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Abstract Background Mycoplasma hyopneumoniae, the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next‐generation vaccines by screening protective antigens is crucial. Objectives The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae. Methods The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX‐6P‐1, pGEX‐6P‐2, pGEX‐5X‐3 or pGEX‐4T‐3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL‐1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera. Results All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty‐one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera. Conclusions The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.

Abstract Vaccination with inactivated bacterin is the most popular and practical measure to control enzootic pneumonia. After immunisation with inactivated bacterin, Mycoplasma hyopneumoniae colonised on the respiratory tract and lung stimulates the humoural immune responses and produces IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG‐ELISA kit cannot distinguish between inactivated bacterin‐induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA‐ELISA method needs to collect nasal swabs, but collecting nasal swabs is not easy to operate. Establishment of a discriminative ELISA detecting humoural IgG from convalescent sera but not hyperimmune sera facilitates to evaluate the natural infection of M. hyopneumoniae after inactivated bacterin vaccination. We expressed and purified a recombinant protein named Mhp366‐N which contains an epitope recognised by the convalescent sera but not hyperimmune sera. The developed discriminative IgG‐ELISA could discriminate between inactivated bacterin‐induced hyperimmune sera and convalescent sera and was reproducible, sensitive and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA‐ELISA method, discriminative IgG‐ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it has limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. The established discriminative IgG‐ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin.