【作者】 王春阳；

【导师】 牛钟相；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2009， 博士

【摘要】 由于畜禽肠道菌群复杂、无菌动物的费用昂贵和高技术要求、限制了乳酸菌等益生菌作用效果及机理的研究,致使乳酸菌等益生菌制剂(微生态制剂)的质量难以控制、合理的开发应用受到了很大的影响。因此,本研究选用肠道结构简单、细胞内部基因调控研究比较深入、在病原菌激活机制、感染过程及免疫反应等诸多方面和哺乳动物存在高度相似性的秀丽隐杆线虫(Caenorhabditis elegans)作为实验动物,以沙门氏菌作为病原指示菌,以乳酸菌为研究益生菌,建立了线虫益生菌实验动物模型,并应用于乳酸菌的研究。本研究首先采用改良线虫液体培养法,成功孵化了秀丽隐杆线虫突变株SS104;在确定了沙门氏菌的最适攻毒量及乳酸菌的最佳保护剂量基础上,建立了秀丽隐杆线虫益生菌动物模型;以线虫的存活率为判定标准,从17株乳酸菌中筛选出了抑菌效果较好的S64株和较差的CL11株菌,并通过对线虫存活率及肠道菌数量检测分析,比较了两株的活菌、灭活菌和培养上清液作用效果的差异;探讨了线虫动物模型筛选的乳酸菌对断奶仔猪和雏鸡生产性能、腹泻率、免疫性能及肠道菌群的影响,从而验证线虫动物模型筛选的乳酸菌对线虫的作用效果与对畜禽作用效果的相似性。秀丽隐杆线虫益生菌动物模型的成功建立为乳酸菌等益生菌的筛选、作用效果及机理研究提供了技术支撑,为微生态制剂的质量控制、开发利用提供了保障。本研究内容共分四部分。试验一秀丽隐杆线虫益生菌动物模型的建立应用改良线虫液体培养法,将同期化的秀丽隐杆线虫(SS104)L4期幼虫孵化、分装于24孔培养板,再将24孔随机分为不同处理组,每组4孔,每孔15-20条线虫;然后以GFP标记沙门氏菌(DT104)作为线虫的致病菌、以乳酸菌(S64株)作为线虫的有益保护菌,探索建立线虫动物模型的条件及效果。结果显示,改良线虫液体培养法操作简单、条件易控制;沙门氏菌浓度在107cfu/ml、108cfu/ml和109cfu/ml均能致死线虫,但以109cfu/ml时致死速度快、致死率最高,于第6d,所有线虫全部死亡(致死率100%);第10d时,浓度为108cfu/ml乳酸菌对线虫存活率达到87.9%,较对照组提高5.1%,而浓度为109cfu/ml的乳酸菌对线虫的存活率仅为63.2%,较对照组下降19.2%;保护实验第9d,攻毒前加入乳酸菌和攻毒后加入乳酸菌对线虫的保护率分别为44.9%和58.1%;说明乳酸菌能够有效保护线虫免受GFP标记沙门氏菌的感染。结果表明,本研究成功建立了秀丽隐杆线虫益生菌动物模型。试验二利用线虫动物模型对乳酸菌的筛选研究首先应用16S rRNA序列分析法对17株乳酸菌株进行了鉴定,在此基础上,以沙门氏菌DT104为病原指示菌,进行了乳酸菌体外抑菌和线虫体内抑菌实验。结果显示,17株乳酸菌株对沙门氏菌的抑菌圈直径在19mm-25mm之间,培养液上清也均能有效抑制沙门氏菌的生长(OD600值在0.058-0.074之间);17株乳酸菌对线虫的保护率为-2.6%-56%不等,其中CL9、L3、LB1、LB4、S64株的保护率分别为59%、53.2%,51.1%、58.7%和55.7%(P﹤0.05),而CL10、CL11、CL12、LB2四株菌株保护率仅为-3.2%、-6.2%,2.2%和2.9%(P﹤0.01)。该研究结果表明,秀丽隐杆线虫动物模型对乳酸菌的筛选效果优于体外抑菌实验筛选法。试验三线虫益生菌动物模型的应用研究——筛选乳酸菌S64株和CL11株作用效果比较研究通过对线虫存活率和肠道菌数的检测,对线虫动物模型筛选的有效乳酸菌株S64和无效菌株CL11的作用效果进行了比较试验,结果显示:(1)S64乳酸菌株组和CL11株组线虫存活率分别为72.2%和25.7%,对线虫的保护率分别为51.7%和2.2%,差异显著(P﹤0.05);灭活S64乳酸菌株组和CL11株组线虫存活率分别为57.4%和25.8%,对线虫的保护率分别为33.9和2.3%,差异显著(P﹤0.05); S64和CL11株乳酸菌培养上清液组线虫存活率分别为36.7%和35.8%,对线虫的保护率分别为13.2%和12.3%,差异不显著(P>0.05)。(2)S64株活菌组线虫肠道内乳酸菌数高于活菌CL11株组,线虫肠道内沙门氏菌数低于活菌CL11株组和对照组,差异显著(P﹤0.05);灭活菌S64株组线虫肠道内沙门氏菌数低于灭活菌CL11株组和对照组,差异显著(P﹤0.05);S64和CL11株培养上清液组线虫肠道内沙门氏菌数均与对照组差异不显著(P>0.05);虫体荧光结果显示,S64株乳酸菌活菌和灭活菌均可有效抑制沙门氏菌在线虫肠道内的生长。试验四线虫益生菌动物模型的应用研究——筛选乳酸菌对畜禽作用效果的研究为探讨线虫动物模型筛选的乳酸菌对畜禽作用的有效性,本研究以断奶仔猪和雏鸡作为实验动物进行了饲喂实验。(1)选用大三元断奶仔猪96头,随机分为8组,分别饲喂乳酸菌S64、LB1、LB2、ALB2、L3和CL11株发酵液体饲料,对照1组饲喂基础颗粒料,对照2组饲喂湿拌料,实验至21d经检测料肉比、腹泻率和肠道菌数,结果显示,乳酸菌ALB2、S64、LB1和L3株发酵饲料组料肉比较对照2组分别下降了49%、47%、36%和24% (P﹤0.05),腹泻指数较对照2组分别下降了50.89%、64.29%、93.51%和32.5%(P﹤0.05),腹泻率较对照2组分别下降了50.01%、81.82%、63.65%和23.72%;CL11和LB2株组料肉比分别提高了4%和12%(P<0.05),腹泻指数分别提高了15%和27.27%(P<0.05),腹泻率分别提高了21.78%和29.95%(P<0.05);对照1组较对照2组料肉比、腹泻指数和腹泻率分别下降了15%、7.14%和16.63%(P>0.05);LB1株发酵组较对照组和LB2发酵组肠道乳酸菌数量显著增加( P < 0. 05),肠杆菌数量显著减少( P < 0. 05)。(2)选用200羽1日龄海兰褐蛋公鸡,随机分成4组,第1、2、3组分别为乳酸菌S64、L3和CL11株饮水组,第4组为空白对照组,对各组鸡抗体效价、免疫器官指数和肠道菌群数进行测定,结果显示,35日龄时,第1、2组血凝抑制效价显著高于对照组和第3组(P﹤0.05),第3组较对照组差异不显著(P>0.05);第1、2组鸡肠道乳酸菌数量较第3组和对照组增加显著(P﹤0.05),肠杆菌数量显著减少(P﹤0.05);第1组胸腺、法氏囊和脾脏指数均显著高于对照组(P﹤0.05),第3组较对照组差异不显著(P>0.05)。ERIC-PCR结果显示,S64株组肠道菌群指纹条带较L3株和CL11株组明显增多,表明S64株更能有效调整鸡肠道菌群平衡。本试验结果显示,线虫动物模型筛选的有效乳酸菌(S64和LB1株)能够明显提高断奶仔猪的生产性能和雏鸡的免疫等功能,表明线虫动物模型筛选的乳酸菌对线虫的作用效果与对畜禽作用效果是相似的。更多还原

