Safety of PRRSV-2 MLV vaccines administrated via the intramuscular or intradermal route and evaluation of PRRSV transmission upon needle-free and needle delivery

Two distinct experiments (Exp) were conducted to evaluate the shedding and efficacy of 2 modified live porcine reproductive and respiratory syndrome virus (PRRSV) type 2 vaccines (MLV) when administered intramuscularly (IM) or intradermally (ID) (Exp A), and the potential of PRRSV transmission using a needle-free device (Exp B). One-hundred fifty-four, 3-week-old castrated-male, pigs were procured from a PRRSV-free herd. In Exp A, 112 pigs were randomly allocated into 4 groups of 21 pigs including IM/Ingelvac MLV (G1), IM/Prime Pac (G2), ID/Prime Pac (G3), and non-vaccination (G4). Twenty-eight remaining pigs were served as non-vaccination, age-matched sentinel pigs. G1 was IM vaccinated once with Ingelvac PRRS MLV (Ing) (Boehringer Ingelheim, Germany). G2 and G3 were IM and ID vaccinated once with a different MLV, Prime Pac PRRS (PP) (MSD Animal Health, The Netherlands), respectively. Following vaccination, an antibody response, IFN-γ-SC, and IL-10 secretion in supernatants of stimulated PBMC were monitored. Sera, tonsils, nasal swabs, bronchoalveolar lavage, urines, and feces were collected from 3 vaccinated pigs each week to 42 days post-vaccination (DPV) and assayed for the presence of PRRSV using virus isolation and qPCR. Age-matched sentinel pigs were used to evaluate the transmission of vaccine viruses and were introduced into vaccinated groups from 0 to 42 DPV. Seroconversion was monitored. In Exp B, 42 pigs were randomly allocated into 5 groups of 3 pigs each including IM/High (T1), ID/High (T2), IM/Low (T3), ID/Low (T4), and NoChal. Twenty-seven remaining pigs were left as non-challenge, age-matched sentinel pigs. The T1 and T2, and T3 and T4 groups were intranasally challenged at approximately 26 days of age with HP-PRRSV-2 at high (106) and low (103 TCID50/ml) doses, respectively. At 7 days post-challenge, at the time of the highest viremia levels of HP-PRRSV-2, T1 and T2, and T3 and T4 groups were IM and ID injected with Diluvac Forte using needles and a need-less device (IDAL 3G, MSD Animal Health, The Netherlands), respectively. Same needles or needle-less devices were used to inject the same volume of Diluvac Forte into sentinel pigs. Seroconversion of sentinels was evaluated. The results demonstrated that PP vaccinated groups (G2 and G3), regardless of the route of vaccination, had ELISA response significantly lower than G1 at 7 and 14 DPV. PP-vaccinated groups (G2 and G3) had significantly higher IFN-γ-SC and lower IL-10 secretion compared to the Ing-vaccinated group (G1). The two different MLV when administered intramuscularly demonstrated the difference in virus distribution and shedding patterns. PP-vaccinated pigs had significantly shortened viremia than the Ing-vaccinated pigs. However, ID-vaccinated pigs had lower virus distribution in organs and body fluids without virus shedding to sentinel pigs. In Exp B, regardless of the challenge dose, sentinel pigs intradermally injected with the same needle-less device used to inject challenged pigs displayed no seroconversion. In contrast, sentinel pigs intramuscularly injected with the same needle used to inject challenged pigs displayed seroconversion. The results demonstrated the transmission of PRRSV by using a needle, but not by using a needle-less device. In conclusion, our results demonstrated that ID vaccination might represent an alternative to improve vaccine efficacy and safety, and may be able to reduce the shedding of vaccine viruses and reduce the iatrogenic transfer of pathogens between animals with shared needles.

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 performed in previously described methods 37 . To challenge pigs, Thai PRRSV-2 (HP-PRRSV-2) isolate FDT10US23, 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 method 9 , was used as a virus inoculum at the fifth passage in MARC-145 cells. 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 2010-2011 13 . Pathogenesis and challenge studies of the challenged isolate were demonstrated according to previous studies 12,13,18,27,38 . 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 immunity 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), at the highest level of viremia, challenged pigs were injected with Diluvac Forte, either by the IM or ID route, using a conventional needle or needle-free device, respectively. The same needle or needle-free device was used to inject Diluvac Forte into sentinel pigs. Seroconversion of the sentinel pigs was determined. This was performed in order to investigate the virus transmission from infected to naïve pigs when using needle or needle-free device. 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. Twenty-eight remaining pigs were served as non-vaccination, age-matched sentinel pigs. 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.
