基于小鼠模型研究肠道菌群紊乱对BVDV易感性的影响

Study on the Effect of Gut Microbiota Disturbance on Susceptibility to BVDV Based on a Mouse Model

本试验通过建立肠道菌群紊乱小鼠模型,旨在探究肠道菌群紊乱对BVDV易感性的影响。肠道菌群紊乱小鼠模型建立试验,共分为2组,采用两性霉素B、硫酸新霉素、氨苄西林、甲硝唑、盐酸万古霉素,以灌胃的方式构建肠道菌群紊乱小鼠模型,对照组给予等体积的生理盐水。连续处理13 d后,无菌收集小鼠粪便,提取细菌DNA,进行16S rRNA测序,检测小鼠肠道菌群多样性和丰度的变化。肠道菌群紊乱对BVDV易感性的影响试验,共分为2组,抗生素处理组和未处理组小鼠均接种10^(5) TCID_(50)的CP型BVDV,于感染第7天,采集小鼠血液和十二指肠,通过qRT-PCR、Western blot方法检测病毒载量。通过苏木精-伊红染色法(HE染色)检测十二指肠病理组织变化。粪菌移植(FMT)移植试验共分为2组,以灌胃等体积生理盐水组为对照,进一步验证恢复肠道菌群对BVDV感染的影响。结果显示,抗生素处理明显降低肠道菌群α多样性,维恩图分析也表明抗生素处理减少肠道菌群OTU数量。β多样性结合柱形图分析进一步表明,抗生素处理组与未处理组小鼠肠道菌群的组成存在明显差异,其中厚壁菌门和拟杆菌门的相对丰度在抗生素处理组中均显著降低,然而变形菌门相对丰度明显增加。对BVDV易感性探究发现,菌群紊乱小鼠血液和十二指肠中BVDV载量均显著高于BVDV感染的菌群正常组小鼠的BVDV载量。Western blot和病理组织学检测也发现,肠道菌群紊乱增加了小鼠十二指肠中BVDV E0蛋白表达水平,加重了十二指肠病理损伤。FMT回补给菌群紊乱小鼠后,BVDV载量显著降低,十二指肠病理变化也明显改善。综上,本研究成功建立肠道菌群紊乱小鼠模型,依据该模型进一步证实了肠道菌群紊乱可以增加BVDV的易感性,而FMT回补具有抑制BVDV感染的作用。这些结果可为防控BVDV感染的微生态制剂研发以及抗病毒药物的筛选奠定基础。

This study aimed to explore the effect of gut microbiota disturbance on susceptibility to BVDV by establishing a mouse model of gut microbiota disturbance.The experiment was divided into two groups.Gut microbiota disturbance mice were treated with amphotericin-B,neomycin trisulfate salt hydrate,ampicillin,metronidazole,and vancomycin hydrochloride by gavage,while mice in control group were given an equal volume of physiological saline.After 13 consecutive days,fecal samples of mice were collected to extract bacterial DNA and 16S rRNA sequencing was performed to detect the changes in the diversity and abundance of gut microbiota in mice.The mice exploring the impact of gut microbiota disturbance on susceptibility to BVDV were divided into 2 groups.Mice in antibiotic treated and untreated groups were intraperitoneally injected with 10^(5) TCID_(50) CP BVDV.The blood and duodenum of mice at day 7 of post-infection were collected,and viral loads were detected by qRT-PCR and Western blot.The pathological changes of duodenum were detected by hematoxylin and eosin staining(HE staining).The experiment of fecal microbiota transplantation(FMT)was divided into two groups.Mice in the experiment group were treated with FMT while and the mice in control group were given the same volume normal saline.Aimed to further verify the effect of restoring gut microbiota on BVDV infection.The results showed that antibiotic treatment significantly reduced theα-diversity of gut microbiota,and Venn diagram analysis also showed that antibiotic treatment reduced the number of OTUs of gut microbiota.Theβ-diversity combined with the histogram analysis further showed that the composition of gut microbiota was significantly different between the antibiotic treated group and the untreated group.The relative abundance of Firmicutes and Bacteroidetes decreased significantly in the antibiotic treated group,while the relative abundance of Proteobacteria increased significantly.Exploring the susceptibility to BVDV showed that viral loads in blood and duodenum of mice with gut microbiota disturbance were significantly higher than that of mice with normal microbiota.It was also showed that t The disturbance of gut microbiota increased the expression of BVDV E0 protein in duodenum of mice,and aggravated duodenal pathological damage were detected by Western blot and histopathological analysis.However,FMT treatment significantly reduced viral loads and improved duodenal pathological damage.The above results indicated that the antibiotic induced the mouse model of gut microbiota disturbance was successfully constructed.On the basis of this model,it was further confirmed that the disturbance of gut microbiota increased the susceptibility to BVDV,and FMT supplementation showed the effect of inhibiting BVDV infection.These studies lay a foundation for developing probiotics for the prevention and control of BVDV infection and the screening of antiviral drugs.