Animals and experimental design
Sixteen 21 days-old Landrace x Large White hybrid female piglets with homogeneous weights and tested as PCV-2 PCR negative with low anti-PCV2 antibody titers were obtained from a commercial pig production farm. Animals were housed in class III biocontainment animal facilities (BSL-3) located at VISAVET Health Surveillance Centre (Madrid, Spain) for acclimatization one week before the experiment. All animals received continuous access to water, non-medicated pig feed and veterinary monitorization. Individuals were randomly assigned to either the immunized group (n=8), which received two oral doses of HIMB with an interval of three weeks (-42 and -21 days pre-infection), or the control group (n=8), which received PBS instead of immunostimulant. Twenty-one days after the second immunization dose (day 0), all animals from both groups were infected by intranasal inoculation with 105 TCID50 of PCV-2 per animal (Figure 1). All pigs were confirmed as PCV-2 PCR negative and PCV-2 ELISA negative before challenge. Animals were handled on 0, 7, 14 and 21 days post-infection (dpi) for blood sampling, rectal temperature and weight measurements.
All piglets were euthanized 21 dpi by captive bolt after sedation with xylazine (Xilagesic 2%, Laboratories Calier, Barcelona, Spain) and were subjected to necropsy to assess PCV-2 gross lesions in palatine tonsil, as well as mandibular, tracheobronchial and mediastinal lymph nodes (LNs), lung (cranial and caudal lobes) and kidney. Typical PCV-2 gross lesions such as lymphadenomegaly, interstitial pneumonia and nephritis were evaluated by experienced pathologists. Lesion severity was graded with a lesion score (0 = absent, 1 = mild, 2 = moderate or 3 = severe).
Samples from palatine tonsil, lung (cranial and caudal lobes), as well as mandibular, tracheobronchial and mediastinal LNs were fixed by immersion in 10% neutral buffered formalin during 24 hours at room temperature (RT), dehydrated in a graded series of ethanol, immersed in xylol, and embedded in paraffin wax using an automatic processor for further histopathology and immunohistochemical studies.
Heat-inactivated Mycobacterium bovis (HIMB) immunostimulant
The oral immunostimulant consisted of 2 mL of sterile PBS containing approximately 107 heat-inactivated colony forming units (CFU)/mL of a field isolate (Strain 1403; spoligotype SB0339) originally obtained from a naturally infected wild boar. HIMB was prepared at Neiker-Tecnicalia (Derio, Spain) following the protocol described by Garrido et al. (2011) [53], except for an extended inactivation step at 83 °C for 45 minutes (min). Bacterial concentration was determined prior to inactivation by measuring Turbidity in a VITEK® DensiCHEK® (BioMerieux) and by plating a serially diluted aliquot onto agar-solidified Middlebrook 7H9 with glycerol (0.2% v/v) and OADC (10% v/v) (Becton Dickinson, Franklin Lakes, NJ, USA).
Porcine circovirus 2
PCV-2 genotype b (Sp-10-7-54-13 strain at 14th passage) was used as the inoculum [54]. This strain was isolated from the lymphoid tissues of a field case of PCV-2-SD in 2006 in Spain. For this study, the PCV-2 strain was propagated in PCV-free PK15 cells in minimal essential medium (MEM) [55] to a titer of 105 TCID50/mL.
Humoral response
Blood was extracted through puncture of the sinus ophthalmicus at intervals of one week upon infection. Blood samples were centrifuged at 400 g for 10 min to extract sera which was further stored at – 20 °C.
Sera were tested in duplicate by means of Ingezim CIRCO IgG ELISA (R.11.PCV.K1; Eurofins Technologies, SA; Madrid, Spain) to detect antibodies against PCV-2 VP2 protein, following manufacturer’s instructions.
Serum samples were also analyzed by ELISA to detect antibodies against Mycobacterium tuberculosis complex using P22 as antigen in an indirect in-house ELISA previously described by Thomas et al. (2019) [56]. The estimated sensitivity and specificity of this ELISA in swine was 84.1% and 98.4%, respectively.
Detection of porcine circovirus 2 infection by real time quantitative PCR (qPCR)
DNA was extracted from 200 μL of serum by using the MagMAX™ Pathogen RNA/DNA Kit (Thermo Fischer Scientific Baltics. Vilnius, Lithuania) following the manufacturer’s instructions. The DNA obtained was suspended in 90 μL of elution solution. The commercial QPCR kit VetMAX™ Porcine PCV2 Quant Kit (Applied Biosystems, Lisseu, France) was used to detect and quantify PCV-2 DNA in serum samples. PCV-2 qPCR results were expressed as PCV-2 copies/mL of serum. A cutoff value of 104 PCV-2 DNA copies/mL was considered the quantification limit of the PCV-2 qPCR. Viremia represented the proportion of positive animals (quantifiable and non-quantifiable) per group, and viral load represented the mean of the quantifiable individuals per group.