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, 1 sentinel pig/group at each day. Each batch of sentinel pig was comingled with vaccinated pigs for 3 consecutive weeks. Sera were collected at 0, and 21 days post-commingling. Commingled pigs were weekly measured PRRSV specific antibody and PRRSV RNA using ELISA and qPCR, respectively. www.nature.com/scientificreports/ 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. Twenty-seven remaining pigs were left as non-challenge, age-matched sentinel pigs. 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, 10 6 TCID 50 /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, 10 3 TCID 50 /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. To mimic vaccine administration, Diluvac Forte, an adjuvant, was used instead of a vaccine. 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. The same device that was used to inject Diluvac Forte to one infected pig were used to administer Diluvac Forte into 2 sentinel pigs. 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 criteria 39 : 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 2000 × 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 4000 × 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 described 40 . 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 described 26 . 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- Table 2. Experimental design of experiment B (Exp B). Five treatment groups included 4 challenged groups and 1 non-challenged group. 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 with the same needle or needle-less device used for inoculating pigs. ID injection was performed using an IDAL 3G needle-free device. www.nature.com/scientificreports/ 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. 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 × 10 6 PBMC were seeded 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 the mock-stimulated well from the PRRSV-stimulated well. Subtracted values were compared between treatment groups.
ELISPOT assay. The number of PRRSV-specific IFN-γ-SC were determined in stimulated PBMC using a commercial ELISPOT IFN-γ kit (ELISpot porcine IFN-γ, R&D Systems, MN, USA). The assay was performed in accordance with manufacturer's instruction and a previously described method 18 . Briefly, 2 × 10 5 PBMC/well were seeded into 96-well plates and stimulated with homologous vaccine viruses at 0.01 MOI for 24 h. Phytohemagglutinin (PHA, 10 μg/ml, Sigma-Aldrich, MO, USA) 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, Germany), 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 10 6 PBMC.
Pathological examination. Three pigs from each vaccinated group were necropsied at 7, 14, 21, 28, 35, and 42 DPV. Macroscopic or microscopic lung lesions associated with PRRSV-induced pneumonia were evaluated as previously described 39 . 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 the affected lung). Sections were collected from all lung lobes as previously described 39 . 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 cells and PAM as previously described 41 . In brief, 100 μl of the filtered clinical sample was incubated in 96-well plates of monolayers of MARC-145 cells 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% CO 2 . 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 described 27 . 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 described 37 .
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/ softw are/ analy tics/ spss/ regis ter/). Viremia. In all vaccinated groups, PRRSV RNA was detected and reached peaks at 7 DPV and gradually decreased to undetectable levels from 14 to 28 DPV (Fig. 1B). All vaccinated groups had no detectable levels of PRRSV RNA from 28 to 63 DPV. At 7 DPV, the IM/Ingelvac MLV (G1) group a had significantly higher (P < 0.05) PRRSV RNA level (246 ± 40.30 copies/ml) than that of the IM/Prime Pac (G2) and ID/Prime Pac (G3) groups. There was no difference between Prime Pac vaccinated groups, regardless to the vaccination routes. At 14 DPV, the ID/Prime Pac (G3) group had the lowest PRRSV RNA level (29.32 ± 12.13 copies/ml) compared to the other two groups and the difference was statistically significance. At 14 DPV, RNA levels in IM/Ingelvac MLV (G1) and IM/Prime Pac (G2) groups were not different.

BAL
Viremia. Following the challenge, PRRSV RNA in challenged groups with high doses rapidly increased and reached peaks at 7 DPC then gradually decreased to negativity from 14 to 28 DPC (Fig. 6B). In contrast, PRRSV RNA in challenged groups with low doses had continually increased and reached peaks at 14 DPC then rapidly decreased to negativity at 28 DPC. The IM/High (T1) and ID/High (T2) groups had significantly higher (P < 0.05) PRRSV RNA (range from 209.51 ± 37.7 to 237.60 ± 47.17 copies/ml) than that of the IM/Low (T3) and ID/Low (T4) groups at 7 DPC (range from 115.12 ± 20.22 to 120.53 ± 45.28 copies/ml). Meanwhile, PRRSV RNA in the IM/Low (T3) and ID/Low (T4) groups were significantly (P < 0.05) higher (range from 32.21 ± 4.21 to 194.43 ± 40.74 copies/ml) than that of the IM/High (T1) and ID/High (T2) groups (range from 19.09 ± 3.03 to 97.00 ± 19.14 copies/ml) at 14 DPC (Fig. 6B).
