产肠毒素大肠杆菌K88诱导仔猪炎症反应的分子机制

为探讨产肠毒素大肠杆菌（ETEC） K88感染仔猪发生炎症反应的分子机制,试验用ETEC K88灌服断奶仔猪,ELISA法检测攻毒后仔猪血清中白细胞介素8（IL-8）含量,实时荧光定量PCR方法检测淋巴结中Toll样受体2（TLR2）、Toll样受体4（TLR4）及其信号通路相关基因（髓样分化因子88（MyD88）、Toll相互作用蛋白（Tollip）、B细胞淋巴瘤因子3（Bcl3））的mRNA相对表达水平。结果发现,仔猪攻毒ETEC K88后6和24 h血清IL-8含量和淋巴结TLR2/4的表达水平均极显著或显著高于对照组（P<0.01;P<0.05）,且感染后24 h显著低于感染后6 h（P<0.05）;仔猪感染ETEC K88后24 h淋巴结中MyD88、Tollip和Bcl3的表达水平均极显著高于对照组（P<0.01）,但是感染后6 h时与对照组相比均无显著差异（P>0.05）。综上所述,ETEC K88感染仔猪可能是通过TLR2/4-MyD88信号通路产生炎症因子IL-8,促使仔猪出现炎症反应,且该炎症反应可能受Tollip和Bcl3蛋白的调控而被减弱。     

Early supplementation with Lactobacillus plantarum in liquid diet modulates intestinal innate immunity through toll-like receptor 4-mediated mitogen-activated protein kinase signaling pathways in young piglets challenged with Escherichia coli K88

This study was conducted to investigate the effects of early supplementation during 4 to 18 d of age with Lactobacillus plantarum (LP) in liquid diets on intestinal innate immune response in young piglets infected with enterotoxigenic Escherichia coli (ETEC) K88. Seventy-two barrow piglets at 4 d old were assigned to basal or LP-supplemented liquid diet (5 × 10¹⁰ CFU·kg⁻¹). On day 15, piglets from each group were orally challenged with either ETEC K88 (1 × 10⁸ CFU·kg⁻¹) or the same amount of phosphate-buffered saline. The intestinal mucosa, mesenteric lymph node (MLN), and spleen samples were collected on day 18. Here, we found that LP pretreatment significantly decreased the mRNA relative expression of inflammatory cytokines (interleukin [IL]-1β, IL-8, and tumor necrosis factor-α), porcine β-defensin 2 (pBD-2), and mucins (MUC1 and MUC4) in the jejunal mucosa in piglets challenged with ETEC K88 (P < 0.05). Moreover, LP significantly decreased the ileal mucosa mRNA relative expression of IL-8 and MUC4 in young piglets challenged with ETEC K88 (P < 0.05). Furthermore, the piglets of the LP + ETEC K88 group had lower protein levels of IL-8, secretory immunoglobulin A, pBD-2, and MUC4 in the jejunal mucosa than those challenged with ETEC K88 (P < 0.05). Besides, LP supplementation reduced the percentage of gamma/delta T cells receptor (γδTCR) and CD172a⁺ (SWC3⁺) cells in MLN and the percentage of γδTCR cells in the spleen of young piglets after the ETEC K88 challenge. Supplementation with LP in liquid diets prevented the upregulated protein abundance of toll-like receptor (TLR) 4, phosphorylation-p38, and phosphorylation-extracellular signal-regulated protein kinases in the jejunal mucosa induced by ETEC K88 (P < 0.05). In conclusion, LP supplementation in liquid diet possesses anti-inflammatory activity and modulates the intestinal innate immunity during the early life of young piglets challenged with ETEC K88, which might be attributed to the suppression of TLR4-mediated mitogen-activated protein kinase signaling pathways. Early supplementation with LP in liquid diets regulates the innate immune response, representing a promising immunoregulation strategy for maintaining intestinal health in weaned piglets.     

TLRs介导的信号通路在马链球菌感染RAW264.7细胞中的作用

为探究Toll样受体(TLRs)介导的信号通路在马链球菌马亚种(S.equi)感染小鼠巨噬细胞RAW264.7中的作用,收集S.equi感染后不同时间点的RAW264.7细胞,提取总RNA并反转录成cDNA,利用实时荧光定量PCR技术检测细胞Toll样受体1、2、6(TLR1、TLR2、TLR6)、接头蛋白骨髓分化蛋白88(MyD88)及细胞因子IL-1、IL-6、IL-10、IL-12、TNF-αmRNA的表达情况。结果显示,S.equi感染RAW264.7细胞后6h时,TLR1、TLR2、TLR6与MyD88mRNA水平均较对照组没有显著差异(P>0.05);感染后12h时,TLR1、TLR2和TLR6mRNA表达量未出现明显上升(P>0.05),而MyD88mRNA水平极显著升高(P<0.01);感染后24h时,TLR1、TLR2和TLR6mRNA表达水平出现极显著升高(P<0.01),MyD88mRNA表达没有显著变化(P>0.05),且IL-10和IL-12mRNA水平与对照组相比极显著升高(P<0.01),IL-1、IL-6和TNF-αmRNA水平均极显著下降(P<0.01)。结果表明,TLRs介导的信号通路参与S.equi感染RAW264.7细胞的免疫应答反应。     

