【作者】 张锦华；

【导师】 杨倩；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2011， 博士

【摘要】 本试验从健康仔猪十二指肠黏膜分离筛选到一株唾液乳酸杆菌(L. sarlivariusB1),体外试验结果表明L. sarlivarius B1产酸能力及与上皮细胞粘附能力均较强。采用新生仔猪为对象,研究L. sarlivarius B1对仔猪黏膜免疫的影响及其作用机制,并以大肠杆菌E. coli k88为指示菌感染仔猪,进一步研究L. sarlivarius B1抵抗大肠杆菌E. coli k88感染的影响。1.猪源乳酸杆菌的分离与鉴定首先从四种不同品种的30日龄仔猪(长白猪、山东莱芜黑猪、杜洛克猪、贵州巴马小香猪)十二指肠黏膜分离到36株乳酸杆菌,然后对36株乳酸杆菌体外产乳酸、在Caco-2细胞表面的粘附力以及抗生素敏感性进行测定。从中筛选出10株产酸能力和粘附力强的的乳酸杆菌,最后通过PCR-DGGE图谱技术对10株纯菌分离株和四个品种猪的十二指肠黏膜乳酸杆菌进行对比,分析所分离的乳酸杆菌在仔猪十二指肠黏膜分布情况,最后选取其中的4株菌进行16S rRNA分子鉴定。结果表明：36株乳酸杆菌产酸性能力和粘附力存在一定的差别,10株产酸能力强的乳酸杆菌与上皮细胞的粘附能力均比参考菌株L.brevis1.2028和L. acidophilus(ATCC4356)强,16S rRNA基因序列分析显示,所测4株乳酸杆菌均与唾液乳酸杆唾液亚种(Lactobacillus salivarius subsp. salivarius)同源性高达98%,部分序列已被Genbank收录,登录号为GQ303717和GQ303718。选取产酸能力与粘附力最强的L. sarlivarius B1为益生菌候选菌株。2. L. salivarius B1对仔猪小肠发育的影响应用筛选出的L. sarlivarius B1饲喂新生仔猪,研究L. sarlivarius B1对仔猪生产性能和小肠发育的影响,试验选择6窝初生重、胎次相近的新生仔猪为研究对象,分成乳酸杆菌组、对照组和商品日粮组。乳酸杆菌组分别于仔猪0、7、11、26日龄灌喂L. salivarius B1(菌液离心后用蔗糖溶液调至活菌数为5×109cfu/m1),对照组则饲喂同体积的蔗糖溶液,乳酸杆菌组和对照组仔猪均从7日龄开始饲喂无抗开食料,商品日粮组则饲喂商品仔猪料。28日龄称重后每窝随机选择2头仔猪宰杀,随后取小肠固定,制作组织切片、染色。测定肠绒毛高度和肠道上皮内淋巴细胞和IgA分泌细胞数量。试验结果显示：L. salivarius B1能够显著地提高仔猪小肠IEL数量(p<0.05)和IgA分泌细胞的面积(p<0.01)；L. salivarius B1还能提高仔猪日增重(p<0.05)和小肠绒毛高度(p<0.05)3. L. salivarius B1在仔猪肠道的定植情况及其对肠道菌群的影响L. salivarius B1饲喂新生仔猪后能够促进小肠发育和增加肠道免疫细胞数量。为进一步研究L. salivarius B1在体内的作用机制,本试验对L. salivarius B1饲喂后在仔猪体内的定植及对肠道菌群组成的影响进行了研究。给新生仔猪饲喂L. salivarius B1后分别于7、14、21日龄采取仔猪新鲜粪便,28日龄每窝随机挑选2头(每组4头)仔猪宰杀,取肠黏膜。采用DGGE方法分析L. salivarius B1在体内的定植情况,利用Real-time PCR技术对肠黏膜中L. salivarius数量进行分析；用传统的活菌计数方法对几种主要的肠道菌计数。结果表明：L. salivarius能够在仔猪哺乳后期自然出现；DGGE图谱显示饲喂L. salivarius B1的7日龄仔猪粪便中出现该菌株相应的条带；在饲喂L. salivarius B1的28日龄仔猪肠黏膜中,L. salivarius,总数显著高于其它组(p<0.05)；7日龄的仔猪粪便和28日龄仔猪肠黏膜中双歧杆菌数量为乳酸杆菌组明显高于对照组(p<0.05)；但对大肠杆菌和肠球菌等肠道菌数量没有明显的影响。本试验结果说明L. salivarius B1饲喂新生仔猪后能够有效地定植于仔猪体内,并且能够增加哺乳前期仔猪粪便和十二指肠局部黏膜双歧杆菌的数量。4. L. salivarius B1早期定植对仔猪肠道β-防御素2和细胞因子分泌的影响新生仔猪肠道细胞能够分泌抗菌肽参与天然免疫反应,β-防御素(pBD-2)是抗菌肽中重要的一种,在仔猪先天性免疫中起着非常重要的作用。给新生仔猪饲喂L. salivarius B1后于7、14、21、28日龄采取仔猪唾液,28日龄每窝随机挑选2头(每组4头)仔猪宰杀取黏膜。应用Real-time PCR方法检测肠黏膜中pBD-2及细胞因子IL-1p、TNF-α和IL-6基因的表达水平,用ELISA方法对不同日龄仔猪唾液中的pBD-2含量进行测定。结果显示：L. salivarius B1早期定植能使十二指肠黏膜pBD-2基因表达水平及含量显著升高(p<0.01)；仔猪唾液中pBD-2的含量随日龄有不同程度的增加,其中在14,28日龄时乳酸杆菌组仔猪唾液中的pBD-2含量极显著增加(p<0.05)。L. salivarius B1早期定植还对回肠IL-6基因表达有明显的促进作用(p<0.05)。本试验的结果表明L. salivarius B1早期定植能够促进肠黏膜细胞功能基因的表达,提高小肠局部黏膜免疫的防御水平。5. L. salivarius B1早期定植对肠道TLR表达的影响Toll样受体(TLRs)是一类主要表达于天然免疫细胞表面的模式识别受体,通过识别与结合相应的配体启动宿主天然抗感染免疫。目前,研究发现哺乳动物体内存在12种TLRs, TLR2主要对革兰氏阳性细菌细胞壁成分进行识别,而TLR9主要识别细菌DNA中非甲基化的CpG基序。本试验主要从基因和蛋白水平研究L.salivariusB1对仔猪小肠TLR2和TLR9表达水平的影响。给新生仔猪饲喂L. salivarius B1后,28日龄每组随机挑选4头仔猪宰杀取肠黏膜,分别应用Real-time PCR方法和Western-Blot方法测定十二指肠和回肠中TLR2和TLR9的基因表达水平和蛋白表达水平。试验结果显示：L.salivariusB1能够显著地增强十二指肠和回肠中的TLR2的基因和蛋白表达(p<0.05),而对TLR9表达基因和蛋白水平均无明显的影响。6. L. salivarius B1对仔猪抵抗E. coli K88感染的影响为进一步探导L. salivarius B1定植后对肠道病原菌的抗感染作用,本试验研究了L. salivarius B1对仔猪抵抗E. coli K88感染的影响。对前期试验中三组仔猪宰杀后余下的仔猪28日龄断奶后的腹泻情况观察一周,35日龄时所有仔猪接种相同剂量的大肠杆菌菌液(E. coli K88,活菌数为108cfu/ml,接种量为2 mL/只),观察仔猪对E. coli K88感染的抵抗能力,并对仔猪肠黏膜受损情况及细胞因子IL-6、TNF-a和IL-1p基因表达进行测定。结果表明：早期接种L. salivarius B1的仔猪腹泻指数明显低于对照组(p<0.05)且康复较快；肠黏膜的IL-61比未接种L.salivarius B1的仔猪高(p<0.05),而炎性因子TNF-α则较低(p<0.05)；回肠肠绒毛受损程度也较轻。说明早期L. salivarius B1勺定植提高了仔猪天然免疫功能,增强仔猪断奶期对病原菌感染的抵抗力,有助于仔猪健康生长。更多还原

