豚丹毒菌YS-19株免疫豚のマイコプラズマ･ハイオニューモニエP97抗原に対する免疫応答の解析

Size: px

Start display at page:

Download "豚丹毒菌YS-19株免疫豚のマイコプラズマ･ハイオニューモニエP97抗原に対する免疫応答の解析"

れいなみやのじょう
7 years ago
Views:

1 Immune responses to the Mycoplasma hyopneumoniae P97 antigen in pigs vaccinated with Erysipelothrix rhusiopathiae YS-19 Mycoplasma hyopneumoniae M. hyopneumoniae M. hyopneumoniae Mycoplasma hyopneumoniae 2 16) M Corresponding author, Mailing address: Yoshihiro SHIMOJI, Research Team for Advanced Biologicals, National Institute of Animal Health, Kannondai, Tsukuba, Ibaraki , JAPAN Tel & Fax: shimoji@affrc.go.jp hyopneumoniae 16) 16) M. hyopneumoniae M. hyopneumoniae 2), 3), 8) M. hyopneumoniae P97adhesin 113,

1 2 3 4 Corresponding author, Mailing address: Yoshihiro SHIMOJI, Research Team for Advanced Biologicals, National Institute of Animal

2 2Erysipelothrix rhusiopathiaeys-19 18) 19) M. hyopneumoniae YS-19 P97 19) YS-19 P97 T Needle-free injector P97 6 YS-19 P97Needlefree injectorys-19 1 YS-19 18) M. hyopneumoniae 12) M. hyopneumoniaee-1 specific pathogen-freespf 9Hanks balanced saline solution (Sigma- Aldrich, Tokyo, Japan) M. hyopneumoniae E-13 1:1 2 M. hyopneumoniae E-1P97 R1R2 2 rp97 Sephacryl S-2GE Healthcare Biosciences, TokyoM. hyopneumoniae Ribonucleotide reductasernrdf 4) rp97 TGENETYX- MACVersion 7.3, Genetyx, Tokyo 3 19) Ficoll-Paque PlusAmersham Pharmacia Biotech, Uppsala, Sweden21 6 /ml 12 mm - 1 mm 5U/ml 5g/mlRPMI164GIBCO BRL, Rockville, MD1l1g/ml rp971l37 5 CO24 3 H- 361.Ci/ well P97 P97 3 H- count per minute: cpmstimulation index: SI 4ELISA ELISArP97 18) 2x1 6 cells/ml1l 5g/ml1l IFN- Biosource International IL-8 Splichal et al., 21) Bull. Natl. Inst. Anim. Health No (January 27)

hyopneumoniae E-1P97 R1R2 2 rp97 Sephacryl S-2GE Healthcare Biosciences, TokyoM. hyopneumoniae Ribonucleotide reductasernrdf 4) rp97 TGENETYX- MACVersion 7.

3 5 1 19) Fig SPF24 8YS-1P97 8; 1 9 CFU/YS-19 8; 1 9 CFU/ CFU 49, 5, 51 M. hyopneumoniae2.1 8 Color Changing Unit [CCU] Fig. 1. Schedules for animal experiments. The numbers below arrows indicate days after first immunization or inoculation. (Experiment 1) Twenty-four SPF pigs were divided into three groups. Groups of eight pigs from groups B and C were immunized intranasally with YS-1 and YS-19, respectively, on days, 16 and 32, while eight pigs in group A were inoculated intranasally with the medium as a control. The pigs in three groups were challenged with a virulent M. hyopneumoniae strain on days days. On day 85, the pigs were necropsied. (Experiment 2) Six germ-free pigs were divided into three groups. On three consecutive days, two pigs from groups A and B were inoculated intranasally with a suspension containing 1.5mg of rp97protein and rnrdf protein, respectively. Two pigs in group A were inoculated PBS. On days 3 and 6, three pigs from each group were necropsied. (Experiment 3) Twenty-one SPF pigs were divided into three groups. Groups of seven pigs from groups B and C were immunized intradermally with a needle-free injector with YS-1 and YS-19, respectively, while seven pigs in group A were inoculated intradermally with the medium as a control. The pigs in three groups were challenged with a virulent M. hyopneumoniae strain on days days. On day 66, the pigs were necropsied. (Experiment 4) Nineteen SPF pigs were divided into two groups. Ten pigs were immunized intradermally with a needle-free injector with YS-19, while nine pigs were inoculated intradermally with the medium as a control. The pigs were challenged with a virulent M. hyopneumoniae strain on days 28-3 days. On day 58, the pigs were necropsied. 113,