Detection of porcine circovirus 2 infection by immunohistochemistry
Immunohistochemistry was performed to detect PCV-2 as previously described in Chianini et al. (2003) [57]. Briefly, tissue sections were deparaffinized with xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by incubation with hydrogen peroxide 3% in distilled water for 30 min. Afterwards, antigen retrieval was performed using 0.1% Protease XIV (Sigma, ref. P5147) in PBS for 8 min at 37ºC. A monoclonal PCV-2 antibody (Ingenasa, M.11.PCV.I36A9) able to recognize the Cap protein was used at 1/1000 dilution in 0.1 M Tris-buffered saline and incubated overnight at 4-8ºC. Dako EnVision System-HRP Labelled Polymer Anti-Mouse (Dako, ref. K4001) was used as secondary antibody and incubated during 60 min at RT. Sections were finally incubated in diaminobenzidine (DAB)–hydro- gen peroxide solution for 10 min, counterstained with Harris’ haematoxylin, dehydrated and coverslipped.
Sections from a previous case of PCV-2-SD diagnosed in Spain were used as positive control and tissues from pigs from an experimental farm with no history of PCV-2 infection as negative controls.
To evaluate the number of immunolabeled cells, the sections were randomly chosen and blinded to which group was being analyzed and counted by an experienced pathologist (J.S.). PCV-2 antigen detection by immunohistochemistry was graded by score as low (1), moderate (2) and intense (3) amount of antigen.
Histopathology
Sections of tissue samples embedded in paraffin wax were cut at 3 µm and stained with hematoxylin and eosin (HE) for further histological examinations. The presence of lymphocyte depletion, infiltrating histiocytic and multinucleated giant cells in LNs and tonsils, the lymph-epithelium necrosis in tonsils, as well as the interstitial pneumonia, type-II pneumocyte hyperplasia and alveolar edema in lungs were graded as absent or 0% (0), mild or 1% to 30% affected (1), moderate or 30% to 70% affected (2), and severe or 70% to 100% affected (3). The referred pathological findings were independently examined by two blinded and experienced observers (E.F-C. and M.A.R.).
α-Gal content in PCV-2
The PCV-2 infective inoculum and an uninfected PK15 cell culture were tested with in-house direct ELISA to detect α-Gal, following the protocol described by Villar et al. (2021) [58] with slight modifications. First, protein concentration in the inoculum and in the cells was determined with the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following manufacturer’s instructions. Thereafter, 96-well plates were coated with four serial dilutions of protein (from 0.1 to 0.00001 µg/µL per well) from the viral inoculum and the PK15 cells in duplicate, as well as five serial dilutions of α-Gal (from 2 x 10-7 to 1.25 x 10-8 µg/µL per well), in carbonate/bicarbonate buffer and incubated overnight at 4°C. Wells were subsequently washed with PBS solution containing 0.05% Tween-20 (PBST) (Tween 20; Sigma-Aldrich Quimica S.A., Madrid, Spain) three times and blocked with 5% skimmed milk powder solution in PBS (SM) for 1 h at RT. Then, wells were emptied and the anti-α-Gal epitope monoclonal antibody (M86; Enzo Life Sciences) was added at 1:100 dilution in PBS and incubated for 1 h at 37°C. After three washes with PBST, goat anti-mouse IgM (μ-chain specific) peroxidase-conjugated antibody (Sigma-Aldrich Quimica S.A.) was added at 1:2000 dilution in PBS. After three washes with PBST, 100 μL of 3,3´,5,5´- Tetramethylbenzidine (Promega, Madison, WI, USA) were incubated for 20 min in the dark at RT. The colorimetric reaction was stopped with H2SO4 3N and the optical density (OD) was measured at 450 nm with a Multiskan FC ELISA reader (Thermo Fisher Scientific). The average value of the blanks (uncoated wells; n = 2) was subtracted from all reads and the mean value of the replicates (wells coated with the same sample at the same protein dilution; n = 2) was calculated. α-Gal content (ng/μg) in each sample (PCV2 inoculum and uninfected PK15 cells) was calculated from an α-Gal standard curve.
Statistical analysis
Statistical analysis was performed using R version 4.0.3 (R Core Team, 2020) using packages lme4 [59] and ggplot2 [60]. All data were assessed to calculate mean ± standard deviation (SD) or error (SE) values. The variables viremia, viral burden, antibody titers, increments in weight gain and increments in body temperature were fitted with linear mixed-effects models including the categorical variables “group” and “day” as fixed factors with interaction, as well as “individual” as random factor [response variable ~ group * day + (1 | individual)]. Differences in macroscopic and microscopic lesion scores between groups were tested through Mann-Whitney U test. P values ≤ 0.05 were considered statistically significant with a confidence level (CL) of 95%.