【Abstract】 The study concerning effection and mechanism of Lactobacillus has been restricted by complicated intestinal microflora of mammal animals and expensive cost on germ-free animals. Caenorhabditis elegans has been used as a laboratory animal model for the fact that there is a broad overlap between mammals and C. elegans about infection mechanism, immune reaction and so on. In this study, Salmonella used for pathogen and Lactobacillus used for probiotic. Caenorhabditis elegans has been used for estabilished a laboratory animal model for Conduct research on probiotic.The C.elegans strain SS104 has been hatched firstly by fluild incubation method, and proper dose of S. typhimurium and Llactobacillus has been confirmed. We have evaluated 17 Lactobacillus isolates from chicken and pig intestine for their ability to protect the worms from Salmonella-induced death in vitro; When tested on C. elegans infected with S. typhimurium DT104, the isolates were, however, differentiated in their ability to protect the nematode from Salmonella-induced death. Two Lactobacillus isolates S64 and CL11 （S64 with full, but CL11 with no protection） were further studied. The study on difference culture fractions （the extracellular culture fluid, live and heat-killed cells） of isolates CL11 and S64 has been designed; We observed the effects of Lactobacillus isolates on performance , diarrhea incidence, immune activities and intestinal microflora of wean piglets and layer chicken in order to evaluate similarity between livestock and C.elegans.The study consists of four parts:1 Establishment of the assay with C. elegans used as an animal moder for study probioticsSynchronizated L4 larvae has been hatched, resuspension in 24-well titre plates with each well containing 2 ml of S medium and 10– 15 worms, and incubated at 25°C during the assay. Each treatment had four wells. To explore the experimental condition of C.elegans used as animal model for evaluate the effect of a Lactobacillus isolate on protecting the nematode from Salmonella-induced death. The result showed : the fluid incubation method of C.elegans was more easy to operate; C.elegans was infected and died with S. typhimurium at 10⁷cfu/ml、10⁸cfu/ml and 10⁹cfu/ml, however, all the worms died in 6^th days at 10⁹cfu/ml;The survival rate （87.9%） of Lactobacillus-treated worms at 108cfu/ml was higher than that of C. elegans fed E. coli OP50 （82.8%） only on 10^th day, but survival rate of C.elegans was 63.2% at 10⁹cfu/ml; The protective rate of Lactobacillus for C.elegans was 50.3% . meanwhile, the green fluorescence was emitted by the GFP- Salmonella of