【Abstract】 In this study, L. sarlivarius B1 was isolated from the intestinal mucosa of a healthy piglet. It was confirmed to possess excellent probiotic properties, such as high lactic acid production and strong adhesion to Caco-2 cells in vitro. The neonatal piglet was used to investigate the effect on functions of mucosal immune and the mechanism of L. sarlivarius B1 in piglets. Furthermore, E. coli k88 infection model was used to study the resistance of L. sarlivarius B1 on infection of intestinal pathogen.1. Isolation and indentification of dominant porcine lactobacillusSome probiotic lactobacilli are known to confer health benefits to host by modulating its immune functions. It has a close relationship between the probiotics properties and its adhesion on the surface of cell. In this study,36 lactobacillus strains was isolated from duodenum mucosa of 30 day-old piglets from four different pig breeds (Landrace piglets, Laiwu black piglets, Duroc piglets and mini piglets of Guizhou).10 strains were selected from 36 lactobacillus strains basing on their lactic acid production, adhesion properties on Caco-2 cell and antimicrobial susceptibility in vitro. The bands of 10 strains and mucosal bacteria from four pig breeds in DGGE profiles were compared. Four lactobacillus strains were molecular indentified by 16S rRNA technology. The results showed that L. salivarius was the predominant group in the duodenum of piglets, so it is a potential candidate probiotic for piglets. The lactic acid production was different among L. sarlivarius strains. L. sarlivarius B1 and L. sarlivarius XI showed strong ability in lactic acid production and adhesion. Analysis of 16S rRNA gene showed that the strains were all related closely to Lactobacillus salivarius subsp. salivarius (similarity> 98%). Some of the gene sequences have been deposited in the GenBank database under accession numbers:GQ303717 and GQ303718. 2. Effect of L. sarlivarius B1 on development of small intestine of pigletsThis study was to investigate the effects of L. sarlivarius B1 on the performance and development of small intestine in piglets. Six litter neonatal piglets, which average birth, weigh and parity were similar, were divided into three groups: experimental groups, control and commercial diet groups. Each piglet in the experimental group received 1 ml of L. salivarius B1 preparation (diluted to 5×109 cfu/ml-1 with sucrose solution) orally on day 0,2 ml on day 7,3 ml on day 11 and 5 ml on day 26. Each piglet in the control group received equivalent volumes of sucrose solution. The piglets in experimental and control groups were fed diet without carrying antibacterial, commercial diet group was fed commercial diet. On day 28, the litter weight of each litter was recorded, and then two piglets from each litter were sacrificed. The duodenum, jejunum and ileum segments were collected and placed in Bouin’s fixative, then histological examination for analyzing villous height, the number of IELs and IgA producing cells. Results show that the daily weight gain and