GB used in this study as a feed supplement was provided by Seobong Biobestech (Korea). The supplement was composed of silicon dioxide (SiO₂, 61.90%), aluminum oxide (Al₂O₃, 23.19%), iron oxide (Fe₂O₃, 3.97%), sodium oxide (Na₂O, 3.36%), and 36 ppm germanium.

Specific pathogen-free female 6-week-old BALB/c mice (DBL, Korea) were used to investigate the effects of GB on host immune responses. The mice were randomly divided into three groups of eight mice each. The control group received a commercial, nutritionally complete, extruded dry rodent feed (Superfeed, Korea). The experiment groups received the same rodent feed supplemented with either 0.1% (0.1% GB-fed group) or 0.3% (w/w) GB (0.3% GB-fed group).

Conventional 6-week-old pigs (Korea) were used to evaluate GB-mediated clearance of PRRSV. Prior to purchase, sera from the littermates were tested and the animals were confirmed to be negative for antibodies against PRRSV using a commercial enzyme-linked immunosorbent assay kit (IDEXX Laboratories, USA). The pigs were randomly divided into two groups of six pigs each. The control group received a commercial and nutritionally complete swine feed (Daehan Livestock and Feed, Korea). The experiment group received the same swine feed supplemented with 0.3% (w/w) GB.

All animals were housed in an air-controlled separate room, and allowed free access to tap water and their particular diets. All animal procedures were approved (no. CNU IACUC-YB-2010-1) by the Institutional Animal Care and Use Committee of Chonnam National University (Korea).

The mice were fed the control or experimental diets for 2 weeks. The animals were then sacrificed and the spleen was collected. The spleens were aseptically removed with scissors and forceps, placed in cold phosphate-buffered saline (PBS), and single-cell suspensions were prepared by pushing the tissue through a 40-µm nylon mesh (BD Biosciences, USA). Erythrocytes in the cell mixture were destroyed by incubation with Tris-NH₄Cl. The lymphocytes were washed with PBS twice prior to resuspension in complete medium consisting of RPMI-1640 medium (Lonza, Switzerland) containing 10% (v/v) fetal bovine serum (FBS; Gibco, USA) and 2% (v/v) antibiotic-antimycotic solution (Lonza, Switzerland). Live cells were detected by staining with trypan blue.

Mouse lymphocyte proliferation was measured as previously described [8]. Cell suspensions were diluted to a final concentration of 3 × 10⁶ cells/mL in complete medium. One hundred µL of the cell suspension and 100 µL complete medium with or without 5 µg/mL concanavalin A (ConA; Sigma-Aldrich, USA) or 50 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich) were added into each well of a 96-well plate (SPL Life Sciences, Korea). The cultures were set up in duplicate. After a 48-h incubation at 37℃ in a 5% CO₂ incubator, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL; Sigma-Aldrich, USA) were added to each well and the plate was incubated for another 4 h. The plate was then centrifuged at 2,000 × g for 10 min and the supernatants were discarded. A total of 200 µL dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) was added to each well and the plate was shaken until the formazan crystals dissolved. The absorbance of each sample was read using a microplate spectrophotometer (Thermo Labsystems, Finland) at 540 nm.

Immunofluorescent staining was performed to measure the CD3+CD4+ T and CD3+CD8+ T cell populations in the spleen of each sacrificed mouse as previously described [7]. Lymphocytes were stained with both phycoerythrin (PE)-conjugated hamster anti-mouse CD3 (BD Biosciences, USA) and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4 (BD Biosciences, USA) antibodies, or FITC-conjugated rat anti-mouse CD8 antibody (BD Biosciences, USA). After incubating for 30 min at room temperature in the dark, the cells were washed twice with PBS and the T lymphocyte subpopulations were analyzed using a FACSort flow cytometer (BD Biosciences, USA). Viable lymphocytes were gated according to forward and side-scatter characteristics (FSC/SSC), and 10,000 events were analyzed to evaluate FITC- and PE-positive signals. Results for each T lymphocyte subpopulation are expressed as percentages of events in the FSC/SSC lymphocyte gate.

All pigs were fed the experimental or control diet for 2 weeks before being experimentally infected with the virus. PRRSV was obtained from the Animal, Plant and Fisheries Quarantine and Inspection Agency (Korea). For the inoculum, a virus stock was propagated in confluent monolayers of MARC-145 cells as previously described [13]. Five mL of the viral culture (1 × 10^6.1 50% tissue culture infective dose/mL) was administered intranasally through each nostril of the pigs. The rectal temperatures of pigs were measured at 0, 3, 6, 9, 12, 14, 21, 24, and 28 days post-infection (DPI). Three pigs from each group were randomly sacrificed at 12 and 28 DPI. Lung and lymphoid tissues (bronchial lymph node, tonsil, and thymus) were then collected.