Lactobacillus-treated worms was more diminish than positive control in the digestive tract of the worms.2 Presreening of Lactobacillus isolates using a laboratory animal model of Caenorhabditis elegans17 Lactobacillus isolates has been confirmed by 16S rRNA analysis method. The spot-on-the-lawn assay demonstrated that all 17 tested Lactobacillus isolates were inhibitive to S. typhimurium . The size of growth inhibition haloes ranged from 19 to 25 mm in diameter and the extracellular culture fluid （ECF） recovered from tested Lactobacillus cultures all had a similar degree of inhibition towards S. typhimurium; When tested on C. elegans infected with S. typhimurium, the isolates were, however, differentiated in their ability to protect the nematode from Salmonella-induced death. On the last day (9^th) of the assays, the protective rate of Lactobacillus ranged from -2.6% to -56%. Among them, five isolates （CL9, L3, LB1, LB4, and S64） provided maximum protection（59%、53.2%,51.1%、58.7% and 55.7% respectively ） （P﹤0.05） to the infected worms that had a similar level of longevity compared to the negative control. The protective rate of two isolates CL10 and CL11 was -3.2% and -6.2%（P﹤0.01） and demonstrated negative effective on C.elegans . Our observations suggest that C. elegans can be used as an animal model for pre-screening probiotics.3 Application research on Caenorhabditis elegans as an animal model for study probaotics——Effection of Lactobacillus isolates S64 and CL11 screened by C.elegans To explore possible mechanisms underlying the protection offered by Lactobacillus different culture fractions, including the ECF, live and heat-killed cells, of Lactobacillus isolate （S64 and CL11） were investigated for their effect on the survival rate C. elegans infected with Salmonella and the colonization of the pathogen in the digestive tract of the nematode. The result demonstrated: （1）On the 9th day in the assay, the survival rate of C.elegans treated with lactobacillus isolate S64 and CL11 live cells was 72.2% and 25.7%（P<0.05）, the protetive rate was 51.7% and 2.2% respectively; The survival rate of C.elegans treated with lactobacillus isolate S64 and CL11 heat-killed cells was 57.4% and 25.8%（P<0.05）,the protetive rate was 33.9% and 2.3% respectively; The survival rate of C.elegans treated with ECF of S64 and CL11 was 36.7% and 35.8%（P>0.05）,the protetive rate was 13.2% and 12.3% respectively. （2） On the 8^th day in the assay , the amount of Lactobacillus in the digestive tract treated with live Lactobacillus isolate S64 was increase distinctly compare to that of treated with live isolate CL11（P<0.05）; The amount of Salmonella treated with live and heat-killed isolate S64 was significantly reduced than that of treated with isolate CL11 and positive control（P<0.05）; The amount of Salmonella treated with ECF of isolate S64 and CL11 was at similar degree（P>0.05）; Meanwhile, the green fluorescence was emitted by the GFP-salmonella of live isolate S64-treated worms was significantly diminished than positive control and isolate CL11-treated worms.4 Application research on Caenorhabditis elegans as an animal model for study probaotics——Effection of Lactobacillus isolates screened by C.elegans on livestockTo explore the effction of effection of Lactobacillus isolates screened by C.elegans on livestock , the feeding Trial had beedn designed using wean piglet and chicken as animal modle.（1） 96 weaned piglets were divided into 8 groups randomly and fed them with ferment liquid feed （FLF） which fermented by Lactobacillus isolates S64、LB1、LB2、ALB2、L3、CL11、dry feed （control 1） and liquid feed （control 2, no lactobacillus） respectively. The result showed: The ratio of feed to meat was reduced 49%、47%、36% and 24%（P<0.05）, diarrhea index was reduced 50.89%、64.29%、93.51% and 32.5%（P<0.05）,Diarrhea incidence was dropped by 50.01%、81.82%、63.65% and 23.72% （P<0.05）, when fed them with FLF fermented by Lactobacillus isolates ALB2、S64、LB1 and L3 respectively; The ratio of feed to meat was increased 4% and12% （P < 0.05）,diarrhea index was increased 15% and 27.27%（P<0.05）,diarrhea incidence increased 21.78% and 29.95%（P<0.05）, when fed them with FLF fermented by Lactobacillus isolates CL11 and LB2 respectively; Lactobacillus isolates LB1 was beneficial to the balance of gut flora than Lactobacillus isolates LB2.（2）A total of 200 newly hatched Layer Chicken were randomly allotted into 4 dietary treatments fed them with Lactobacillus isolates S64, L3 and CL11. On 35d in the assay, the hemagglutination inhibition antibody potency of Newcastle disease vaccine immunized of Lactobaccilus isolates S64 and L3 increased distinctly compare to negative control and isolate CL11（P<0.05）; The amounts of Lactobaccilus in the Cecal contents significantly increased （P<0.05）, and the amounts of E.coli and and Salmonella significantly reduced （P<0.05） when fed them with Lactobacillus isolate S64 and L3. Immune organ index of layer chicken when fed them with Lactobacillus isolate S64 improved significantly（P<0.05）. The data of the treatment fed with Lactobacillus isolate CL11 was similar to negative control（P>0.05）.The intestinal flora of the chickens fed Lactobacillus isolates S64 was changed clearly using ERIC-PCR based fingerprintsThese data demonstrated Lactobacillus isolates S64 and LB1 which screened by C.elegans was beneficial to production performance of wean piglets and to immunologic function of chickens. The result showed that the effection of Lactobacillus is similar between C.elegans and livestocks, and C.elegans can be used for study probiotic as a laboratory animal model.更多还原