villous height of piglets was increased by L. sarlivarius B1 (p<0.05). The number of IELs were also significantly increased (P<0.05), and similar results were observed in the area of the IgA-producing cells (P<0.05). The results demonstrate that L. salivarius B1 is beneficial for the performance of piglets and development of small intestine.3. The colonization of L. salivarius B1 on intestinal tract and its effect on the intestinal microfloraL. salivarius B1 has showed the probiotics properties in newborn piglets, improving the performance and intestinal morphology. A further study on the mechanism of L. salivarius B1 in vivo was performed. The aim of this study was to investigate the colonization of L. salivarius B1 in piglets and its effect on the composition of the intestinal flora. The fresh feces of four piglets from each group were collected on day 7,14, and 21. On day 28, two piglets per litter (four piglets per group) were selected randomly and sacrificed, the mucosa of duodenum, jejunum and ileum were collected. DGGE was used to check the colonization of L. salivarius B1 in vivo; the number of L. salivarius in mucosa samples was detected by Real-time PCR. Identification and enumeration of intestinal microorganisms was used by traditional method. The results showed that:L. salivarius occurs naturally at late lactation of piglets. The faces of 7 day-old piglets in experimental groups showed the band of L. salivarius in DGGE profile, but no band appeared in the other groups; the number of L. salivarius of mucosa samples in experimental groups was significant higher than that in control group(p<0.05). For the number of three major intestinal bacteria, only the bifidobacterial count in the feces of the piglets on day 7 and intestinal mucosa on day 28 changed, the number in experimental group was higher than control group (p <0.05). The results show that L. salivarius B1 can colonize in the intestinal tract of piglets after administration and increase bifidobacterium count in faces at early breast-feeding and duodenal mucosa on day 28, but no significant effect was observed on the number of E. coli and Enterococcus4. Effect on the secretion ofβ-defensins and cytokine by early colonization of L. salivarius B1An innate immune response of neonatal piglets is the secretion of antimicrobial peptides (AMPs). AMPs known as defensins play an important role in the innate immune response of the intestine. Some investigations have demonstrated that Lactobacillus can induce enterocyte to produceβ-defensin. In this study, the secretion of pBD-2 and cytokine influenced by L. salivarius B1 were studied. On day 7,14,21, and 28, saliva of two piglets from each litter were collected. On day 28, two piglets per litter (four piglets per group) were selected randomly and sacrificed, the mucosa of duodenum and ileum were collected. The secretion of pBD-2 and cytokines (IL-1β, TNF-a and IL-6) were measured. The results showed that the gene expression and contents of pBD-2 in duodenal mucosa was increased significantly by L. salivarius B1 (p<0.01). The pBD-2 levels in saliva were increased in different degree after the mucosa exposure