唾液乳酸杆菌B1对仔猪肠道A型产气荚膜梭菌增殖及毒力基因表达调控的研究

A型产气荚膜梭菌（CpA）广泛存在于自然环境和人畜肠道，有时能引起人类和动物疾病。外毒素是其致病的重要因子，其中β2毒素与新生仔猪坏死性肠炎（NEC）密切相关，然而，健康仔猪肠道也能分离到β2毒素阳性的CpA（CpA-cpb2+）。CpA致病机制目前还不明了。笔者在前期研究中已发现乳酸杆菌能预防NEC的发生，机理尚不明确。本项目拟从临床分离筛选CpA-cpb2+菌株，采用仔猪回肠结扎法和建立活体感染模型，通过生物发光图像（BLI）技术实时监测活体内乳酸杆菌对荧光素酶嵌合的CpA-cpb2+增殖影响；同时结合qRT-PCR和Western-blot技术检测β2毒素的表达，以揭示乳酸杆菌调控CpA机理；同时，采用细菌16SrDNA宏基因组高通量测序技术，研究携带CpA-cpb2+但临床上表现为NEC或健康状态的仔猪肠道菌群组成差异，解析肠道菌群对仔猪NEC的影响，为生态防御仔猪NEC提供理论基础。

Clostridium perfringenstypeA (CpA) widely exists in natural environment and intestinal tract of human beings and animals. CpA leads to many diseases, especially gastroentiritis. It is generally recognized that exotoxins produced by CpA are critical pathogenic factors of the disease. Of which, the β2 toxin, one of important exotoxins, is closely associated with the necrotizing enterocolitis (NEC)of neonatal piglets. However, the CpA harboring cpb2 gene could also be isolated from healthy piglets, andthe pathogenic mechanisms of CpA are roughly unknown yet. The occurring of NEC caused by cpb2-positive CpA perhaps associates with the micro-ecological environment of intestinal tract of piglets. The author observed that Lactobacillus could prevent the NEC of piglets in pilotstudy. In this study, we attempt to use a field isolated CpA-cpb2+strain as a backbone to construct a chimeric Luciferase-CpA-cpb2+which will be employed for a mouse model establishement by utilizing a Bioluminescence imaging (BLI) technology, and thus the proliferation of CpA-cpb2+ modulated by lactobacillus in intestinal tract of mouse will be investigated. Meanwhile，the expression of β2 gene and toxin are measured by real-time PCR and other conventional tests. In order to elucidatethe constitution and diversitiesof intestinal microbe-flora and hunt new probiotics against NEC, 16SrDNA-based metagenomics and other state-of the-art technologies will be used to analyse the difference in composition of gut flora between diseased and healthy piglets carrying CpA-cpb2+. The expected findings achieved from this study will provide insightinto the effects of Lactobacillus salivarius B1 on the proliferation and expression of virulence genes of CpA-cpb2+, the theoretical foundation for ecological prevention of NEC of piglets.