Mycoplasma bovis(M.bovis)is an important pathogen of bovine respiratory disease complex(BRDC),mainly causing pneumonia,mastitis,arthritis,keratoconjunctivitis,otitis,and genital disorders,leading to high economic losses in dairy and beef cattle production.Lack of small animals'models have greatly hindered the progress of effective vaccines and drugs for pre-vention and control in Mycoplasma bovis.In this study,we have established an in vitro infection model of M.bovis in Precision-cut lung tissue slices(PCLS).We collected bovine lungs from ap-parently healthy two-months cattle after slaughter,filled the lung airway with low melting point agarose,transferred it to the vibratome tissue slicer and then made the 250 μm-thickness preci-sion cut lung slices.After M.bovis wildtype strain HB0801(P1)and its attenuated strain P150 at a MOI of 108 CFU infected,quantitative real-time PCR,lactate dehydrogenase cytotoxicity as-say,3D fluorescence microscope panoramic scanning,immunohistochemistry analysis were per-formed.The results showed that cultured PCLS remained 60％cell viability for at least 72 h and maintained normal structural integrity including edge sharpness and alveolar structure well in un-infected PCLS.After infected with wildtype and attenuated M.bovis strains respectively,we found that both M.bovis could survive and proliferate in PCLS,tropism to alveolar and bronchi-al space,and showed cytotoxic reactions until 18 h post-infection.We also observed that after 36 h incubation with M.bovis wildtype strain P1 and its attenuated strain P150,cultured PCLS pro-duced pro-inflammatory factors IL-1β and IL-8 in PCLS,and M.bovis wildtype strain HB0801 could induce significantly higher level than P150(P＜0.05).In conclusion,this study provides a model and method to investigate M.bovis infection in vitro which can mimic M.bovis infection in vivo.It is expected to provide reference for subsequent research on host-pathogen interaction in M.bovis.
关键词
牛支原体/肺组织精准切片模型/体外感染/炎性反应
Key words
Mycoplasma bovis/precision-cut lung tissue slices/infected in vitro/inflammatory response
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