Immunostimulatory effects of the anionic alkali mineral complex Barodon on equine lymphocytes

Abstract

Previous studies have shown that the anionic alkali mineral complex BARODON has an immunoenhancing effect on pigs as an adjuvant and as a nonspecific immunostimulant. Likewise, the equine immune system has been defined with various monoclonal antibodies specific to equine leukocyte differentiation antigens to determine the possibility of enhancing equine resistance to respiratory diseases and promoting other immunostimulatory effects with the application of BARODON. Compared with the control group, after 3 weeks of treatment, BARODON-treated groups showed higher proportions of cells (P < 0.05) expressing major histocompatibility complex class II and CD2, CD4(+), CD4(+) CD25(+), CD8(+), and CD8(+) CD25(+) T lymphocytes, dendritic cells, and surface immunoglobulin M(+) B lymphocytes in peripheral blood, as well as enhanced cell proliferative responses with phytohemagglutinin and increased phagocytic activity against Streptococcus equi and Staphylococcus aureus strains with high antibiotic resistance, the bacteria frequently identified as etiologic agents of equine respiratory diseases at the Seoul Race Park in Seoul, Korea. This study shows that BARODON may act as an immunostimulator and can be an effective alternative to antimicrobial feed additives for nonspecific improvements in equine immune responses, particularly against respiratory diseases.

FIG. 1.

PCR products from Streptococcus equi subsp. equi and Staphylococcus aureus bacteria isolated from nasal swab specimens of horses with IURD, the phagocytic strains used in this study, amplified with corresponding species-specific primer pairs. Lanes: M, 100-bp DNA ladder (Takara Bio Inc., Otsu, Shiga, Japan); 1, S. equi subsp. equi isolate using sodA primer sets (230 bp of PCR product) and seeI primer sets (520 bp of PCR product); 2, S. equi subsp. equi ATCC 33398 using sodA primer sets and seeI primer sets; 3, S. equi subsp. zooepidemicus ATCC 43079 using sodA primer sets and seeI primer sets (notice the negative reactions of S. equi subsp. zooepidemicus with seeI primer sets, which are specifically reactive to S. equi subsp. equi); 4 and 7, negative control (distilled water); 5, S. aureus isolate using nuc primer sets and Sa-442 primer sets; 6, S. aureus ATCC 25923 using nuc primer sets and Sa-442 primer sets.

FIG. 2.

Representative dot plot profiles of PBMC from a control horse (A to C) and a BARODON-treated horse (D to F) labeled with two MAbs (Table 1) for equine CD2 (EqCD2) and EqCD5 (B and E) or for granulocytes and monocytes (G/M) and MHC-II (C and F) before (time zero, T0) 3 days of culture in RPMI alone or with PHA. In profiles A and D, the quadrants show the division between small lymphocytes in gate 1 (R1), large lymphocytes and monocytes in gate 2 (R2), and granulocytes in gate 3 (R3), as well as the relative frequencies of cells in each gate at T0. Profiles B and E, with gates placed only on 1, 2, and 3, show the frequency of EqCD2⁻ EqCD5⁻ cells (lower left quadrant), EqCD2⁺ EqCD5⁻ cells (upper left quadrant), EqCD2⁺ EqCD5⁺ cells (upper right quadrant), and EqCD2⁻ EqCD5⁺ cells (lower right quadrant). Profiles C and F, with gates on 1, 2, and 3, show the frequency of G/M⁻ MHC-II⁻ cells (lower left quadrant), G/M⁺ MHC-II⁻ cells (upper left quadrant), G/M⁺ MHC-II⁺ cells (upper right quadrant), and G/M⁻ MHC class II⁺ cells (lower right quadrant).

FIG. 3.

Summary of FC analysis of R1 plus R2 (lymphocytes) (A), R2 (blasting and proliferating cells) (B), R3 (granulocytes) (C), CD2⁺ cells (all thymocytes, T lymphocytes, and NK cells) (D), MHC-I⁺ cells (E), MHC-II⁺ cells (F), dendritic cells (G), and sIgM⁺ B lymphocytes (H) in peripheral blood with gates placed only on 1, 2, and 3 at time zero. Horses were fed daily both with 6 to 7 kg of Omolene feed, which had already been mixed with 0.05% Barodon F. Gold, and with an additional 60 ml of Barodon-biogenic feed containing 10% Barodon F. Gold (Tx-1; n = 6). Another group was fed only Omolene feed (Tx-2; n = 6), while a third group was fed only Barodon-biogenic feed (Tx-3; n = 6). The fourth group, an untreated control group, was fed general feed with no BARODON (Control; n = 6). Significant differences between control animals and animals treated with BARODON are as indicated in the figure (a, P < 0.01; b, 0.01 < P < 0.05). See Materials and Methods for details on the analysis of subsets by selective gating used to show the frequency of each cell population with gates placed on 1, 2, and 3.

FIG. 4.

Representative dot plot profiles of PBMC at time zero with gates placed only on 1 and 2 from a control horse (A, B, E, and F) and a BARODON-treated horse (C, D, G, and H) labeled with two MAbs (Table 1) for equine CD4 (EqCD4) or EqCD8 and EqCD25 (IL-2Rα) at time zero (A to D) and after 3 days (3D) culture with PHA (E to H). The profiles A, C, E, and G show the frequency of EqCD4⁻ EqCD25⁻ cells (lower left quadrant), EqCD4⁺ EqCD25⁻ cells (upper left quadrant), EqCD4⁺ EqCD25⁺ cells (upper right quadrant), and EqCD4⁻ EqCD25⁺ cells (lower right quadrant). In profiles B, D, F, and H, the quadrants show the frequency of EqCD8⁻ EqCD25⁻ cells (lower left quadrant), EqCD8⁺ EqCD25⁻ cells (upper left quadrant), EqCD8⁺ EqCD25⁺ cells (upper right quadrant), and EqCD8⁻ EqCD25⁺ cells (lower right quadrant).

FIG. 5.

Summary of FC analysis of CD4⁺ (A), CD4⁺ CD25⁺ (B), CD8⁺ (C), and CD8⁺ CD25⁺ (D) T lymphocytes and the CD4/CD8 ratio (E) from the proportions of CD4⁺ and CD8⁺ T lymphocytes in peripheral blood with gates placed only on 1 and 2 at time zero. Horses in one group were fed Omolene feed as well as Barodon-biogenic feed (Tx-1; n = 6), group two was fed only Omolene feed (Tx-2; n = 6), group three was fed only Barodon-biogenic feed (Tx-3; n = 6), and the untreated control group was fed general feed with no BARODON (Control; n = 6). Significant differences between control animals and animals treated with BARODON are as indicated in the figure (a, P < 0.01; b, 0.01 < P < 0.05). Detailed information on how much BARODON was included in each feed and how subsets were analyzed by selective gating, which was used to show the frequency of each cell population, is provided in Materials and Methods.

FIG. 6.

Summary of FC analysis of CD4⁺ (A), CD4⁺ CD25⁺ (B), CD8⁺ (C), and CD8⁺ CD25⁺ (D) T lymphocytes in peripheral blood with gates placed only on 1 and 2 and blast, proliferating lymphocytes (E) in peripheral blood with a gate placed only on 2 after 3 days of culture in RPMI alone or with PHA. Horses in one group were fed both Omolene feed and Barodon-biogenic feed (Tx-1; n = 6), while a second group was fed only Omolene feed (Tx-2; n = 6) and a third group was fed only Barodon-biogenic feed (Tx-3; n = 6). The untreated control group was fed general feed with no BARODON (Control; n = 6). Significant differences in the proportions of CD4⁺ (A), CD4⁺ CD25⁺ (B), CD8⁺ (C), and CD8⁺ CD25⁺ (D) T lymphocytes and cells in gate R2 (E) after culture in RPMI alone and with PHA, between control horses and BARODON-treated horses, are as indicated in the figure (*PHA = 3 days of PHA − 3 days of RPMI; a, P < 0.01; b, 0.01 < P < 0.05). See Materials and Methods for detailed information on the analysis of subsets by selective gating, used to show the frequency of each cell population.

FIG. 7.

Representative dot plot profiles of PBMC with gates placed only on 1, 2, and 3 at time zero from a control horse (A) and a BARODON-treated horse (B) labeled with one MAb (Table 1) for granulocytes and monocytes (G/M) following phagocytosis with opsonized Streptococcus equi subsp. equi, which had been previously stained overnight with PI, and a summary of FC analysis of PBMC following phagocytosis with nonopsonized (C) or opsonized (D) S. equi subsp. equi and with nonopsonized (E) or opsonized (F) Staphylococcus aureus. Profiles A and B show the frequency of S. equi-negative G/M⁻ cells (lower left quadrant), S. equi-negative G/M⁺ cells (upper left quadrant), S. equi-positive G/M⁺ cells (upper right quadrant), and S. equi-positive G/M⁻ cells (lower right quadrant). Horses in one group were fed both Omolene feed and Barodon-biogenic feed (Tx-1; n = 6), while horses in the second group were fed only Omolene feed (Tx-2; n = 6) and horses in the third group were fed only Barodon-biogenic feed (Tx-3; n = 6). The untreated control group was fed general feed with no BARODON (Control; n = 6). Significant differences between the phagocytic capabilities of control animals and animals treated with BARODON are as indicated in the figure (a, P < 0.01; b, 0.01 < P < 0.05). Detailed information on the concentration of BARODON in each feed and instructions on how to perform phagocytosis and opsonization, stain S. equi subsp. equi and S. aureus with PI, and analyze subsets by selective gating to determine the frequency of each cell population are provided in Materials and Methods.

FIG. 8.

Relationship between the amount of BARODON (in grams) fed per horse and the prevalence of IURD from 2004 to 2005 at selected stables in the Seoul Race Park.