HomeScienceUsing a concurrent challenge with porcine circovirus 2 and porcine reproductive and...

Using a concurrent challenge with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus to compare swine vaccination programs

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Experimental design

All animal procedures in the present study were conducted in accordance with the recommendation 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 (protocol number 2031015). 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).

Seventy-two, castrated male, PRRSV-free with 3-week-old pigs were purchased from PRRSV-free commercial herds. Upon arrival, pigs were acclimated for 3 days and tested for the presence of PRRSV and other pathogens with real-time PCR and ELISA kits. Pigs were randomly allocated into 8 treatment groups with 9 pigs each: IMPP0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, IDPP0/PCVMH0, IMTRF0, NV/CH, and NV/NC (Table 1).

Table 1 Experimental design in the present study.

Pigs in the IMPP0/PCVMH0 and IMPP0/PCVMH7 groups were intramuscularly (IM) vaccinated once with a 1 ml dose of Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, Boxmeer, The Netherlands) at 0 days post-vaccination (DPV), followed by a single IM vaccination once with a 2 ml dose of Porcilis PCV M Hyo (MSD Animal Health, Boxmeer, The Netherlands), either at 0 or at 7 DPV, respectively. In contrast, pigs in the IDPP0/PCVMH0 and IDPP0/PCVMH7 groups were intradermally (ID) vaccinated once with a 0.2 ml dose of Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, Boxmeer, The Netherlands) at 0 DPV, followed by concurrent ID vaccination once with a 0.2 ml dose of Porcilis PCV ID (MSD Animal Health, Boxmeer, The Netherlands) and a 0.2 ml dose of Porcilis M Hyo ID ONCE (MSD Animal Health, Boxmeer, The Netherlands), either at 0 or at 7 DPV, respectively. ID vaccination was performed using IDAL 3G needle-free device (MSD Animal Health, Boxmeer, The Netherlands).

Pigs in the IMING0/PCVMH7 group were IM vaccinated once with a 2 ml dose of Ingelvac PRRS MLV (PRRSV-2 MLV, Boehringer Ingelheim, Rhien, Germany) at 0 DPV. Subsequently, pigs in this group were IM vaccinated once with a combined dose of 1 ml Ingelvac CircoFLEX (Boehringer Ingelheim, Rhien, Germany) and 1 ml of Ingelvac MycoFLEX (Boehringer Ingelheim, Rhien, Germany) at 7 DPV. Pigs in the IMTRF0 group were IM vaccinated once with a 2 ml dose of combined products of Ingelvac PRRS MLV (PRRSV-2 MLV, Boehringer Ingelheim, Rhien, Germany), Ingelvac CircoFLEX (Boehringer Ingelheim, Rhien, Germany), and Ingelvac MycoFLEX (Boehringer Ingelheim, Rhien, Germany) at 0 DPV. Pigs in the NV/CH and NV/NC groups were left unvaccinated.

At 28 DPV (0 days post-challenge, DPC), pigs were intranasally inoculated with a 4 ml of mixed cell culture inoculum containing HP-PRRSV-2 (FDT10US23 isolate, fifth passage of MARC-145 cells at 105.6 TCID50/ml) and PCV2 (S1918STC isolate, third passage of PK-15 cells at 105.0 TCID50/ml), with 2 ml/nostril. Pigs in the NV/NC group were left as a non-vaccination, non-challenge control group. All pigs were housed in separated rooms with separated airflow spaces. Clinical signs and rectal temperatures were daily recorded throughout the experiment.

Blood samples were collected at 0, 7, 14, 21 and 28 DPV, 3, 5, 7, 10, 14, 21 and 28 DPC. Sera were separated and assayed for the presence of PRRSV- and PCV2-specific antibodies using ELISA kits. Genomic copy numbers of PRRSV and PCV2 were quantified using RT-qPCR. Peripheral blood mononuclear cells (PBMC) were isolated and used for in vitro stimulation to measure IL-10 secretion using an ELISA kit and virus-specific IFN-γ-secreting cells (IFN-γ-SC) using an ELISPOT assay. At 7 DPC, three pigs from each group were euthanized and necropsied. Pneumonic lung lesions were evaluated as previously described44. Tissues were collected, PRRSV- and PCV2-specific antigens were evaluated using immunohistochemistry (IHC).

Vaccines, vaccination, and viruses

Vaccines used for vaccination were two of each PRRSV, PCV2, and Mycoplasma (M.) hyopneumoniae vaccines. PRRSV vaccines used for vaccination were two PRRSV-2 MLVs including Prime Pac PRRS (MSD Animal Health, Boxmeer, The Netherlands) and Ingelvac PRRS MLV (Boehringer Ingelheim, Rhien, Germany). Prime Pac PRRS is available in two different preparations for intramuscular (IM) or intradermal (ID) vaccination. For PCV2 and M. hyo vaccination, vaccines were including Porcilis PCV ID (MSD Animal Health, Boxmeer, The Netherlands) and Ingelvac CircoFLEX (Boehringer Ingelheim, Rhien, Germany), Porcilis M Hyo ID ONCE (MSD Animal Health, Boxmeer, The Netherlands), Porcilis PCV M Hyo (MSD Animal Health, Boxmeer, The Netherlands) and Ingelvac MycoFLEX (Boehringer Ingelheim, Rhien, Germany), respectively. Dosage and administration routes were applied following the manufacturer’s instructions.

