OIE Reference Laboratory for Contagious Bovine Pleuropneumonia, Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise "G. Caporale", Campo Boario, 64100, Teramo, Italy.
Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d'Accio, 64100, Teramo, Italy.
Vet Res. 2018 Jan 12;49(1):5. doi: 10.1186/s13567-017-0500-z.
Contagious bovine pleuropneumonia (CBPP) is a severe disease caused by Mycoplasma mycoides subsp. mycoides (Mmm). Knowledge on CBPP pathogenesis is fragmented and hampered by the limited availability of laboratory animal and in vitro models of investigation. The purpose of the present study is to assess respiratory explants as useful tools to study the early stages of CBPP. Explants were obtained from trachea, bronchi and lungs of slaughtered cattle, tested negative for Mycoplasma spp. and for the major bacterial and viral respiratory pathogens. The interaction of Mmm with explant cells was studied by immunohistochemistry (IHC), double-labelling indirect immunofluorescence (DLIIF) and laser scanning confocal microscopy (LSCM). Mmm capability to survive and proliferate within the explants was evaluated by standard microbiological procedures. Finally, the putative cellular internalization of Mmm was further investigated by the gentamicin invasion assay. IHC and DLIIF indicated that Mmm can colonize explants, showing a marked tropism for lower airways. Specifically, Mmm was detected on/inside the bronchiolar and alveolar epithelial cells, the alveolar macrophages and the endothelial cells. The interaction between Mmm and explant cells was abolished by the pre-incubation of the pathogen with bovine anti-Mmm immune sera. Mmm was able to survive and proliferate in all tracheal, bronchial and lung explants, during the entire time course of the experiments. LSCM and gentamicin invasion assay both confirmed that Mmm can enter non-phagocytic host cells. Taken together, our data supports bovine respiratory explants as a promising tool to investigate CBPP, alternative to cattle experimental infection.
传染性牛胸膜肺炎（CBPP）是由丝状支原体丝状亚种（Mmm）引起的严重疾病。对 CBPP 发病机制的了解是零散的，并受到实验室动物和体外研究模型的有限可用性的阻碍。本研究旨在评估呼吸外植体作为研究 CBPP 早期阶段的有用工具。外植体取自屠宰牛的气管、支气管和肺，经支原体属和主要细菌和病毒呼吸道病原体检测均为阴性。通过免疫组织化学（IHC）、双标记间接免疫荧光（DLIIF）和激光扫描共聚焦显微镜（LSCM）研究了 Mmm 与外植体细胞的相互作用。通过标准微生物学程序评估了 Mmm 在外植体中存活和增殖的能力。最后，通过庆大霉素侵袭试验进一步研究了 Mmm 的细胞内吞作用。IHC 和 DLIIF 表明 Mmm 可以定殖外植体，对下呼吸道表现出明显的亲嗜性。具体而言，Mmm 被检测到在支气管和肺泡上皮细胞、肺泡巨噬细胞和内皮细胞上/内部。病原体与牛抗 Mmm 免疫血清预孵育可消除 Mmm 与外植体细胞的相互作用。Mmm 能够在所有气管、支气管和肺外植体中存活和增殖，在整个实验过程中。LSCM 和庆大霉素侵袭试验均证实 Mmm 可以进入非吞噬宿主细胞。总之，我们的数据支持牛呼吸道外植体作为研究 CBPP 的有前途的工具，可替代牛的实验感染。
