Comparative study on the inactivation and immunogenicity of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis vaccines prepared using different inactivants.

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The present study aimed to investigate the optimal inactivants and inactivation conditions for preparing inactivated vaccines of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Mycoplasma inactivation was performed using formaldehyde, thimerosal, β-propiolactone (BPL), and binary ethylenimine (BEI) and compared. The results showed that M. hyopneumoniae was completely inactivated when incubated with 0.01% formaldehyde for 24h or 0.02% formaldehyde for 12h at 37°C, with 0.0008% thimerosal for 12h at 37°C, with 0.02% BPL for 24h or 0.1% BPL for 12h at 4°C, or with 0.004% BEI for 24h or 0.5% BEI for 12h at 37°C. M. hyorhinis was completely inactivated when incubated with 0.01% formaldehyde for 24h or 0.02% formaldehyde for 12h at 37°C, with 0.004% thimerosal for 24h or 0.02% thimerosal for 12h at 37°C, with 0.1% BPL for 12h at 4°C, or with 0.004% BEI for 24h or 0.5% BEI for 12h at 37°C. Next, the immunogenicity of the mycoplasmas after inactivation was evaluated by immunizing BALB/c mice. Immunization of mice with a high dose (106 color-changing units [CCU] per dose) of M. hyopneumoniae and M. hyorhinis vaccines inactivated with all inactivants led to high levels of serum IgG antibodies. M. hyopneumoniae vaccines inactivated with formaldehyde induced significantly higher titers of antibodies than vaccines inactivated with other inactivants, whereas M. hyorhinis vaccines inactivated with BEI induced significantly higher titers of antibodies than vaccines inactivated with thimerosal. However, in mice immunized with a low dose of mycoplasmas (104 CCU per dose), only M. hyopneumoniae vaccines inactivated with formaldehyde and BEI and M. hyorhinis vaccines inactivated with formaldehyde, BPL, and BEI led to significant levels of serum IgG antibodies. Among these groups, the antibody levels in the formaldehyde-inactivated vaccine group were higher than those in the other inactivant groups. This study provides a reliable basis for inactivation during large-scale production of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis inactivated vaccines.