马链球菌对树突状细胞Toll样受体和MyD88及细胞因子mRNA表达的影响

为探究马链球菌马亚种(Streptococcus equi subsp.equi,S.equi)对小鼠骨髓源树突状细胞(bone marrow derived dendritic cells,BMDCs)TLR1、TLR2、TLR6、MyD88、IL-1、IL-6、IL-10、IL-12、TNF-αmRNA表达的影响,用GM-CSF、IL-4细胞因子刺激小鼠骨髓细胞使其诱导分化成未成熟的BMDCs,感染S.equi后16、20、26h,采用SYBR GreenⅠ实时荧光定量PCR检测感染组及未感染组细胞TLRs、MyD88和细胞因子mRNA的表达情况。结果显示,S.equi感染BMDCs后16、20、26h,TLR1、TLR2、TLR6、MyD88mRNA的表达量均显著或极显著的高于未感染组(P0.05或P0.01)。S.equi感染DCs后16、26h,IL-1的分泌量较未感染组没有变化(P0.05),IL-10、TNF-α的分泌量显著或极显著增加(P0.05或P0.01)。IL-6在S.equi感染DCs后16h的分泌量显著增加(P0.05),但感染后26h无显著变化(P0.05)。IL-12在S.equi感染DCs后16h的表达没有变化(P0.05),但感染后26h分泌量有所增加(P0.05)。说明S.equi具有调节DCs TLRs、MyD88和细胞因子表达的能力,从而介导机体的免疫应答反应。     

Inflammation-associated responses in piglets induced with post-weaning colibacillosis are influenced by dietary protein level

Forty piglets (average body weight = 5.32 kg) were used to investigate the effect of dietary crude protein (CP) content on immunological responses following a challenge with enterotoxigenic Escherichia coli (ETEC) K88. Pigs, housed 4 per pen, were randomly allotted to 2 diets: 1) a high, 225 g/kg CP diet (HCP) or 2) a low, 176 g/kg CP diet (LCP) supplemented with crystalline amino acids. Pigs were orally challenged with 6 mL of an ETEC K88 suspension containing 10¹⁰ cfu/mL on d 8 after weaning. Blood samples were collected from 10 pigs (1 pig/pen) on d 7 (at weaning), −24 h, 8 h, 72 h and 7 d after the challenge for determination of plasma urea N (PUN) and serum concentrations of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) and haptoglobin (Hp). Tumor necrosis factor alpha, IL-1β and Hp were measured as indicators of inflammatory responses. The concentrations of serum TNF-α at 8 h, 72 h and 7 d after challenge were similar to the level observed at 24 h before challenge but higher (P < 0.05) than the weaning level. Pigs fed the LCP diet had lower (P = 0.032) concentrations of IL-1β (72 vs. 116 pg/mL) at 8 h post-challenge compared with those fed the HCP diet. Likewise, pigs fed the LCP diet tended to have lower (P = 0.088) concentration of Hp (9 vs. 25 mg/dL) compared with those fed the HCP diet at 8 h post-challenge. Compared with the weaning concentration, PUN concentration at 72 h after ETEC challenge was higher (P < 0.05) in pigs fed the HCP diet. The results indicate that the LCP diet supplemented with crystalline amino acids reduced inflammatory responses, as indicated by serum IL-1β, in piglets infected with ETEC K88.     

基于RAGE-TLR4串扰的高糖影响牛肺泡巨噬细胞促炎细胞因子释放的分子机制

本试验旨在研究高浓度葡萄糖对牛肺泡巨噬细胞（BAMs）促炎细胞因子IL-1β、IL-6及TNF-α释放的影响及其机制是否与RAGE-TLR4相关信号通路串扰有关。将BAMs随机分为正常糖组（NG）、高糖组（HG）、高糖+RAGE抑制剂组（H+F）、高糖+TLR4抑制剂组（H+T）及DMSO组,处理12 h后收集上清及下层细胞。采用qRT-PCR和Western blot检测细胞RAGE、TLR4、MyD88、NF-κB p65的mRNA及蛋白表达情况,ELISA检测上清TNF-α、IL-1β、IL-6浓度。结果表明,高糖极显著上调RAGE、TLR4、MyD88和NF-κB p65基因、蛋白表达水平以及上清液中IL-1β、IL-6、TNF-α浓度（P<0.01）;RAGE抑制剂与TLR4抑制剂均极显著抑制高糖引起的RAGE、TLR4、MyD88和NF-κB p65基因、蛋白表达水平上调以及IL-1β、IL-6、TNF-α释放（P<0.01）,即RAGE与TLR4均在激活RAGE/TLR4/MyD88/NF-κB炎症信号通路中发挥调控作用。综上所述,高糖能够通过RAGE-TLR4串扰引起牛肺泡巨噬细胞释放促炎细胞因子IL-1β、IL-6及TNF-α,进一步阐明了高糖促进牛肺泡巨噬细胞炎症反应的分子机制。     