to L. salivarius B1, the pBD-2 level in saliva on day 14,28 in experimental group was significant higher than control group (p<0.05); otherwise, the expressed of IL-6 gene in ileum was promoted by L. salivarius B1. It concluded that the early colonization of L. salivarius B1 can promote the expression of functional genes in intestinal immunocompetent cells, and to improve the defense of intestinal mucosal immune in local.5. Effect on the expression of TLRs in intestine by early colonization of L. salivarius B1L. salivarius is one of resident bacteria in healthy piglets. In theory, L. salivarius B1 was recognized via innate immunity mechanisms when it came into the intestine of piglet which immune systems was immature. TLRs are a group of pattern recognition receptors (PRRs) that expressed on the surface of immunocompetent cells. 12 TLRs have been described in mammals, TLR2 recognizes a variety of components on the cell wall of microbial, TLR9 recognizes bacterial DNA containing CpG motifs. The aim of the present study was to investigate the effects of L. salivarius B1 on the expression of TLR. On day 28, the piglets were sacrificed and their intestinal mucosa samples were immediately collected for extraction of total RNA and protein. The expression of TLR2 and TLR9 gene was detected by the method of Real-time PCR, and the expression of TLR2 and TLR9 protein were determined by Western-Blot. The results showed that the similar results were obtained for the same parts of the sample both using the method of Real-time PCR in gene level and Western-Blot in protein level. L. salivarius B1 can significantly enhance the expression of TLR2 in duodenum and ileum at the gene and protein levels (p<0.05), while no obvious different was observed in the expression of TLR9.6. Effect of L. salivarius B1 on resistance to E. coli challenged pigletsL. salivarius B1 can colonize in intestinal tract of piglets and improve the intestinal innate immune function of piglets. To study the role of anti-infective of L. salivarius B1 to pathogens, diarrhea of piglets in three groups were investigated during a week post-weaning. Piglets was inoculated with E. coli K88 while 35 days old, the diarrhea resistance to infection of E. coli K88 were investigated at weaning. The damage of intestinal mucosa by E. coli K88 and the expression of cytokines IL-6, TNF-a and IL-1βgene were determined. The results showed that the diarrhea index of piglets in experimental group was significant lower than other groups during the stage of weaning (p<0.05). The piglets administration with L. salivarius B1 showed stronger inhibitory on the infection of E. coli K88, i.e. diarrhea index was significantly decreased and piglets faster recovery; the expression of IL-6 gene was higher than that of other two groups (p<0.05), while the inflammatory factor TNF-a was lower than that of control group (p<0.05); damage of intestinal villus was slighter than that of in control group. It can be concluded that early administration of L. salivarius B1 might facilitate health of piglets and promote intestinal natural immune responses and resistance to pathogen infection.更多还原