Groups of eight pigs from groups B and C were immunized intranasally with YS-1 and YS-19, respectively, on days, 16 and 32, while eight pigs in group A were inoculated intranasally with the medium as

4 Student s unpaired t-test 22 12Phosphate-buffered saline PBSrP97M. hyopneumoniae rnrdf1.5mg3 43 5ml PBS 1l Shandon Inc., Pennsylvania, USA Dif Quick 334 SPF21 7YS-17; 2.2x 1 1 CFU/ YS-197; 2.5x 1 1 CFU/ Needle-free injectormed-e-jet Co., USA 1 24, 25, 26 M. hyopneumoniae4.1 8 CCU SPF 19 9YS-191; 1.x 1 1 CFU/ Needle-free injector 128, 29, 3M. hyopneumoniae 2.x1 8 CCU 28 1YS-19 1 YS-1P97 YS-19 YS-19 YS-19 rp97 YS rP97 SI M. hyopneumoniae 371Fig. 2 rp97 M. hyopneumoniae 24 IFN- Figure 3YS-19 52YS YS-1 IFN- 24 rp97il-8 Fig. 2. Proliferation of peripheral blood mononuclear cells (PBMC) in immunized and non-immunized pigs. On days, 16, 24, 37(a), 52(b) and 71(c), PBMC from the pigs were stimulated with rp97 protein. The data for on days, 16 and 24 are not shown. The results are expressed as the mean of stimulation index (SI)SEM. Bull. Natl. Inst. Anim. Health No (January 27)

x1 8 CCU 28 1YS-19 1 YS-1P97 YS-19 YS-19 YS-19 rp97 YS-19 337rP97 SI M. hyopneumoniae 371Fig. 2 rp97 M. hyopneumoniae 24 IFN- Figure 3YS-19 52YS-1 371 YS-1 IFN- 24 rp97il-8 Fig. 2. Proliferation of peripheral blood mononuclear cells (PBMC) in immunized and non-immunized pigs.

5 Fig. 3. Production of IFN-from PBMC in immunized and non-immunized pigs. On days 24, 52(a) and 71(b), PBMC from the pigs were stimulated with rp97 protein. The data for on day 24 are not shown. The results are expressed as the mean of SISEM. IL-8 Fig Table 1M. hyopneumoniae 2 YS-12YS-194 IFN- rp97 IFN- Fig. 5IL-8 IL-8 Fig. 6 2P97 2 IL-8rP97 Table 1. Sequences of the peptides used for stimulation of PBMC Peptide Sequence DNKIKGILPQPPA PAAKPEAAKPEAAKPETAKP PVAAKPETTKPVATNTN INVFLELVHQSEYE EYEEQKIIKELDKT QFQEVKVASDQYQK PEAAKPEAAKPEAAKP PEAAKPEAAKP rp973 rnrdf PBS Fig. 74 Fig. 4. Production of IL-8 from PBMC in immunized and non-immunized pigs. On day 24, PBMC from the pigs were stimulated with rp97 protein. The data for one pig in the YS-19-immunized group was not included. 113,

Sequences of the peptides used for stimulation of PBMC Peptide 1 2 3 4 5 6 7 8 Sequence DNKIKGILPQPPA PAAKPEAAKPEAAKPETAKP PVAAKPETTKPVATNTN INVFLELVHQSEYE EYEEQKIIKELDKT QFQEVKVASDQYQK

6 Fig. 5. Production of IFN-from PBMC in immunized and non-immunized pigs. On day 85, PBMC from the pigs were stimulated with peptides and rp97 protein. 1, peptide 1; 2, peptide 2; 3, peptide 3; 4, peptide4; 5, peptide5; 6, peptide 6; 7, peptide 7; 8, peptide 8; P, rp97; C, control. Fig. 6. Production of IL-8 from PBMC in immunized and non-immunized pigs. On day 85, PBMC from the pigs were stimulated with peptides and rp97 protein. 1, peptide 1; 2, peptide 2; 3, peptide 3; 4, peptide4; 5, peptide5; 6, peptide 6; 7, peptide 7; 8, peptide 8; P, rp97; C, control. Bull. Natl. Inst. Anim. Health No (January 27)

7 Fig. 7. Cells observed in the bronchoalveolar lavage fluid after intranasal inoculation of PBS (a), rnrdf (b) and rp97 protein (c). 3Needle-free injectorys-19 3 YS-1 YS-19Needle-free injectorfig. 8 YS-19Needle-free injector 2345rP97 YS-19 M. hyopneumoniae 3 SIFig YS-19 P97 4Needle-free injectorys-19 4 YS-19 Needle-free injector1 9.2YS- 19.2p<.1 Table 2 Fig. 8. The needle free-injector used for intradermal immunization of YS-19 strain. Fig. 9. Proliferation of PBMC in immunized and nonimmunized pigs. On days, 2, 3 and 45, PBMC from the pigs were stimulated with rp97 protein. The data for on days, 2 and 45 are not shown. The results are expressed as the mean of SI SEM. 113,

9 1945 YS-19 P97 4Needle-free injectorys-19 4 YS-19 Needle-free injector1 9.2YS- 19.2p<.1 Table 2 Fig. 8.