脂多糖刺激对断奶仔猪肌肉炎症和蛋白质降解相关基因表达的影响

本试验旨在研究脂多糖(LPS)刺激后不同时间断奶仔猪肌肉炎症和肌肉蛋白质降解相关基因表达的变化规律。选择42头(7.1±0.9)kg杜×长×大三元杂断奶仔猪,按注射LPS之前(0 h)和注射LPS后1、2、4、8、12、24 h随机分为7个处理,每个处理6头猪。预试14 d后,腹腔注射100μg/kg体重的LPS。按以上时间点将仔猪屠宰,取背最长肌样品待测。结果表明:背最长肌炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6的mRNA表达量在注射LPS 1～2 h后达到峰值;Toll样受体4信号通路关键基因Toll样受体4(TLR4)、骨髓分化因子88(MyD88),核苷酸结合寡聚域信号通路关键基因核苷酸结合寡聚域2(NOD2)、受体互作蛋白激酶2(RIPK2)的mRNA表达量在注射LPS 2～4 h后达到峰值;肌肉蛋白质降解相关基因叉头转录因子-1(FOXO-1)、FOXO-4、肌肉环指蛋白1(MuRF1)、肌萎缩F-box(MAFbx)的mRNA表达量在注射LPS 12 h后达到峰值。可见,LPS刺激诱导肌肉释放大量炎性细胞因子,使TLR4和NOD炎症信号通路关键基因及肌肉蛋白质降解相关基因mRNA显著表达。     

Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets

Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC). Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05). Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal. Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E. coli and the number of bacteria adhering to 250 micron villus brush border was counted. Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection. Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001). The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E. coli.     

日粮添加枯草芽胞杆菌对大肠埃希菌感染仔猪免疫功能的影响

本试验旨在研究饲粮中添加枯草芽胞杆菌对大肠埃希菌K88感染仔猪血清免疫功能的影响。选用平均体重6.8kg±0.5kg的去势"杜×长×大"三元杂交公猪8头,随机分为2组,每头猪单栏饲养,对照组饲喂基础饲粮,试验组饲喂基础饲粮+0.1%枯草芽胞杆菌,每头猪灌服致病性大肠埃希菌K88菌液(1×1011 CFU)。采用ELISA方法检测血清中细胞因子和猪瘟抗体水平。结果表明,采食添加枯草芽胞杆菌的处理组仔猪比对照组仔猪血清IL-10水平提高(P0.01),血清IL-8和TNF-α水平降低(P0.01),猪瘟抗体阻断率提高(P0.05)。结果提示,仔猪采食含枯草芽胞杆菌的饲粮时,能够调节机体免疫功能,缓解致病性大肠埃希菌K88感染造成的炎症反应。研究工作为枯草芽胞杆菌的推广应用提供了试验依据。     

Experimental colibacillosis in gnotobiotic piglets exposed to 3 enterotoxigenic serotypes

Three strains of enterotoxigenic Escherichia coli (ETEC) (064:KSNT, K88ac; 020:KSNT, K88ac and 08:K85ab, K99) originally cultured from outbreaks of diarrhoea in piglets a few hours old, were administered orally to gnotobiotic piglets. There was a marked age-related difference in the clinical response to infection between the 3 strains although they all produced heat-stable toxin. All 3 strains produced severe clinical signs of depression, anorexia, vomiting, diarrhoea, followed by dehydration and death in one-day-old piglets. In piglets infected at 3 days of age the two K88+ ETEC caused diarrhoea and death but the K99+ ETEC induced moderate diarrhoea only. In piglets infected at 7 days of age, the 064 strain produced severe diarrhoea and death, and 020 strain caused mild diarrhoea in 3 of 6 piglets with one death while the 08 strain caused no illness. Pathological changes in the intestinal tract associated with these infections were minimal, or absent. Immunofluorescent staining with homologous hyperimmune sera demonstrated adherence of the 3 ETEC strains to the brush border of small intestinal epithelial cells. Fluorescing organisms were observed in all infected piglets irrespective of the severity of clinical signs but the degree and extent of colonisation varied with the age of the piglets and the infecting strain. This may explain the difference in clinical response between the 3 strains.     