Briefly, a 2 ml dose of Ingelvac PRRS MLV (batch number 2451281A) and a 1 ml dose of Prime Pac PRRS (batch number A605CE04) was used for IM vaccination, respectively. A 0.2 ml dose of Prime Pac PRRS MLV (batch number A605CE04) was used for ID vaccination using IDAL 3G needle-free device. A 1 ml dose of Ingelvac CircoFLEX (batch number 3091225A), a 1 ml dose of Ingelvac MycoFLEX (batch number 2730552A), and a 2 ml dose of Porcilis PCV M Hyo (batch number A099A01) were used for IM vaccination. A 0.2 ml dose of Porcilis PCV ID vaccine (batch number A020A01), and Porcilis M Hyo ID ONCE vaccine (batch number A027B01) were used for ID vaccination. ID vaccination was performed using IDAL 3G needle-free device (MSD Animal Health, Boxmeer, The Netherlands).

The combined vaccine was prepared as follow: Ingelvac MycoFLEX (batch number 2730552A) and Ingelvac CircoFLEX (batch number 3091225A) were mixed, and the mixture was then used to rehydrate Ingelvac PRRS MLV (batch number 2451281A). This was done in place of the Ingelvac PRRS MLV accompanying vaccine diluent and against the manufacturer’s mixing condition. A 2 ml dose of combined vaccine was used for IM vaccination.

Homologous vaccine viruses, highly pathogenic (HP)-PRRSV-2 and PCV2 isolates 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 virus-specific IFN-γ-SC and IL-10 secretion were performed in previously described methods24,25. To challenge pigs, a mixed cell culture supernatant containing Thai HP-PRRSV-2 isolate FDT10US23 (fifth passage in MARC-145 cells) and PCV2 isolate S1918STC (third passage in PK-15 cells) was used as a virus inoculum. The isolate FDT10US23 is an HP-PRRSV-2 variant genetically classified in the sublineage 8.7/HP-PRRSV-2 based on international systematic classification, according to the previously described method45. The ORF5 genome sequence is available in GenBank under accession number JN255836. The isolates FDT10US23 and S1918STC were isolated from swine herds experiencing PRRS outbreaks in the western region of Thailand during 2010–201146,47. Pathogenesis and challenge studies of the challenged isolate were demonstrated according to previous studies25,30,46,48,49.

Clinical signs and rectal temperature recording

Clinical sings and rectal temperature were monitored daily post-vaccination (DPV) and post-challenge periods (DPC) following two consecutive weeks by the same personnel at the same time. The severity of respiratory disease was evaluated using a scoring system for each pig following stress induction as previously described44: 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.

Antibody detection

PRRSV- and PCV2-specific antibodies were measured using commercially available ELISA kits: IDEXX PRRS X3 Ab test (IDEXX Laboratories Inc., MA, USA) and BioCheck PCV2 ELISA (BioCheck BV, Reeuwijk, The Netherlands), and serum neutralization (SN) assay. The ELISA assays were performed following the manufacturer’s recommendations. Sera were considered positive for PRRSV if the S/P ratio was greater than 0.4 and positive for PCV2 if the titer was greater than 1070, respectively.

Isolation of porcine peripheral blood mononuclear cells (PBMC)

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using gradient density centrifugation (Lymphosep™, Biowest, MO, USA) as previously described39. Isolated PBMC were counted by an inverted microscope, and cell concentrations were accessed in cRPMI-1640 medium [RPMI-1640 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 was used for in vitro stimulation for IL-10 production and enzyme-linked immunospot (ELISPOT) assay as described below.

Porcine interleukin-10 (IL-10)

Porcine IL-10 concentration in the supernatant of stimulated PBMC was quantified using a porcine ELISA IL-10 kit (R&D System, MN, USA) under the manufacturer’s instructions and previously described methods43,48. In brief, total 2 × 106 PBMC were seed into 96-well plates and cultured in vitro for 24 h with either homologous vaccine viruses at 0.01 multiplicity of infection (MOI), phytohemagglutinin (PHA, 10 μg/ml, Sigma-Aldrich, MO, USA), or MARC-145 cell lysate (mock suspension). In each pig, the levels of porcine IL-10 secretion were calculated by subtracting the value of mock-stimulated well from the PRRSV-stimulated well. Subtracted values were compared between treatment groups.

ELISPOT assay

The number of virus-specific IFN-γ-SC were determined in stimulated PBMC using a commercial ELISPOT IFN-γ kit (ELISpot porcine IFN-γ, R&D System, MN, USA). The assay was performed in accordance with manufacturer’s instructions and a previously described method43,48. Briefly, 2 × 105 PMBC/well were seeded into 96-well plates and stimulated with either homologous vaccine viruses or heterologous viruses (FDT10US23 and S1918STC isolates) at 0.01 MOI for 24 h. Phytohemagglutinin (PHA) and cRPMI-1640 were used as positive and negative controls, respectively. The spots were counted by an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH, Strassberg, Germany). The background values were subtracted from the respective count of the stimulated cells and the immune response was expressed as the number of virus-specific IFN-γ-SC per 106 PBMC.

