J Vet Sci.  2017 Sep;18(3):327-332. 10.4142/jvs.2017.18.3.327.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice

• Yim SH
• Hahn TW
• Joo HG

• KMID: 2412450
• DOI: http://doi.org/10.4142/jvs.2017.18.3.327

Abstract

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Keyword

Bordetella bronchiseptica; Mycoplasma hyopneumoniae; antigen-specific immune response; bone marrow cells; vaccine adjuvant

MeSH Terms

Animals
Antibodies, Bacterial/immunology
Antibody Formation/drug effects
Antigens, Bacterial/*immunology/pharmacology
Bordetella bronchiseptica/*immunology
Cytokines/metabolism
Female
Immunoglobulin G/*immunology
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Mycoplasma hyopneumoniae/*immunology
Nitric Oxide/metabolism
Antibodies, Bacterial
Antigens, Bacterial
Cytokines
Immunoglobulin G
Nitric Oxide

Figure

• 

  Fig. 1 The effect of Bordetella (B.) bronchiseptica Ag and Mycoplasma (M.) hyopneumoniae antigen (Ag) on the cell metabolic activity of bone marrow cells (BMs). BMs were cultured for 4 days with 0 to 10 µg/mL M. hyopneumoniae Ag and 0.4 µg/mL B. bronchiseptica Ag (A) or with 0 to 10 µg/mL B. bronchiseptica Ag and 10 µg/mL M. hyopneumoniae Ag (B). MTT solution was treated at a concentration of 0.5 mg/mL. The optical density (OD) was measured at 570 nm by using a microplate reader. All values are presented as mean ± SD values from four individual wells.

• 

  Fig. 2 Bordetella (B.) bronchiseptica antigen (Ag) enhances the proliferation of bone marrow cells (BMs). Carboxyfluorescein succinimidyl ester (CFSE)-stained BMs were treated with 5 µg/mL Mycoplasma (M.) hyopneumoniae Ag and 1 µg/mL B. bronchiseptica Ag for 4 days. Stained cells were analyzed by flow cytometry as described in Materials and Methods. The number in each dot plot or histogram indicates the percentage of normal cell size (A) or the percentage proliferating cells with low fluorescence intensity (B). MH, M. hyopneumoniae Ag-treated BMs; BB, B. bronchiseptica Ag-treated BMs; MHBB, both MH and BB Ags-treated BMs.

• 

  Fig. 3 Bordetella (B.) bronchiseptica antigen (Ag) promotes the production of tumor necrosis factor alpha (TNF-α) in bone marrow cells (BMs). After 3 days of treatment with 0 to 10 µg/mL Mycoplasma (M.) hyopneumoniae Ag and 1 µg/mL B. bronchiseptica Ag, supernatants of the treated BMs were collected and used for measurement of TNF-α. Optical density was measured at 450 nm by a microplate reader. All values are presented as mean ± SD values from four individual wells. ND, not detectable. *p < 0.05 compared to control (0 µg/mL M. hyopneumoniae Ag), ***p < 0.001 compared between two treatments (with or without B. bronchiseptica Ag).

• 

  Fig. 4 Mycoplasma (M.) hyopneumoniae antigen (Ag) and Bordetella (B.) bronchiseptica Ag promote the production of nitric oxide (NO) in bone marrow cells (BMs). After 3 days of treatment with 0 to 10 µg/mL M. hyopneumoniae Ag and 0.4 µg/mL B. bronchiseptica Ag, supernatants of the treated BMs were collected and used to measure NO level. Optical density was measured at 570 nm by a microplate reader. All values are presented as mean ± SD values from four individual wells. *p < 0.05, **p < 0.01, and ***p < 0.001, respectively, compared to control (0 µg/mL M. hyopneumoniae Ag). #p < 0.05 and ##p < 0.01, respectively, compared between two treatments (with and without B. bronchiseptica Ag).

• 

  Fig. 5 Bordetella (B.) bronchiseptica antigen (Ag) treatment increases Mycoplasma (M.) hyopneumoniae Ag-specific immunoglobulin (Ig)G. Mice were injected with phosphate-buffered saline (PBS), M. hyopneumoniae Ag (10 µg/mouse), or M. hyopneumoniae Ag + B. bronchiseptica Ag (5 µg/mouse). After 4 weeks, blood was collected and sera were used for measuring Ag-specific IgG via enzyme-linked immunosorbent assay (ELISA). For this assay, the ELISA module was coated with 5 µg/mL M. hyopneumoniae Ag (A) or B. bronchiseptica Ag (B). The antibody titer was determined at an optical density (OD) of 450 nm. MH, M. hyopneumoniae Ag-treated BMs; BB, B. bronchiseptica Ag-treated BMs; MHBB, both MH and BB Ags-treated BMs. All values are presented as mean ± SD values from four individual wells. *p < 0.05, **p < 0.01, and ***p < 0.001, respectively, compared to the lowest serum concentration (5−10 dilution). #p < 0.05, ##p < 0.01 and ###p < 0.001, respectively, compared between two treatments (MH and MHBB).

Reference

1. Audibert FM, Lise LD. Adjuvants: current status, clinical perspectives and future prospects. Immunol Today. 1993; 14:281–284.

2. Avron A, Gallily R. Mycoplasma stimulates the production of oxidative radicals by murine peritoneal macrophages. J Leukoc Biol. 1995; 57:264–268.

3. Calabro S, Tortoli M, Baudner BC, Pacitto A, Cortese M, O’Hagan DT, De Gregorio E, Seubert A, Wack A. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine. 2011; 29:1812–1823.

4. Chanter N, Magyar T, Rutter JM. Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Res Vet Sci. 1989; 47:48–53.

5. Chi Y, Lu C, Han JH, Hahn TW. [Efficacy of atropic rhinitis vaccine in pigs]. Korean J Vet Res. 2004; 40:707–717.

6. Haesebrouck F, Pasmans F, Chiers K, Maes D, Ducatelle R, Decostere A. Efficacy of vaccines against bacterial diseases in swine: what can we expect. Vet Microbiol. 2004; 100:255–268.

7. Horiguchi Y. Swine atrophic rhinitis caused by pasteurella multocida toxin and bordetella dermonecrotic toxin. Curr Top Microbiol Immunol. 2012; 361:113–129.

8. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol. 1997; 15:323–350.

9. O'Connor BP, Gleeson MW, Noelle RJ, Erickson LD. The rise and fall of long-lived humoral immunity: terminal differentiation of plasma cells in health and disease. Immunol Rev. 2003; 194:61–76.

10. Parish CR. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol. 1999; 77:499–508.

11. Simionatto S, Marchioro SB, Maes D, Dellagostin OA. Mycoplasma hyopneumoniae: from disease to vaccine development. Vet Microbiol. 2013; 165:234–242.

12. Wangoo A, Brown IN, Marshall BG, Cook HT, Young DB, Shaw RJ. Bacille Calmette-Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-γ). Clin Exp Immunol. 2000; 119:92–98.

13. Yim SH, Joo HG. [T helper 1-type immunogenicity of Mycoplasma hyopneumoniae antigen on mouse spleen cells]. J Biomed Res. 2013; 14:55–59. Korean.

14. Yim SH, Joo HG. [Stimulatory effects of Bordetella bronchiseptica antigen on bone marrow cells and immune memory responses]. Korean J Vet Res. 2014; 54:203–208. Korean.

15. Zhao Z, Wang C, Xue Y, Tang X, Wu B, Cheng X, He Q, Chen H. The occurrence of Bordetella bronchiseptica in pigs with clinical respiratory disease. Vet J. 2011; 188:337–340.