
PRRSV isolate
|
Diluvac Forte injection
|
IM/High (T1)
|
3
|
Yes
|
High dose group for IM injection
|
4 ml/pig, at 106 TCID50/ml
|
HP-PRRSV-2 (FDT10US23)
|
1 ml/pig, IM
|
ID/High (T2)
|
3
|
Yes
|
High dose group for ID injection
|
4 ml/pig, at 106 TCID50/ml
|
HP-PRRSV-2 (FDT10US23)
|
0.2 ml/pig, ID
|
IM/Low (T3)
|
3
|
Yes
|
Low dose group for IM injection
|
4 ml/pig, at 103 TCID50/ml
|
HP-PRRSV-2 (FDT10US23)
|
1 ml/pig, IM
|
ID/Low (T4)
|
3
|
Yes
|
Low dose group for ID injection
|
4 ml/pig, at 103 TCID50/ml
|
HP-PRRSV-2 (FDT10US23)
|
0.2 ml/pig, ID
|
NoChal (T5)
|
3
|
No
|
Negative control
|
-
|
-
|
-
|
At 7 DPC, the IM/High (T1) and IM/Low (T3) groups were IM injected with 1 ml of Diluvac Forte (batch no G197A01, MDS Animal Health, Boxmeer, The Netherlands) using a conventional needle (G18, 1"). The ID/High (T2) and ID/Low (T4) groups were ID injected with 0.2 ml of Diluvac Forte using IDAL 3G needle-free vaccinator (MSD Animal Health, Boxmeer, The Netherlands). The same conventional needles or needle-free device were used to inject the same volume of Diluvac Forte (MSD Animal Health, Boxmeer, The Netherlands) to sentinel pigs with the same route of injection (1 injected pig to 2 sentinels). The NoChal group was left as a negative control. Blood samples were collected from sentinel pigs at 0, 7, 14, 21, and 28 days post-injection (DPI). Injected sentinel pigs were weekly measured PRRSV-specific antibody and PRRSV RNA using ELISA and qPCR, respectively.
Clinical evaluation. Clinical signs were monitored daily post-vaccination and post-challenge periods for two consecutive weeks by the same person at the same time. The severity of clinical respiratory disease for each pig was evaluated using a scoring system following stress induction as previously described criteria23: 0 = normal, 1 = mild dyspnea and/or tachypnea when stressed, 2 = mild dyspnea and/or tachypnea when at rest, 3 = moderate dyspnea and/or tachypnea when stressed, 4 = moderate dyspnea and/or tachypnea when at rest, 5 = severe dyspnea and/or tachypnea when stressed, and 6 = severe dyspnea and/or tachypnea when at rest.
Clinical sample collection. Blood was collected from pigs in serum separation tubes (Monovette, Sarstedt, Numbrecht, Germany) and centrifuged at 2,000⋅g for 10 min at 20 °C. Sera were stored in 1 ml aliquots at -80 °C until used. Nasal swabs, tonsils, and feces were collected using individually packaged sterile swabs which were placed in Dulbecco’s modified eagle’s medium (DMEM, Gibco, MA, USA) supplemented with 5⋅ antibiotics (100⋅ Antibiotic-antimycotics, Gibco, MA, USA). Tonsils and feces were weighed and mixed with DMEM medium (10% weight by volume). Then, samples were homogenized and centrifuged at 4,000⋅g for 10 min. Homogenates were filtered through 0.2 µm pore size filters, treated with 5⋅ antibiotics overnight at 4 °C, and kept at -80 °C until used. Urines were collected from bladders (10 ml/pig), filtered through 0.2 µm pore size filters, and kept at -80 °C until used. Bronchoalveolar lavage (BAL) was performed aseptically at necropsy as previously described24. In brief, 50 ml of lavage fluid consisting of DMEM with 5⋅ antibiotics was gently dispensed and aspirated several times into the lungs. The BAL was kept at -80°C until used.
Antibody detection. PRRSV-specific antibodies were measured using a commercial ELISA kit (IDEXX PRRS X3 Ab test, IDEXX Laboratories Inc., MA, USA). The assay was performed following the manufacturer’s recommendation. Sera were considered positive for PRRSV antibody if the S/P ratio was greater than 0.4.