Seroconversion of sentinel pigs. None of the sentinel pigs injected with Diluvac Forte via ID route was seropositive (Table 4). In contrast, sentinel pigs injected via IM route had seroconversion from 14 to 28 DPI, regardless of doses of PRRSV challenge. At 14 DPI, 4 and 2 of 6 sentinel pigs of the IM/High (T1) and IM/Low (T3) groups were seropositive, respectively. All 6 sentinel pigs both IM/High (T1) and IM/Low (T3) groups were seropositive at 21 and 28 DPI.

Discussion
A conventional needle is a routine procedure for vaccine administration in swine industry in Southeast Asian countries. Many problems including broken needles in swine meat, muscle damage, abscesses, and infection and transmission by other pathogens, PRRSV, for instance, were found. In addition, a recent outbreak of highly infectious swine pathogen, African Swine Fever virus (ASFV) in Asia has raised a concern of transmission over a conventional needle. The use of a shared needle under this circumstance could be the risk of virus transmission not only PRRSV but also ASFV. The reduction of careless needle stick injuries to improve carcass quality and prevention of virus transmission are highly concern. In the present study, we would like to emphasize the safety and pathogen transmission during vaccine administration of intradermal vaccination using a needle-free device. PRRSV infection was used as a model. Therefore, the two studies were conducted to investigate the safety of vaccination with two PRRSV-2 MLV, either by IM or ID routes, in terms of the immune response, vaccine virus distribution in tissues and organs, and shedding patterns to sentinel pigs. The transmission of virus through a needless device and conventional needle were additionally investigated. Following vaccination, it was demonstrated that different PRRSV-2 MLV had different outcomes of the induction of an immune response and safety. It was notable that there were differences in early antibody detection between two PRRSV-2 MLV. ID Prime Pac PRRS vaccinated pigs showed delayed antibody response compared to that of Ingelvac PRRS MLV vaccinated pigs and IM Prime Pac vaccinated pigs. However, an ELISA response has long been recognized for its un-relationship with protection, unlike a response measured by viral neutralization assay. However, major findings in the present study are the induction of IFN-γ-SC and IL-10. The IFN-γ-SC in the ID Prime Pac vaccinated group had significantly higher frequencies than the other two IM vaccinated groups following vaccination. Increased IL-10 production was observed in all vaccinated groups following vaccination, and the ID vaccinated group had significantly lowest IL-10 levels compared to that of both IM vaccinated groups. For the evaluation of the safety following vaccination, our findings showed that Prime Pac vaccinated pigs had lower levels of viremia, decreased the frequency of PRRSV distribution in organs, and shortened virus shedding compared to that of the Ingelvac PRRS MLV vaccinated pigs. Interestingly, an ID vaccination was best able to shorten the viremic phase, reduced the frequency of PRRSV distribution in organs, and shorten virus shedding compared to that of the intramuscular route. Our findings demonstrate that PRRSV distribution was found in intramuscular vaccinated groups but not in the intradermal vaccinated group. In addition, the use of conventional needles led to biological and mechanical transmissions of PRRSV between vaccinated or infected pigs, and naïve pigs but the transmission was not observed in the use of a needle-free device. ID vaccination might represent an alternative www.nature.com/scientificreports/ to improve vaccine efficacy and safety, and may be able to reduce the shedding of vaccine viruses and reduce the iatrogenic transfer of pathogens between animals with shared needles. For the humoral immune response, the results of the present study demonstrated that the induction of the humoral immune response of PRRSV MLV was different, regardless of vaccination routes. In agreement with our previous study, antibody response against PRRSV following vaccination with PP was delayed and reached values above the cut-off levels at 21 DPV 37 . The differences in the induction of PRRSV specific antibody response as measured by ELISA between IM and ID vaccinated pigs are in accordance with findings from previous studies 26,27 . The mechanisms responsible for the induction of humoral immune response were not fully understood but the specific virus isolate used for the vaccines might play an important role.