8 Table 2. Effect of intradermal vaccination with the needle-free injector of E. rhusiopathiae YS-19 on development of pneumonia Treatment group and pig number Control Dorsal 29.3* Percentage of lung pneumonic (%) Ventral Average YS-19-immunized group * The value indicates the proportions of the lung surfaces with pneumonic lesions. Messier et al., 9) M. hyopneumoniae M. hyopneumoniae Thacker et al., 22) IFN- M. hyopneumoniae TIFN- YS-192 P97 M. hyopneumoniae rp97 19) YS-19P97 M. hyopneumoniae M. hyopneumoniaep97 M. hyopneumoniae P97 YS-19 P97 M. hyopneumoniae IFN- 13M. hyopneumoniae YS-19 M. hyopneumoniae Bull. Natl. Inst. Anim. Health No (January 27)

6 6.9 17.4 13.9 17.7 Average 2.8 24. 6.4 19.3 11.4 18.7 YS-19-immunized group 251 252 253 254 255 256 257 258 259 26 2.5 2.3 2.4 * The value indicates the proportions of the lung surfaces with pneumonic lesions.

9 1YS-193 P97 3rP97 M. hyopneumoniae rp97ifn- 3YS-191P97 M. hyopneumoniae rp97 3Needle-free injector4ys-191 M. hyopneumoniae YS-19 P97 King et al., 6 P97 M. hyopneumoniaep97 P97 1 P97M. hyopneumoniae 16) rp97il-8 M. hyopneumoniae IL-8 IL-8 13) 16) M. hyopneumoniae IL-8 mrna 1), 7), 14), 15), 2), 23) rp97 P97 M. hyopneumoniae P97 11) IL IL-8 rp97 LPS in vitrolps BrP97 IL-8 P97IL-8 YS-19 Needlefree injector YS-19 P97 17) P97 Needle-free injector Needle-free injector Asai, T., Okada, M., Ono, M. et al.:. Increased levels of tumor necrosis factor and interleukin 1 in bronchoalveolar lavage fluids from pigs infected with Mycoplasma hyopneumoniae. Vet. Immunol. Immunopathol. 38, (1993). 2 Blanchard, B., Vena, M.M., Cavalier, A. et al.: Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. Vet. Microbiol. 3, (1992). 113,

hyopneumoniae P97 11) IL-8 2 2 IL-8 rp97 LPS in vitrolps BrP97 IL-8 P97IL-8 YS-19 Needlefree injector YS-19 P97 17) P97 Needle-free injector Needle-free injector 1415 1 Asai, T., Okada, M., Ono, M.

