基于16S rRNA测序分析PRRSV感染仔猪肺和肠道中微生物菌群的变化

Changes of PRRSV Infection on Microorganisms in Lungs and Intestines of Piglets based on 16S rRNA Sequencing

本试验旨在探究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)感染对仔猪肺、肠道中的菌群影响以及肺和肠道的组织学变化。试验选取35日龄断奶健康仔猪14头,适应性饲养7 d,随机分为感染组(n=7)和对照组(n=7),感染组仔猪接种2 mL 1×10^(5) TCID 50·mL^(-1) PRRSV-JTS毒株病毒液,对照组仔猪接种2 mL DMEM培养基。感染组3头仔猪分别于10、12和19 d死亡,将存活的感染组与对照组仔猪于21 d后处死,并取肺、不同肠段组织样品以及肠道内容物。采用免疫组化染色和HE染色观察肺和肠道组织病理学变化,并基于16S rRNA基因的高通量测序分析仔猪肺和各肠段菌群结构。结果表明,感染组仔猪肺部和肠道均有病毒分布;与对照组仔猪相比,感染组仔猪肺部和肠道均存在明显的炎症反应。微生物组成结构及多样性分析显示,感染组仔猪Chao1、ACE指数在肺中上升,在仔猪各肠段中下降,Shannon、Simpon指数在肺中升高,在大多数肠道中下降。在门水平上,感染仔猪肠道变形菌门等有害微生物比例增加,而厚壁菌门等有益微生物比例下降;在科水平上,感染组肺、空肠中巴斯德菌科,十二指肠乳杆菌科,回肠肠杆菌科,盲肠、结肠、直肠中的瘤胃菌科相对比例有明显的下降,假单胞菌科在肺和十二指肠中的比例明显上升;在属水平上,感染组有益菌属乳酸菌属水平显著下降,β多样性证明小肠与大肠聚类效果具有一致性。综上,PRRSV致仔猪肺部病变和肠道炎症,影响肺部和肠道菌群组成、丰度和功能。

The purpose of this study was to investigate the effect of porcine reproductive and respiratory syndrome virus infection on the flora in the lungs and intestines of piglets and the histological changes in the lungs and intestines.Fourteen weaned healthy piglets of 35 day old were randomly divided into infection group(n=7)and control group(n=7)after 7 days of adaptive feeding.The piglets in the infection group were inoculated with 2 mL 1×10^(5) TCID 50·mL^(-1) PRRSV JTS virus solution,and the piglets in the control group were inoculated with 2 mL DMEM.Three piglets in the infection group died on 10,12,and 19 days,respectively.The surviving piglets in the experimental group and the control group were euthanized on 21 days,and lung,intestinal tissue samples and intestinal contents were collected.Immunohistochemical staining and HE staining were used to observe the histopathological changes of lung and intestine,and high-throughput sequencing based on 16S rRNA Gene was used to analyze the flora structure of piglet lung and intestine.The results showed that the virus was distributed in the lungs and intestines of piglets in the infection group;Compared with the control group,there were significant inflammatory reactions in the lungs and intestines of the infected group.The analysis of microbial composition,structure and diversity showed that the indexes of Chao1 and ACE of piglets in the infection group increased in the lung and decreased in the intestine,while the indexes of Shannon and Simpon increased in the lung and decreased in most intestines.At the phylum level,the proportion of harmful microorganisms such as Proteus increased,while the proportion of beneficial microorganisms such as Firmicutes decreased;In the infection group,the relative proportion of pasteurebacteriaceae,duodenal lactobacilliaceae,ileal Enterobacteriaceae,cecum,colon and rectum decreased significantly,and the proportion of Pseudomonas in lung and duodenum increased significantly;In terms of genus level,the level of beneficial bacteria and lactic acid bacteria in the infection group decreased significantly,theβdiversity proves that the clustering effect of small intestine and large intestine is consistent.It can be seen that PRRSV can cause lung lesions and intestinal inflammation in piglets,and affect the composition,abundance and function of lung and intestinal flora.