Pneumonia caused by Mesomycoplasma hyopneumoniae (Mhp) is a respiratory disease with high morbidity and low mortality that typically presents in growing pigs. Although often subclinical, the disease can significantly affect the pig farming industry economically due to decreased growth rates and inefficient feed conversion. Effective control of Mhp depends on the detection of dominant strains prevalent in infected animals, which vary in virulence. However, traditional culture methods for diagnosing Mhp are laborious and slow, whereas multi-locus sequence typing, another possible method, requires identifying several genes. This study introduces a novel pair of polymerase chain reaction (PCR) primers for the rapid detection and genetic evolution analysis of Mhp strains to facilitate improved vaccine selection. The genetic evolutionary tree established using the PCR amplification fragment was highly similar to the genetic evolutionary tree established using whole-genome sequences. Analysis of 131 samples from Guangxi and Hunan slaughterhouses revealed a 30.53 % prevalence of Mhp. High-throughput sequencing has shown that Mhp has a diverse bacterial population in clinically collected samples. The prevalence of major strains may vary among regions. Additionally, the strains of Mhp vaccines sold may differ significantly from the strains prevalent on farms. In summary, this work has designed a pair of primers that will be useful for detecting the diversity of Mhp and for targeted prevention and control.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world\'s swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.