Lung and lymphoid tissues were homogenized (10%, w/v) in minimum essential medium alpha modification (alpha-MEM; Welgene, Korea) containing 2% (v/v) antibiotic-antimycotic solution (Lonza, Switzerland). The tissues were homogenized using a Tissue Tearor homogenizer (Biospec Products, USA) and then centrifuged at 4,000 × g for 30 min at 4℃. The supernatant was passed through a nonpyrogenic syringe filter with 0.2-µm pores (Pall Corporation, USA). The resulting suspensions were used as inocula to determine the viral titer using a plaque assay [14]. Briefly, each suspension was serially diluted 10-fold in alpha-MEM (Welgene, Korea) containing 2% (v/v) antibiotic-antimycotic solution (Lonza, Switzerland). One mL of each dilution was used to inoculate confluent monolayers of MARC-145 cells prepared in a 6-well plate (SPL Life Sciences). The cells were incubated with the inoculum in 5% CO₂ at 37℃ for 1 h with manual rocking every 10 min. The inoculum was then replaced with an overlay medium consisting of alpha-MEM (Welgene, Korea) supplemented with 5% (v/v) FBS (Lonza, Switzerland), 2% (v/v) antibiotic-antimycotic solution (Lonza, Switzerland), and 1.6% (w/v) carboxymethylcellulose sodium salt (Sigma-Aldrich, USA). The cells were further incubated for up to 5 days in 5% CO₂ at 37℃ until plaques were clearly observed. After overnight fixation of the monolayers in 10% (v/v) neutral buffered formalin, the supernatant was removed and 1% (w/v) crystal violet (Sigma-Aldrich, USA) was added to each well. The plates were kept at room temperature for 30 min, washed with tap water until the excess crystal violet was removed, inverted over absorbent paper, and allowed to dry. The plaques were counted and viral titers are expressed as plaque forming unit (pfu)/gram tissue. Each sample was tested in duplicate.

Lung and lymphoid tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into sections (5-µm thick), and stained with hematoxylin and eosin (H&E) for histopathological examination. Microscopic lesions were evaluated using a previously described scoring system [18]. In sections of lung, the presence and severity of type 2 pneumocyte hypertrophy and hyperplasia, alveolar septal infiltration by inflammatory cells, peribronchial lymphoid hyperplasia, volume of alveolar exudate, and degree of inflammation in the lamina propria of the bronchi and bronchioles were scored on a scale ranging from 0~6 (0, normal; 1, mild and multifocal; 2, mild and diffuse; 3, moderate and multifocal; 4, moderate and diffuse; 5, severe and multifocal; or 6, severe and diffuse). Sections of lymphoid tissues were evaluated for lymphoid depletion using a scale ranging from 0~3 (0, normal; 1, mild lymphoid depletion with loss of overall cellularity; 2, moderate lymphoid depletion; and 3, severe lymphoid depletion with loss of lymphoid follicular structure) and the presence of inflammation with a scale ranging from 0~3 (0, normal; 1, mild histiocytic-to-granulomatous inflammation; 2, moderate histiocytic-to-granulomatous inflammation; and 3, severe histiocytic-to-granulomatous inflammation with replacement of follicles). Scores representing the microscopic lesion severity in sections of lymphoid tissues were calculated as the sum of the individual scores for both parameters. All histological evaluations were performed in a blinded manner.

All data are expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests was used to analyze results from the mouse model. Student's t-test was performed to analyze data from the swine experiments. A Mann-Whitney U test (nonparametric test) was also used to compare microscopic lesion scores (ordinal data) for tissues from the experimentally PRRSV-infected pigs. All statistical analyses were performed using SPSS version 17.0 software (SPSS, USA). P-values < 0.05 were considered statistically significant.

Lymphocytes were incubated with or without mitogens (ConA or LPS) and lymphocyte proliferation was measured as the OD₅₄₀ value using an MTT assay. Lymphocyte proliferation in response to ConA was significantly enhanced (p < 0.05) in the GB-fed groups (0.745 ± 0.084 and 0.762 ± 0.093 for the 0.1% and 0.3% GB-fed groups, respectively) compared to the control group (0.650 ± 0.044). However, proliferation of the unstimulated or LPS-treated lymphocytes did not vary among the groups (Fig. 1).

Percentages of CD3+CD8+ T lymphocytes in spleens from the GB-fed groups (12.82 ± 1.39% and 13.96 ± 1.60% for the 0.1% and 0.3% GB-fed groups, respectively) were significantly increased (p < 0.001) compared to those observed in the control group (9.63 ± 0.61%). However, no significant differences in the percentages of CD3+CD4+ T lymphocytes in the spleen were observed among the groups (Fig. 2).

Mean rectal temperature of the pigs gradually increased up to 14 DPI. The temperature subsequently declined until the end of the experiment. Rectal temperature of the GB-fed group was lower than that of the control group throughout the experimental period. In particular, a significant difference in rectal temperature (p < 0.05) was observed between the groups at 12 and 14 DPI (Fig. 3).

At 12 DPI, viral titer of the lungs was significantly decreased (p < 0.05) in the GB-fed group (3.19 ± 1.14 log pfu/g tissue) compared to the control group (5.73 ± 0.41 log pfu/g tissue). Moreover, viral titers of the lymphoid tissues including bronchial lymph node, tonsil, and thymus were also significantly decreased (p < 0.05) in the GB-fed group compared to the control group (Fig. 4). However, viral titers of lungs and lymphoid tissues from both groups were almost under the limit of detection (10 pfu/g tissue) at 28 DPI (data not shown).

At 12 DPI, the experimentally PRRSV-infected pigs developed interstitial pneumonia characterized by type 2 pneumocyte hypertrophy and hyperplasia along with alveolar wall thickening by macrophages and lymphocytes. These lesions were milder in the GB-fed group (Fig. 5B) compared to the control group (Fig. 5A). Additionally, lymphoid depletion was less severe in the GB-fed animals, and clearer separation between the cortex and medulla of the thymus was observed (Fig. 5D) compared to the control group (Fig. 5C). Scores for microscopic lesions in the collected tissue samples are shown in Fig. 5E. Scores for the lung microscopic lesions in the GB-fed pigs were significantly decreased compared to ones for the control group (p < 0.05). Moreover, scores for the lymphoid microscopic lesions in the GB-fed group were also lower compared to those for the control group although this difference was not significant. Lesions in the lungs and lymphoid tissues of both groups were almost fully healed at 28 DPI (data not shown).