10 3 DeBey, M.C., Jacobson, C.D., & Ross, R.F.: Histochemical and morphologic changes of porcine airway epithelial cells in response to infection with Mycoplasma hyopneumoniae. Am. J. Vet. Res. 53, (1992). 4 Fagan, P. K., Djordjevic, S.P,, Eamens, G.J., et al.: Molecular characterization of a ribonucleotide reductase (nrdf) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia. Infect Immun. 64, (1996). 5 Hsu, T. & Minion, F.C.: Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect. Immun. 66, (1998). 6 King, K.W., Faulds, D.H., Rosey, E.L. et al.: Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1 s vaccine potential. Vaccine 15, (1997). 7 Lorenzo, H., Quesada, O., Assuncao, P., et al.: Cytokine expression in porcine lungs experimentally infected with Mycoplasma hyopneumoniae. Vet. Immunol. Immunopathol. 19, (26). 8 Mebus, C.A. & Underdahl, N.R.: Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae. Am. J. Vet. Res. 38, (1977). 9 Messier, S., Ross, R.F. & Paul, P.S.: Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae. Am. J. Vet. Res. 51, (199). 1Minion, F.C., Adams, C., & Hsu, T.: R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia. Infect. Immun. 68, (2). 11Minion, F.C., Lefkowitz, E.J., Madsen, M.L., et al.: The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J. Bacteriol. 21, (24). 12Mori, Y., Yoshida, Y., Kuniyasu C. et al.: Improvement of complement fixation test antigen for the diagnosis of Mycoplasma hyopneumoniae infection. Natl. Inst. Anim. Health Q. (Tokyo) 23, (1983). 13Narita, M., Tanaka, H., Yamada, S., et al.: Significant role of interleukin-8 in pathogenesis of pulmonary disease due to Mycoplasma pneumoniae infection. Clin. Diagn. Lab. Immunol. 8, (21). 14Okada, M., Asai, T., Ono, M. et al: Cytological and immunological changes in bronchoalveolar lavage fluid and histological observation of lung lesions in pigs immunized with Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate. Vaccine. 18, (2). 15Rodriguez, F., Ramirez, G.A., Sarradel, J., et al.: Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with Mycoplasma hyopneumoniae. J. Comp. Pathol. 13, (24). 16Ross, R.F.: Diseases of swine. In: Mycoplasmal diseases. (Straw, B.E., D Allaire, S., Mengeling, W.L., et al., eds.), 8th ed., , Iowa State University Press. Iowa (1999). 17Shimoji, Y., Yokomizo, Y., & Mori, Y.: Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages. Infect. Immun. 64, (1996). 18Shimoji, Y., Oishi, E., Kitajima, T. et al.: Erysipelothrix rhusiopathiae YS-1 as a live vaccine vehicle for heterologous protein expression and intranasal immunization of pigs. Infect. Immun. 7, (22). 19Shimoji, Y., Oishi, E., Muneta, Y. et al.: Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine. Vaccine. 21, (23). 2Sohn, M.H., Lee, K.E., Choi, S.Y., et al.: Effect of Mycoplasma pneumoniae lysate on interleukin-8 gene expression in human respiratory epithelial cells. Chest. 128, (25). 21Splichal, I., Muneta, Y., Mori, Y., et al.: Development and application of a pig IL-8 ELISA detection system. J. Immunoassay Immunochem. 24, (23). 22Thacker, E.L., Thacker, B.J., Kuhn, M., et al.: Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacteria to pigs. Am. J. Vet. Res. 61, (2). 23Thanawongnuwech, R., Thacker, B., Halbur, P. et al.: Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Clin. Diagn. Lab. Immunol. 11, (24). 24Zhang, Q., Young, T.F. & Ross, R.F.: Identification and characterization of a Mycoplasma hyopneumoniae adhesin. Infect. Immun. 63, (1995). Bull. Natl. Inst. Anim. Health No (January 27)

: Molecular characterization of a ribonucleotide reductase (nrdf) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia.

11 Summary Immune responses to the Mycoplasma hyopneumoniae P97 antigen in pigs vaccinated with Erysipelothrix rhusiopathiae YS-19 We investigated immune responses to the P97 antigen of Mycoplasma hyopneumoniae in pigs vaccinated with Erysipelothrix rhusiopathiae YS-19 strain expressing a recombinant protein of the P97 antigen on the cell surface. In the pigs that were intranasally immunized three times with the YS-19 stain, the P97-specific serum antibodies were not detected throughout the experimental period. However, after the third immunization, peripheral blood mononuclear cells (PBMC) from the YS-19-immunized pigs significantly proliferated when stimulated with the P97 protein, compared to the cells from non-immunized pigs. Furthermore, when stimulated with the P97 protein, production of IFN-, an indicator of cell-mediated immune responses, from PBMC of the YS-19-immunized pigs was observed after the challenge. Irrespective of the immunization of pigs with the YS-19 strain, PBMC produced IL-8 with stimulation with P97 protein, and exuded polymorphonuclear leukocytes were observed in the bronchoalveolar lavage fluid from the pigs that had been intranasally inoculated with P97 protein. In an experiment in which a needle-free injector was used for intradermal immunization, PBMC from the pigs immunized with the YS-19 strain significantly proliferated with stimulation with the P97 protein after a challenge with a virulent M. hyopneumoniae strain. In pigs immunized with the YS-19 strain using the needle-free injector, the severity of pneumonic lung lesions caused by M. hyopneumoniae infection was significantly reduced. Key words: Erysipelothrix rhusiopathiae YS-19, mycoplasmal pneumonia of swine, P97 antigen, vaccination 113,

In the pigs that were intranasally immunized three times with the YS-19 stain, the P97-specific serum antibodies were not detected throughout the experimental period.

日本消化器外科学会雑誌第29巻第9号

日本消化器外科学会雑誌第29巻第9号 Table 1 Oligonucleotide primers used for RT-PCR and internal probes used for Southern blot hybridization Cytokine Primer Sequence (5'-3') 5' 3' Internal probe s' 3' Internal probe 5' 3' Internal probe