ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2018, Vol. 49 ›› Issue (2): 378-387.doi: 10.11843/j.issn.0366-6964.2018.02.017

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The Pathogenic Mechanism of Mycoplasma Hyopneumoniae Strains with Different Virulence on 3D Cultured Swine Tracheal Epithelial Cells

FENG Yan-yan1,2, WANG Hai-yan2, LIU Bei-bei2, WEI Yan-na2, ZHANG Zhen-zhen2, BAI Yun2, NI Bo2, SHAO Guo-qing2, LEI Zhi-hai1*, FENG Zhi-xin2*   

  1. 1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
    2. Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
  • Received:2017-06-26 Online:2018-02-23 Published:2018-02-23

Abstract:

To study the pathogenic mechanism of Mycoplasma hyopneumoniae (Mhp), an air-liquid interface (ALI) cultivation model of continuous STEC cell line, closer to the status in vivo, was developed for Mhp infection. Mhp strains with different virulence (virulence strains NJ, intermediate virulence strain AH, and attenuated strain 168L) were selected for respective infection with the same dose. The integrity of cell monolayer and the vitality of the infected cells were respectively evaluated by TEER value, Alamar Blue assay and lactate dehydrogenase (LDH) release assay. The ability of the differentiated cells to secrete MUC5B mucin against Mhp infection was measured by laser confocal and fluorescence spectrophotometer. To further study the pathogenic mechanism of Mhp infection, the cellular oxidation system of the infected STEC cells, including the secretion of NO and endogenous ROS were detected, respectively. The effect of NAC antioxidant on cell damage of Mycoplasma infection group was observed and was further demonstrated. The results revealed that the infection of virulence strains NJ and intermediate virulence strains AH could significantly roughen, disorder or damage the microvillus and cilia on the surface of STEC cells, but not attenuated strains 168L. The resistance of cell monolayer was decreased after infection, which was positively correlated with the virulence of Mhp strains. Mhp strains NJ and AH could significantly reduce the vitality of the infected cells using Alamar Blue and LDH release assays, especially at 48 h post-infection. Mhp strains could also promote the differentiated cells to secrete MUC5B mucus against Mhp infection, and the stronger the virulence of the strain was, the more mucous was secreted. It was found that except the 168L strain, AH strain and NJ strain could both significantly stimulate STEC cells to secrete NO and endogenous ROS, which could cause the oxidative stress reaction of cells significantly. After the anti-oxidation treatment of NAC, the ROS and mucus secretion of virulence strains NJ, intermediate virulence strains AH and the attenuated strains 168L groups decreased significantly, while the cell activity and resistance value increased significantly, while control group were not significantly changed. This study has been proved that Mhp infection can comprehensively disorder the growth characteristics, vitality and secretory function of the host cells, which are positively correlated with the virulence of Mhp strains. Moreover, the pathogenic mechanism of Mhp infection is closely related to the ability to induce the oxidative stress to host cells.

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