Quantification of PRRSV RNA

Total RNA was extracted from sera using NucleoSpin RNA Virus (Macherey–Nagel, Duren, Germany) according to the manufacturer’s instructions. The RNA quality was measured using a NanoDrop spectrophotometer (Colibri spectrophotometer, Titertek Berthold, Pforzheim, Germany). Copy number of PRRSV RNA in sera and nasal swabs was quantified using probed-based real-time PCR as previously described48. The reaction was carried out in QuantStudio 3 Real-time PCR machine (Thermo-Fisher Scientific, MA, USA).

Quantification of PCV2 DNA

Total DNA was extracted from sera using NucleoSpin Tissue (Macherry–Nagel, Duren, Germany) according to the manufacturer’s instructions. The DNA quality was measured using a NanoDrop spectrophotometer (Colibri spectrophotometer, Titertek Berthold, Pforzheim, Germany) and quantified using QuantStudio 3 Real-time PCR machine (Thermo-Fisher Scientific, MA, USA). The capsid gene of PCV2-specifc primers were used as follow: forward primer 5′-GTGCCCGCTGCCACATCGAG-3′; reverse primer 5′-CAAAAGTTCAGCCAGCCCGCGGA-3′. The reaction contained 0.1 μg of DNA, 0.2 μM of each primer, 2′ qPCRBIO SyGreen Mix (PCR Biosystems, London, UK), and deionized water to yield a 20 μl final volume. The thermal profile was 94 °C for 4 min, followed by 35 cycles of 94 °C for 45 s, 60 °C for 30 s, and fluorescence acquisition at 72 °C for 45 s. pGEM-T Easy Vector (Promega, WI, USA) containing an inserted capsid gene was used to construct plasmid standard. A standard curve was generated using serial diluted plasmid standards of 100–107 copies/μl. Copy number of PCV2 DNA was calculated using standard curve method.

Pathological examination

Pigs from each group were necropsied at 7 DPC. Macroscopic and microscopic lung lesions associated with PRRSV-induced pneumonia were evaluated as previously described44. For macroscopic lung lesions, each lung lobe was assigned a number to represent 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 affected lung). Additionally, the size of lymph nodes was scored ranging from 0 (normal) to 3 (four times the normal size) according to previously described method50. Sections were collected from all lung lobes and lymph nodes as previously described44,50 and fixed with 10% neutral buffered formalin for 7 days. Tissues were routinely processed and embedded in paraffin in an automated tissue processor. Sections were cut 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.

Immunohistochemistry (IHC)

Immunohistochemistry for PRRSV antigens in lung sections were performed using PRRSV-specific monoclonal antibody as previously described25. In brief, tissues were processed and placed on Superfrost Plus slides (Thermo Fisher Scientific, MA, USA). Sections were deparaffinized, rehydrated, and treated with proteinase K (Thermo Fisher Scientific, MA, USA). Endogenous alkaline phosphatase was quenched with 0.3% hydrogen peroxide. Slides were treated with BSA and incubated with monoclonal antibody overnight. After washing, PRRSV antigens were visualized by secondary antibody HRP-conjugated (Agilent, Santa Clara, CA, USA) and counterstained with Meyer’s hematoxylin. To obtain quantitative data, slides were analyzed with the NIH Image J 1.50i Program (https://rsb.info.nih.gov/ij). In each slide, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm2) was determined as previously described25,51,52. The mean values were calculated.

PCV2 antigens were analyzed by IHC using paraffin-embedded blocks of tissues including lungs, tonsils, tracheobronchial, and inguinal lymph nodes. In brief, tissues were processed and placed on Superfrost Plus slides (Thermo Fisher Scientific, MA, USA). Sections were deparaffinized, rehydrated, and treated with proteinase K (Thermo Fisher Scientific, MA, USA). Endogenous alkaline phosphatase was quenched with 0.3% hydrogen peroxide. Slides were treated with BSA and incubated with PCV-2 specific monoclonal antibody (PCV-2-A, RIT, SD, USA) overnight. After washing, PCV2 antigens were visualized by secondary antibody HRP-conjugated (Agilent, Santa Clara, CA, USA) and counterstained with Meyer’s hematoxylin.

The scoring of PCV2 antigen was performed according to previously described50. Assessment of staining for PCV2 antigen was done in a blinded fashion and scores ranged from 0 to 3 (0 = negative; 1 = less than 10% of the lymphoid follicles have cells with PCV2 antigen staining; 2 = 10–50% of the lymphoid follicles contain cells with PCV2 antigen staining; 3 = more than 50% of the lymphoid follicles contain cells with PCV2 antigen. Mean values were calculated.

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 analyzed using IBM SPSS Statistic for Windows version 22.0 (IBM, Armonk, NY, USA) (https://www.ibm.com/software/analytics/spss/register/).


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