【作者】 任继平；

【导师】 王九峰；

【作者基本信息】 中国农业大学 ， 临床兽医学， 2014， 博士

【摘要】 目的：益生菌芽孢杆菌已被用于预防断奶仔猪腹泻,但芽孢杆菌调节宿主肠道健康机制尚不明确,尤其是在我国逐渐推广的大肠杆菌F4受体阴性猪上。适度的机体免疫应答及稳定的肠道微生物菌群能抵御致病菌感染。本研究的目的是通过比较不同剂量枯草芽孢杆菌(Bacillus subtilis)与地衣芽孢杆菌(Bacillus licheniformis)混合制剂对肠道菌群多样性影响及其免疫调控效应,探究芽孢杆菌制剂对受体阴性断奶仔猪F4+产肠毒素大肠杆菌(F4+Enterotoxigenic Escherichia coli,F4+ETEC)感染的预防机制。方法：32头21日龄断奶F4受体阴性仔猪随机分为4组,每组8头：1)CONT：第1-8天每天连续灌服10mL无菌生理盐水；2)ETEC：第一周每天连续灌服10mL无菌生理盐水,第8天灌服10mL浓度为10×109CFU/mL的F4+ETEC;3)LDBE:第一周连续灌服溶解于10mL生理盐水的低剂量芽孢杆菌制剂(3.9×107CFU/mL),第8天灌服与ETEC组等量F4+ETEC;4)HDBE：第一周连续灌服溶解于10mL生理盐水的高剂量芽孢杆菌制剂(78×107CFU/mL),第8天灌服与ETEC组等量F4+ETEC。第15天,仔猪全部屠宰。记录采食量、体重及直肠温度。通过平板计数和高通量DNA测序方法分析粪样及肠段内容物菌群结构多样性；H&E染色分析空肠及回肠炎症程度；血液样品进行血常规检查,ELISA检测血清中IL-10及TNF-a的浓度。结果：高、低剂量芽孢杆菌制剂均显著抑制F4+ETEC引起的仔猪采食量下降。低剂量芽孢杆菌制剂显著抑制F4+ETEC引起的粗蛋白质和总能消化率的下降,同时显著减缓F4+ETEC引发的发热及空肠肠炎。高、低剂量芽孢杆菌制剂明显抑制F4+ETEC感染后12,48,96及144h引起的杆状核嗜中性粒细胞升高。感染后6h,高剂量芽孢杆菌制剂显著抑制F4+ETEC引起的血清TNF-α浓度升高。低剂量芽孢杆菌制剂在感染后12h显著抑制F4+ETEC引起的血清IL-10浓度下降。相比于F4+ETEC,高剂量芽孢杆菌制剂显著升高第9天粪样中乳酸杆菌及双歧杆菌的数量,而低剂量芽孢杆菌制剂在第12天显著升高粪样中乳酸杆菌的数量。第12天高剂量芽孢杆菌制剂组仔猪粪样肠杆菌的数量显著低于其他三组。高、低剂量芽孢杆菌制剂在12天显著降低粪样中肠球菌的数量。与F4+ETEC相比,高剂量芽孢杆菌制剂显著升高了盲肠、结肠中乳酸杆菌数量,而低剂量芽孢杆菌制剂仅升高结肠中乳酸杆菌的数量。相比于对照组及ETEC组,高、低剂量芽孢杆菌制剂组仔猪在回肠、盲肠及结肠中有更高的双歧杆菌和更低的肠杆菌数量。结论：高、低剂量芽孢杆菌制剂均可以缓解F4+ETEC引起的F4受体阴性仔猪生长性能下降,但仅低剂量芽孢杆菌制剂抑制养分消化率的降低,同时减轻F4+ETEC引起的发热及肠炎。高剂量芽孢杆菌制剂对调节菌群平衡更有效,但影响机体免疫应答,增加了断奶仔猪发生感染性肠炎的风险。更多还原

