新生仔猪肠道菌群的形成及其对CpA菌β2毒素作用影响的研究

Healthy and mature intestinal flora plays an important role in preventing the diseases of newborn piglets. However, it is not clear which factors affect the formation of intestinal flora and what the healthy intestinal standard of piglets is. The microecology of human mother and infant is a hot research area at present, and newborn piglets are usually used as biomedical models for the study of intestinal flora of infants. In our previous study, a preliminary result on healthy intestinal Biomarker has been established basing on the analysis of intestinal flora of healthy and diarrhea newborn piglets in Jiangxi Province. Meanwhile, it is demonstrated that Lactobacillus salivarius had a negative effect on the expression of β 2 toxin gene of Clostridium perfringens type A (CpA) . This study intends to further explore the intestinal microflora formation process of newborn piglets affected by the microbiota present in the mothers’ gut, vagina, oral cavity, and mammary areola, and its effects on β 2 toxin of CpA in piglets. The probiotics will be screened from the vaginal or intestinal dominant flora of sows, using a method of internal transcribed spacer (ITS) sequencing and the strain level diversity. In order to creat different intestinal flora of newborn piglets, maternal vaginal and intestinal probiotics were inoculated before and after their birth. The quantum of CpA and their β2 toxin were detected to clarify the pathogenic mechanism of β2 toxin carrying by CpA in piglets and optmize a Biomarker for the healthy intestinal microflora of newborn piglets in Jiangxi.

健康成熟的肠道菌群对预防新生仔猪疾病有重要作用，而影响仔猪肠道菌群形成的因素和判断仔猪健康肠道的依据并不清楚。人类母婴微生态是当前研究热点，且新生仔猪通常作为婴儿肠道菌群研究的生物医学模型。本课题组在前期研究中，通过对江西地区健康和腹泻新生仔猪肠道菌群差异分析，初步建立新生仔猪健康肠道生物标记（Biomarker），并发现唾液乳杆菌对A型产气荚膜梭菌（CpA) β2毒素基因表达有负调节作用。本研究拟进一步探讨母猪阴道、肠道、口腔和乳头菌群等对仔猪肠道菌群形成过程中的影响及其对CpAβ2毒素作用，采用内转录间隔区（ITS）测序和种下多样性分型对母猪肠道、阴道优势菌属进行测序，依据测序分析结果针对性地筛选、分离母源益生菌株。在仔猪出生前、后分别以母源益生菌干扰形成不同的新生仔猪肠道菌群。测定不同菌群组成下CpA菌数和β2毒素，揭示CpAβ2致病机制和完善江西地区新生仔猪健康肠道Biomarker。

本项目对江西地区健康和腹泻新生仔猪肠道菌群和代谢组学差异分析，研究了新生仔猪健康肠道菌群与差异代谢产物，以及腹泻仔猪致病共生菌谱，探讨母猪阴道、肠道菌群等对仔猪肠道菌群形成过程中的影响及其对CpAβ2毒素作用，依据测序分析结果针对性地筛选、分离母源益生菌株，研究其有效代谢产物。结果显示：健康仔猪肠道差异代谢产物关联到的菌群主要为Lactobacillus, Clostridium sensu stricto_1和肠球菌，依据结果在健康仔猪肠道分离筛选到了唾液乳酸杆菌H1,并对其有效代谢产物进行了研究，QE 鉴定获取活性物质的氨基酸序列信息，经过与 H1 全基因组测序结果进行匹配，初步鉴定该物质为 50S 核糖体 L35 蛋白。同时研究了产气荚膜梭菌Beta2毒素作用机制及建立了beta2毒素多克隆抗体ELISA和电化学免疫检测方法。揭示肠道益生菌对CpAβ2致病调节机制。完成并发表了8篇学术论文，其中2篇SCI，培养了7位硕士研究生，申请了3项发明专利，获得了1项发明专利授权。