牛支原体体外肺精准切片感染模型的建立

doi:10.11843/j.issn.0366-6964.2024.10.035

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4638-4645.doi: 10.11843/j.issn.0366-6964.2024.10.035

牛支原体体外肺精准切片感染模型的建立

张慧¹(), 路豆昆², 张怡秋², 赵刚³, 陈颖钰², 陈曦², 胡长敏², 郭爱珍²^,*()

1. 西南民族大学畜牧兽医学院, 成都 610041
2. 华中农业大学动物医学院, 武汉 430070
3. 宁夏大学生命科学学院, 银川 750021

收稿日期:2023-12-13 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 郭爱珍 E-mail:dkyzhanghui@163.com;aizhen@mail.hzau.edu.cn
作者简介:张慧(1986-), 女, 山西定襄人, 助理研究员, 博士, 主要从事预防兽医学研究, E-mail: dkyzhanghui@163.com
基金资助:
西南民族大学中央高校基本科研业务费专项资金资助(ZYN2023045);西南民族大学引进高层次人才科研资助金资助(RQD2023031);宁夏回族自治区重点研发计划项目(2021BEF02028);宁夏回族自治区重点研发计划项目(2023BCF01038)

Establishment of Cattle Precision-Cut Lung Tissue Slices (PCLS) Models Infected with Mycoplasma bovis in vitro

Hui ZHANG¹(), Doukun LU², Yiqiu ZHANG², Gang ZHAO³, Yingyu CHEN², Xi CHEN², Changmin HU², Aizhen GUO²^,*()

1. College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China
2. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
3. School of Life Sciences, Ningxia University, Yinchuan 750021, China

Received:2023-12-13 Online:2024-10-23 Published:2024-11-04
Contact: Aizhen GUO E-mail:dkyzhanghui@163.com;aizhen@mail.hzau.edu.cn

摘要/Abstract

摘要：

本研究旨在建立一种牛支原体体外感染模型-牛肺组织精准切片(precision-cut lung tissue slices, PCLS)感染模型。采集2月龄犊牛新鲜肺脏，经低熔点琼脂糖灌注、固定后，使用振动切片机制成厚度为250 μm的牛PCLS，利用牛支原体野毒株HB0801(P1)及其传代致弱株P150以10⁸ CFU·孔^-1的剂量进行体外感染，分别经qRT-PCR、乳酸脱氢酶(LDH)细胞毒性试验、3D荧光显微镜全景扫描、免疫组化等技术评估牛支原体在PCLS中的感染效果。结果显示正常PCLS在体外培养72 h后，细胞活力可维持在60%以上，组织形态学观察PCLS边缘清晰，肺泡结构正常；qRT-PCR检测牛支原体强、弱菌株在PCLS中的基因拷贝数，发现二者均可存活和增殖，免疫组化观察牛支原体主要定位于PCLS的肺泡腔周围以及支气管间隙，LDH试验检测发现牛支原体强、弱菌株感染18 h出现细胞毒性反应，二者均可诱导PCLS产生促炎因子IL-1β和IL-8，且强毒株诱导水平显著高于弱毒株(P < 0.05)。本研究初步建立了牛支原体PCLS体外感染模型，提供了一种可模拟牛支原体体内感染试验的离体模型，为今后研究牛支原体与宿主相互作用提供借鉴和参考。

关键词: 牛支原体, 肺组织精准切片模型, 体外感染, 炎性反应

Abstract:

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 prevention 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 apparently 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 precision 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 assay, 3D fluorescence microscope panoramic scanning, immunohistochemistry analysis were performed. 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 uninfected 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 bronchial 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 produced 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.

Key words: Mycoplasma bovis, precision-cut lung tissue slices, infected in vitro, inflammatory response

中图分类号:

S852.62

张慧, 路豆昆, 张怡秋, 赵刚, 陈颖钰, 陈曦, 胡长敏, 郭爱珍. 牛支原体体外肺精准切片感染模型的建立[J]. 畜牧兽医学报, 2024, 55(10): 4638-4645.

Hui ZHANG, Doukun LU, Yiqiu ZHANG, Gang ZHAO, Yingyu CHEN, Xi CHEN, Changmin HU, Aizhen GUO. Establishment of Cattle Precision-Cut Lung Tissue Slices (PCLS) Models Infected with Mycoplasma bovis in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4638-4645.