【Abstract】 Aims:Although the probiotic Bacillus has been used as an promising alternative to protect piglets from post-weaning diarrhea, the mechanism of probiotic in regulating the intestinal health still remains elusive, especially involving with F4receptor-negative weaning piglets. Moderate immune responses and stable gut microbiota community possesses the capacities in defensing the exogenous pathogens-induced infections. The present study aimed at exploring the preventing mechanism of the amalgam of Bacillus subtilis and Bacillus licheniformis in an F4Enterotoxigenic Escherichia coli (F4+ETEC) infection model of F4receptor-negative weaning piglets by comparing the dose effects on gut microbial diversity and immune regulation.Methods:Exact21-day-old weaning F4receptor negative-piglets were randomly divided into four groups and each group is comprised of eight individuals:1) CONT, piglets orally administered with10mL of sterile physiological saline daily during first8days;2) ETEC, pigs orally administered with10mL sterile physiological saline every day for the first week and then orally challenged with10mL F4+ETEC culture (1.0×109CFU/mL) on day8;3) LDBE, pigs orally administered with low dose of Bacillus amalgam dissolved in10mL sterile physiological saline (3.9×107CFU/mL) every day for the first week and then orally challenged with10mL of F4+ETEC culture (1.0R109CFU/mL) on day8; and4) HDBE, pigs orally administered with high dose of Bacillus amalgam dissolved in10mL sterile physiological saline (7.8×10’CFU/mL) every day for the first week and then orally challenged with10mL of F4+ETEC culture (1.0×109CFU/mL) on day8. Piglets were euthanized on day15. Feed intake, body weight and rectal temperature were recorded regularly. The fecal and intestinal contents samples were analysed for microbial diversity by plate counting and high-throughput DNA sequence methods. The jejunal and ileal enteritis were assessed by H&E straining. Blood samples were analysed for routine examination and Serum IL-10and TNF-a concentrations were detected by ELISA.Results:Administration of both low and high dose of Bacillus significantly inhibited the F4+ETEC-induced decrease of daily feed intake. The low dose of Bacillus remarkly attenuated the decline of crude protein and gross energy degestibility, as well as ameliorated the fever and jejunum inflammation induced by F4+ETEC.The surging percentage of banded neutrophils induced by F4+ETEC in12,48,96and144h following challenge were attenuated by both low and high dose of Bacillus. Administration of the high dose of Bacillus remarkly inhibited the elevation of TNF-a concentration induced by F4+ETEC in6h. The IL-10concentrations of piglets administrated with low dose of Bacillus were lower than piglets challenged with F4ETEC in12h. Compared to exposure to F4ETEC, administration of high dose of Bacillus increased the fecal Lactobacillus and Bifidbacteria counts on day9, whereas low dose of Bacillus only multiplied the fecal Lactobacillus number on day12. The fecal Coliformis numbers of piglets administrated with high dose of Bacillus was significantly lower than either of piglets with other treatments. The low and high dose of Bacillus administration lessened the numbers of fecal Enterococci compared to CONT and ETEC treatments on day12. Compared with F4+ETEC challenge, high dose of Bacillus augmented the Lactobacillus numbers in caecum and colon, while low dose of Bacillus boosted the Lactobacillus numbers only in colon. Piglets administrated with low or high dose of Bacillus had higher Bifidbacteria number and lower Coliformis in ileum, caecum and colon than piglets with CONT and ETEC treatments.Conclusions:Both low and high dose of Bacillus ameliorated the decline of growth performance induced by F4+ETEC in F4receptor-negative weaning piglets, but the low dose of Bacillus were more efficient to inhibit the decrease of nutrient digestibility and attenuated F4+ETEC-induced fever and intestinal inflammation. Although high dose of Bacillus was associated with better regulation of microbial balance, it may impose negative effects in immune responses and thus increased the risk of being infected.更多还原