Regarding cell-mediated immune response, delivery of antigen through the intradermal route could induce T cell polarization via the Th1 pathway, favoring the induction of IFN-γ which was observed in the present study. The ID-vaccinated pigs showed a significantly higher IFN-γ-SC response than that of the IM-vaccinated pigs. These findings are in agreement with previous reports in which the ID-vaccinated pigs induce relatively more IFN-γ-SC than that of the IM-vaccinated pigs [25][26][27] . One possible factor associated with this finding is the presence Another possibility of higher IFN-γ-SC in the ID-vaccinated group could be due to the lower IL-10 production. Our results showed that the IL-10 concentration was lower in ID-vaccinated pigs than in the IM-vaccinated pigs following vaccination. These findings are in agreement with previous reports in which both ID-and IMvaccination induce IL-10 production, but the ID-vaccinated pigs induced significantly lower IL-10 levels than that of the IM-vaccinated pigs 26,27 . Nevertheless, the delivery of antigen through the intradermal route could target dendritic cells. IL-10 is a cytokine of the Th2 response. The delivery through this route could induce T cell polarization through the Th1 pathway, favoring other cytokines that act against Th2 cells 43 . The differences in IL-10 production among the PRRSV MLV vaccination group are not surprising. Previous reports demonstrated that PRRSV isolates vary in the degree of IL-10 production both in vivo and in vitro 44,45 . Additionally, the induction of IL-10 might depend on the virus isolate used in the experiment and the vaccine [46][47][48] . However, the mechanism of IL-10 induction following vaccination by IM and ID routes are not understood.
The results of the present study demonstrated that some differences can be established in the safety of the 2 PRRSV MLV compared, although not all parameters were evaluated. Consequently, no differences in the induction of clinical signs were observed among vaccinated groups. Also, no clinical signs were recorded for any pig throughout the experiment. Although the macroscopic lung lesions can be considered similar between vaccinated groups, the microscopic lung lesions increased over time until 28 DPV and resolved at 35 DPV. This finding is in accordance with previous reports in which indicate that lung lesions generated by PRRSV-2 MLV isolates tend to be more persistent and last longer than those produced by PRRSV-1 MLV isolates 20,49 . Therefore, it is assuring that the patterns of lung lesions observed in the present study are more relevant to the virus isolate used for the vaccine than the changes occurring through the attenuation process 20 . Nonetheless, since the pathophysiological characteristics of virus isolates used for the vaccine could not be determined in the present study, the impact of the attenuation process in the occurrence and progression of lung lesions cannot completely ruled out.
Our results showed that the microscopic lung lesions of pigs vaccinated with Ingelvac PRRS MLV were significantly higher compared to PP vaccinated pigs on 21 DPV. These findings might be due to the difference in the intrinsic characteristics of the parent strains and, in particular, in their pneumotropism and pathological effects in the host might be related to these differences, that have been previously described 39,50 . Contrarily, the attenuation process might have altered the tropism of the vaccine viruses and restricted their ability to replicate in the lungs, and cause lung pathology. Remarkably, the frequency of virus isolation from the lung obtained in the present study was relatively lower than that of the other studies carried out with wide-type viruses 49,51 , indicating that the attenuation process of the vaccine virus might affect the cell tropism in the lung.
In addition to their ability to induce an immune response, the shedding patterns of PRRSV-2 MLV were evaluated using three different measurements, including the duration of viremia, the distribution of vaccine virus in organs, and the infection in sentinel pigs. Following vaccination, differences in the magnitude of viremia and the percentages of PRRSV-positive samples between PRRSV-2 MLV vaccinated groups were observed. Although the dynamics of viremia were similar among vaccinated groups, the magnitude of viremia of pigs vaccinated with Ingelvac PRRS MLV was statistically higher than those of the PP vaccinated pigs. In the same way, the percentage of PRRSV-positive tissue samples was higher in pigs vaccinated with Ingelvac PRRS MLV than in pigs vaccinated with the PP. These findings suggest that the viremic phase and the vaccine virus distribution in tissues were associated with the virus isolates use as vaccines 37 . However, the virus distribution in organs and the shedding patterns of the vaccine virus over the long period of time are limited, the shedding of vaccine virus could be occurred beyond our desired time point. Notably, the ID-vaccinated pigs show no PRRSV-positive samples regardless of the detection analyses, although the magnitude of viremia of the ID-vaccinated pigs was similar to the IM-vaccinated pigs. The discrepancy between viremia and virus isolation could be due to the fact that the ability to replicate in the target cells of the vaccine virus is restricted or that the quantity of virus in serum was lower than the limit of detection of the qPCR. To postulate, additional studies are needed.
