Quantitative detection and inactivation of Mycoplasma hyopneumoniae.

Abstract

Mycoplasmal pneumonia of swine (MPS) caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) is a chronic respiratory infectious disease of swine and has a significant impact on the economy. The main clinical symptom of the disease is cough, accompanied by reduced growth performance. Inactivated vaccine is used extensively to control M. hyopneumoniae infection worldwide. This study focused on a quantitative detection method and inactivation kinetics of M. hyopneumoniae in the process of vaccine production. In this study, an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was established and optimized using elongation factor thermo unstable (EF-Tu) as the target protein. EF-Tu protein is abundant on the surface of M. hyopneumoniae. The ic-ELISA assay was used to quantify the M. hyopneumoniae cultures at different growth stages, and compared with the traditional color changing unit (CCU) assay. The ic-ELISA assay results obtained for a growth curve were similar to that of the CCU assay indicating a strong correlation in the log phase. Also, a linear regression equation was established between the ic-ELISA and CCU assays in the log phase. The ic- ELISA method was used to evaluate the relative potency values of different batches of cultures over the internal reference vaccine to determine whether the cultures meet the antigen amount requirements for vaccine preparation. Compared with the CCU assay, the established ic-ELISA assay can quantitatively detect the antigen content in the process of vaccine production more quickly. Furthermore, this study also systematically compared the inactivation effects of, formaldehyde, thimerosal, β-propiolactone (BPL) and binary ethylenimine (BEI) on M. hyopneumoniae. Complete inactivation were achieved by 0.01% formaldehyde for 24 h at 37°C, 0.0008% thimerosal for 12 h at 37°C, or 0.02% BPL for 24 h at 4°C or 0.004% BEI for 24 h at 37°C. The immunogenicity of the antigens after inactivation was detected by mice immune test. The IgG antibody level of the mice immunized with the vaccine inactivated by 0.01% formaldehyde for 24 hours was significantly higher than those of the mice immunized with the vaccines inactivated by other solutions. The data indicated that formaldehyde can be used to inactivate M. VIII hyopneumoniae before vaccine preparation. In a conclusion, this study provides important reference value for the antigen quantitative detection and inactivation of M. hyopneumoniae for vaccine production.