图/表 5

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参考文献 17

1	VALERIS-CHACIN R , POWLEDGE S , MCATEE T , et al. Mycoplasma bovis is associated with Mannheimia haemolytica during acute bovine respiratory disease in feedlot cattle[J]. Front Microbiol, 2022, 13, 946792. doi: 10.3389/fmicb.2022.946792
2	AMMAR A M , ABD EL-HAMID M I , MOHAMED Y H , et al. Prevalence and antimicrobial susceptibility of bovine Mycoplasma species in Egypt[J]. Biology (Basel), 2022, 11 (7): 1083.
3	石磊, 龚瑞, 尹争艳, 等. 肉牛传染性牛支原体肺炎流行的初步诊断[J]. 华中农业大学学报, 2008, 27 (4): 572. doi: 10.3321/j.issn:1000-2421.2008.04.029
	SHI L , GONG R , YIN Z Y , et al. Preliminary diagnosis of cattle infectious Mycoplasma bovis pneumonia[J]. Journal of Huazhong Agricultural University, 2008, 27 (4): 572. doi: 10.3321/j.issn:1000-2421.2008.04.029
4	GANDHI N N , INZANA T J , RAJAGOPALAN P . Bovine airway models: approaches for investigating bovine respiratory disease[J]. ACS Infect Dis, 2023, 9 (6): 1168- 1179. doi: 10.1021/acsinfecdis.2c00618
5	NIZAMOGLU M , JOGLEKAR M M , ALMEIDA C R , et al. Innovative three-dimensional models for understanding mechanisms underlying lung diseases: powerful tools for translational research[J]. Eur Respir Rev, 2023, 32 (169): 230042. doi: 10.1183/16000617.0042-2023
6	LYONS-COHEN M R , THOMAS S Y , COOK D N , et al. Precision-cut mouse lung slices to visualize live pulmonary dendritic cells[J]. J Vis Exp, 2017, 5 (122): 55465.
7	WATSON C Y , DAMIANI F , RAM-MOHAN S , et al. Screening for chemical toxicity using cryopreserved precision cut lung slices[J]. Toxicol Sci, 2016, 150 (1): 225- 233. doi: 10.1093/toxsci/kfv320
8	TEMANN A , GOLOVINA T , NEUHAUS V , et al. Evaluation of inflammatory and immune responses in long-term cultured human precision-cut lung slices[J]. Hum Vaccin Immunother, 2017, 13 (2): 351- 358. doi: 10.1080/21645515.2017.1264794
9	崔红亮, 张洪亮, 汪孟航, 等. 猪繁殖与呼吸综合征病毒在猪肺组织精细切片中感染特性的研究[J]. 中国预防兽医学报, 2020, 42 (8): 747- 752.
	CUI H L , ZHANG H L , WANG M H , et al. Study on the infection characteristics of porcine reproductive and respiratory syndrome virus in porcine precision-cut lung slices[J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42 (8): 747- 752.
10	WELDEAREGAY Y B , MVLLER S , HÄNSKE J , et al. Host-pathogen interactions of Mycoplasma mycoides in caprine and bovine precision-cut lung slices (PCLS) models[J]. Pathogens, 2019, 8 (2): 82. doi: 10.3390/pathogens8020082
11	LU D K , ZHANG H , ZHANG Y Q , et al. Secreted MbovP0145 promotes IL-8 expression through its interactive β-actin and MAPK activation and contributes to neutrophil migration[J]. Pathogens, 2021, 10 (12): 1628. doi: 10.3390/pathogens10121628
12	任泽民, 姜勇, 巴晓亮, 等. 牛支原体单克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2012, 39 (7): 58- 61.
	REN Z M , JIANG Y , BA X L , et al. Preparation and identification of monoclonal antibodies against Mycoplasma bovis[J]. China Animal Husbandry & Veterinary Medicine, 2012, 39 (7): 58- 61.
13	NEUHAUS V , DANOV O , KONZOK S , et al. Assessment of the cytotoxic and immunomodulatory effects of substances in human precision-cut lung slices[J]. J Vis Exp, 2018, 9 (135): 57042.
14	ALSAFADI H N , UHL F E , PINEDA R H , et al. Applications and approaches for three-dimensional precision-cut lung slices. Disease modeling and drug discovery[J]. Am J Respir Cell Mol Biol, 2020, 62 (6): 681- 691. doi: 10.1165/rcmb.2019-0276TR
15	GAUDINO M , LION A , SAGNÉ E , et al. The activation of the RIG-I/MDA5 signaling pathway upon influenza D virus infection impairs the pulmonary proinflammatory response triggered by Mycoplasma bovis superinfection[J]. J Virol, 2023, 97 (2): e0142322. doi: 10.1128/jvi.01423-22
16	HERMEYER K , BUCHENAU I , THOMASMEYER A , et al. Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: characterization of lung pathology, persistence of variable surface protein antigens and local immune response[J]. Acta Vet Scand, 2012, 54 (1): 9. doi: 10.1186/1751-0147-54-9
17	QIN L , MENG F D , HE H J , et al. Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo[J]. Infect Immun, 2023, 91 (12): e0027323. doi: 10.1128/iai.00273-23

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牛支原体体外肺精准切片感染模型的建立

Establishment of Cattle Precision-Cut Lung Tissue Slices (PCLS) Models Infected with Mycoplasma bovis in vitro

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