An HP-PRRSV-2 isolate was used as a challenge virus because it has been a predominant virus in Asian countries 12 , and its virulence was well recognized. The results generated herein could be valuable information to www.nature.com/scientificreports/ swine farms in this region. The HP-PRRSV-2 strain used for the challenge in the present study was grouped in the sublineage 8.7/HP-PRRSV-2. Although the HP-PRRSV isolate has been well studied, in terms of pathogenesis and virulence, virus shedding and transmission into other pigs or environment have not been characterized yet. The transmission of virus to other pigs infected with genetically distinct PRRSV isolates could potentially results in recombination. The recombination between field PRRSV strains could resulted in the greater PRRSV pathogenicity. The recombination between HP-PRRSV-2 and the NADC30 isolate that has been confirmed as highly pathogenic 52 or an HP-PRRSV-2 vaccine-like strain circulating in the field followed by PRRSV vaccination has been reported 53 . According to recent findings 22 , recombination events may occur between MLV and field strains of the virus. The reduction of MLV shedding post-vaccination may be able to reduce the potential of this recombination event occurring. Several other studies have also documented known vaccine strains recombining with field strains to form more pathogenic strains 54 . PRRSV is known to have a high degree of survival in the environment 55 . Working on the assumption that survival is similar to that of wild-type isolates, vaccine viruses shed into the environment may become longer, be re-ingested by swine and play a part in new recombination isolates arising on the farm. However, we acknowledge that type of PRRSV MLV may have been attenuated through cell culture passage and hence have a decreased level of survival in the environment. Additional studies to investigate the survival of the vaccine virus in the environment and the duration of vaccine virus shedding are needed. Besides, our results suggest that the lower the vaccine virus shedding of the PRRSV MLV vaccine, the lesser the recombination events between the vaccine strain and wild-type isolates.
Regarding the immunological and virological outcomes of the HP-PRRSV-2 challenge, it was evident that the high dose challenged pigs developed rapidly viremia and a PRRSV-specific antibody response compared to the low dose challenged pigs. These finding demonstrates that the onset of the immune response and viremia was associated with challenge dose 56 . Supporting the case for intradermal needle-free vaccination, the Exp B illustrates the cessation of iatrogenic pathogen transfer between swine using a jet injector needle-free device (IDAL 3G), using PRRSV as a model in swine vaccinated with shared needles. Jet injection devices have been trialed in human medicine and utilize a high-pressure fluid jet, propelled by a spring or gases, to breach the skin, before introducing the vaccine antigen through a low-pressure jet 57 . Theoretically, there is no possibility of pathogens being transferred from animal to animal using this device. However, it has been documented in the past that certain devices can transfer pathogens due to liquid splashing back into the device as the low-pressure stream ceases 58,59 . It is likely that differences exist across devices due to different design mechanics and that not all devices are hence equal. In conventional swine farming, needles are often shared between pigs that are being vaccinated. Needles shared in such a manner have been documented in human medicine to be capable of transmitting pathogens such as HIV 60 , and it is likely that for swine it is no different. Unfortunately, this is common practice on commercial swine operations due to the need for worker speed and efficiency. It is important to note that this study only assessed the potential for the vaccination 'jet' to transmit diseases and that great care was taken not to contaminate the device exterior between animals with fecal matter or other secretions. Given the highly transmissible nature of PRRSV in vivo 34 , it could be imagined that in a conventional situation, extraneous contamination of the devices may cause transmission of PRRSV via other routes, such as the oral-fecal route.

Conclusions
The present studies illustrated that different PRRSV-2 MLV had similar patterns of the induction of antibody response, but the differences were observed in the early phase following vaccination. The intradermal vaccination might represent an alternative to improve vaccine safety, as it induced lower IL-10 levels and more IFN-γ-SC as well as the reduction of virus shedding within the herd and reduce the iatrogenic transfer of pathogens between animals with shared needles.