| In order to evaluate the effects of the attenuated Mycoplasma hyopneumoniae Strain immunized by intramuscular and intrapulmonic route and with adjuvant CpG by intranasal route on local respiratory tract and systemic immune responses.36newborn piglets were randomly divided into6groups, and each pig was immunized on being7days old. The pigs from intranasal vaccination groups were immunized again3days later. Good management practices, housing conditions were applied. All the piglets were slaughtered on42day post-immunization(DPI). To investigate the local cellular immunity in respiratory tract, the numbers of intraepithelial lymphocytes(IELs) in trachea and tracheal fork, the levels of Cytokine IL-6, IL-10and IFN-y in nasal secretion and the areas of CD4+and CD8+T cells in lung and hilar lymph node were detected. To investigate the local humoral immunity in respiratory tract, the areas of IgA and IgG secreting cells in nasal mucosa and trachea, the levels of Mycoplasma hyopneumoniae specific SIgA antibody in nasal secretion and in the tissues of nasal mucosa, trachea and lung were detected. To investigate the systemic immunity, the level of Mycoplasma hyopneumoniae specific IgG antibody in serum was detected.1. The effects of the attenuated Mycoplasma hyopneumoniae strain immunized by intranasal route on the local cellular immunity in respiratory tractHE coloration method was applied to show IELs in respiratory tract. The results showed that the number of IELs in trachea and tracheal fork increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG(P<0.01), the number of IELs in tracheal fork was also increased after intramuscular and intrapulmonic immunized with the attenuated Mycoplasma hyopneumoniae strain (P<0.05)ELISA kits were applied to detect the levels of Cytokine IL-6, IL-10and IFN-γ. The results showed that the levels of IL-6, IL-10and IFN-y increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG(P<0.01), the levels of IL-6and IL-10increased significantly after intramuscular vaccination(P<0.01), and the levels of IL-6and IFN-y increased significantly after intrapulmonic vaccination on3DPI(P<0.01). On5DPI, the levels of IL-6, IL-10and IFN-y increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG and intrapulmonic vaccination(P<0.01). The leval of IFN-y was also increased after intramuscular vaccination(P<0.01), but the level was lower than that of intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG group.SABC immunohistochemical technique was applied to show CD4+and CD8+T cells in respiratory tract. The results showed that the areas of CD4+and CD8+T cells in lung and hilar lymph node were increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG(P<0.01), and so were intramuscular and intrapulmonic vaccination (P<0.05or P<0.01). The areas of CD4+and CD8+T cells in hilar lymph node were also increased after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain alone, but the number was lower than that of intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG group.The results of three essays above prompted that intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG may have increased significantly the local cellular immunity in respiratory tract of pigs.2. The effects of the attenuated Mycoplasma hyopneumoniae strain immunized by intranasal route on the local humoral immunity in respiratory tractIndirect enzyme-linked immuno-technique was applied to detect the level of Mycoplasma hyopneumoniae specific SIgA antibody in nasal secretion. The results showed that the level of Mycoplasma hyopneumoniae specific SIgA antibody in nasal secretion was increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG on7and14DPI(P<0.01). On21and35DPI, intrapulmonic immunization could significantly increase the level of Mycoplasma hyopneumoniae specific SIgA antibody in nasal secretion(P<0.01). On28DPI, intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG and intrapulmonic immunization both could increase the level of Mycoplasma hyopneumoniae specific SIgA antibody in nasal secretion(P<0.05or P<0.01).SPA-HRP immunohistochemical technique was applied to show IgA and IgG secreting cells. The results showed that the areas of IgA and IgG secreting cells increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG(P<0.01). Intramuscular vaccination could also increase the areas of IgA and IgG secreting cells(P<0.01), but the areas in intramuscular vaccination group were lower than that of intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG group. No significant changes were found in intrapulmonic group.Indirect enzyme-linked immuno-technique was applied to detect the level of Mycoplasma hyopneumoniae specific SIgA antibody in nasal mucosa, trachea and lung. The results showed that the level of SIgA in nasal mucosa and lung increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain alone or coupled with CpG(P<0.05), whereas intramuscular and intrapulmonic vaccination could also increase the level of SIgA in nasal mucosa and lung. The level of SIgA in trachea increased significantly after intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with CpG and intramuscular vaccination(P<0.01).The results of three essays above prompted that intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with adjuvant CpG may have increased significantly the local humoral immunity in respiratory tract of pigs.3. The effects of the attenuated Mycoplasma hyopneumoniae strain immunized by intranasal route on the systemic immunityELISA kit was applied to detect the level of Mycoplasma hyopneumoniae specific IgG in serum. The results showed that in the early days after immunization, the level of IgG antibody in control group, intranasal immunized with CpG group and intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain alone group was higher than that of intramuscular and intrapulmonic vaccination group and intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with CpG group. As the pigs grew older, the level of IgG antibody decreased gradually. The IgG antibody in intrapulmonic vaccination group maintained a low level, while the IgG level of intramuscular vaccination group peaked on14DPI and then decreased gradually. The IgG level of intranasal immunized with the attenuated Mycoplasma hyopneumoniae strain coupled with CpG group decreased on21DPI and then increased gradually, and peaked on35DPI. The IgG levels of other groups all decreased gradually as the piglets grew older. The results indicated that intranasal immunization with the attenuated Mycoplasma hyopneumoniae strain coupled with CpG could improve the systemic immunity level at a certain extent. |
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