Peripheral blood mononuclear cells (PBMC) isolation. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using gradient density centrifugation (Lymphosep, Biowest, MO, USA) as previously described25. Isolated PMBC were counted by an inverted microscope, and concentrations were accessed in cRPMI-1640 [RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 50 µg/ml of gentamycin]. The viability of isolated PBMC was determined by Trypan blue (Sigma-Aldrich, MO, USA) staining and more than 90% viability were used for in vitro stimulation for IL-10 production and enzyme-linked immunospot (ELISPOT) assay as described below.
Quantification of porcine IL-10. Following vaccination, porcine IL-10 concentration in the supernatant of stimulated PBMC was quantified using a porcine ELISA IL-10 kit (R&D Systems, MN, USA) under the manufacturer’s instructions. In brief, 2 x 106 PBMC were seeded into 96-well plates and cultured in vitro for 24 h with either homologous viruses at 0.01 multiplicity of infection (MOI) or phytohemagglutinin (PHA, 10 µg/ml, Sigma-Aldrich, MO, USA).
ELISPOT assay. The number of PRRSV-specific IFN-γ-SC were determined in PBMC using commercial ELISPOT IFN-γ kit (ELISpot porcine IFN-γ, R&D Systems, MN, USA), processed according to manufacturer’s instruction and previously described19. Briefly, 2 x 105 PBMC were seeded into 96-well plates and stimulated with homologous viruses at 0.01 MOI for 24 h. The linear response was tested between 0.01 to 0.1 MOI. Phytohemagglutinin (PHA, 10 µg/ml, Sigma-Aldrich, MO, USA) and cRPMI-1640 were used as positive and negative controls, respectively. The spots were counted, and the background values were subtracted from the respective count of the stimulated cells and the immune response was expressed as the number of IFN-γ-SC per 106 PBMC.
Pathological examination. Three pigs from each vaccinated group were necropsied at 7, 14, 21, 28, 35, and 42 DPV. lung lesions, either macroscopic or microscopic lung lesions associated with PRRSV-induced pneumonia were evaluated as previously described23. For macroscopic lung lesions, each lung lobe was assigned a number to reflex the approximate percentage of the volume of the entire lung and the percentage of the volume from each lobe added to the entire lung score (range from 0 to 100% of the affected lung). Sections were collected from all lung lobes as previously described23. Lung tissues were fixed in 10% neutral buffered formalin for 7 days and routinely processed and embedded in paraffin in an automated tissue processor. Sections were cut at 5 µm and stained with hematoxylin and eosin (H&E). For microscopic lung lesions, the lung sections were examined in a blinded manner and given an estimated score of the severity of interstitial pneumonia. In brief, 0 = normal; 1 = mild interstitial pneumonia; 2 = moderate multifocal interstitial pneumonia; 3 = moderate diffuse interstitial pneumonia, and 4 = severe diffuse interstitial pneumonia. The mean values of the microscopic lung lesion score of each group were calculated.
Virus isolation. Virus isolation was performed in MARC-145 and PAM as previously described26. In brief, 100 µl of the filtered clinical sample was incubated in 96-well plates of monolayers of MARC-145 and PAM for 60 min at 37 °C to facilitate adsorption, washed twice with DMEM supplemented with 3% FBS (Gibco, MA, USA). Then, the plates were incubated for 3 days at 37 °C in a humidified atmosphere containing 5% CO2. Media were removed and cells were fixed with a cold acetone-methanol solution for 10 min and then air-dried. The virus was detected in a monolayer by indirect microscopy using PRRSV-specific monoclonal antibody (mAb) SR-30 (RTI, South Dakota, USA).
Quantification of PRRSV RNA and RT-PCR. Total RNA was extracted from clinical samples using NucleoSpin Virus (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instruction. The RNA quality was measured using a NanoDrop spectrophotometer (Colibri spectrometer, Titertek Berthold, Pforzheim, Germany). Copy number of PRRSV RNA in serum was quantified using probed-based real-time PCR as previously described20. The reaction was carried out in QuantStudio 3 Real-time PCR machine (Thermo-Fisher Scientific, MA, USA). To detects the presence of virus in clinical samples, extracted RNA was converted into cDNA and used for PCR which was performed using GoTaq Green Master Mix (Promega, WI, USA). Primer specific for the ORF5 gene and detection conditions were followed as previously described17.
Statistical analysis. Analysis of variance (ANOVA) was performed to determine if there were significant differences among groups for each day separately. If the P-value for an ANOVA table was less than or equal to 0.05, the difference between treatment groups was evaluated using a multiple comparison test. All data were performed using IBM SPSS Statistic software version 22 (IBM Corporation, WA, USA)