无菌大鼠深二度烫伤感染模型机体炎性反应的研究

为探究无菌大鼠深二度烫伤感染模型机体炎性反应，试验利用无菌大鼠分别建立深二度烫伤损伤模型和烫伤后绿脓杆菌感染模型，比较这两种模型和SPF级大鼠深二度烫伤模型在愈合过程、炎性反应及病理变化等方面的异同。20只6周龄雌性Wistar无菌大鼠，在超净工作台内采用恒温恒压烫伤仪94℃作用8 s造成大鼠深二度烫伤，随机分为2组，一组伤口感染浓度为1×10⁵ CFU/mL绿脓杆菌菌液0.5 mL，一组不做处理，创面保持无菌状态，SPF级Wistar大鼠在同样条件下烫伤，创面不做处理。分别观察3组大鼠的伤口结痂时间、脱痂时间和愈合时间，并在烫伤前（0 h），烫伤后24 h、3 d和7 d时，取血清，检测炎性因子肿瘤坏死因子-α（TNF-α）、白细胞介素1α（IL-1α）、粒细胞-巨噬细胞刺激因子（GM-CSF）的变化。结果显示，感染绿脓杆菌大鼠组开始结痂所需时间显著长于SPF级大鼠组和无菌大鼠组（P<0.05）；而脱痂及愈合所需时间显著变短（P<0.05）；感染绿脓杆菌组大鼠烫伤24 h时，皮下组织中可见淋巴细胞，有慢性炎性反应；3 d时，真皮深层有化脓性感染，横纹肌周围可见大量炎细胞，烫伤7 d时，皮下组织中有轻微血管扩张充血，化脓性感染减轻，无菌烫伤大鼠与SPF级烫伤大鼠皮肤在烫伤后一周内有轻度炎性反应。3组大鼠烫伤后，血清GM-CSF、IL-1α及TNF-α水平均较烫伤前显著升高（P<0.05）；烫伤24 h时，感染绿脓杆菌大鼠组和无菌大鼠组血清IL-1α水平显著高于SPF级大鼠组（P<0.05）；无菌大鼠组血清GM-CSF水平显著高于SPF级大鼠组和感染绿脓杆菌大鼠组（P<0.05）；烫伤3 d时，染绿脓杆菌大鼠组和无菌大鼠组血清IL-1α水平显著高于SPF大鼠组（P<0.05），无菌大鼠组血清GM-CSF水平显著高于染绿脓杆菌大鼠组（P<0.05）。综上所述，无菌大鼠对烫伤刺激反应迅速强烈，血清中炎性因子迅速升高，SPF级大鼠深二度烫伤后炎性反应缓慢；无菌大鼠深二度烫伤伤口感染1×10⁵ CFU/mL绿脓杆菌，脱痂时间和愈合时间反而缩短。     

Differential Expression Analysis of MUC4 and MUC13 Genes Between Resistant and Sensitive Weaned Meishan Piglets to ETEC F18

MUC4 and MUC13 genes as important candidate genes for enterotoxigenic Escherichia coil (ETEC) F4 resistance,may play an important role in the process of against ETEC F18 infection in weaned piglets. In this study,ETEC F18-resistant and -sensitive weaned Meishan piglets were used,and the expression levels of MUC4 and MUC13 genes in 11 tissues (heart,liver,spleen,lung,kidney,stomach,muscle,thymus,lymph nodes,duodenum and jejunum) were determined by quantitative Real-time PCR. The results showed that MUC4 and MUC13 genes were broadly expressed with different expression levels in all the 11 tissues. In the thymus and lymph tissues,the expression of MUC4 gene in resistant piglets was significantly higher than that in sensitive piglets (P<0.05);In the lung tissue,theMUC13 gene expression level in resistant individuals was significantly higher than that in sensitive individuals (P<0.05),and in the intestinal tissues of duodenum and jejunum, the expression level of MUC13 gene was relatively higher in resistant individuals. Thus we speculated that the high expression of MUC4 gene in immune tissues and MUC13 gene in intestinal tissues might improve the immune ability of piglets,protect and lubricate the intestinal tract, and resist ETEC F18 infection.     

Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.     

表达Ⅰ型耐热肠毒素的重组大肠杆菌诱发仔猪小肠炎症的分子机制研究

本试验旨在研究表达Ⅰ型耐热肠毒素(STa)的重组大肠杆菌诱发7日龄仔猪小肠炎症反应。选取24头7日龄健康仔猪,随机分为4个处理组:对照组(人工乳)、STa组(人工乳+2×10~9 CFU重组菌LMG194-pBAD-STa)、LMG194组(人工乳+2×10~9 CFU大肠杆菌LMG194)和K88组(人工乳+2×10~9 CFU大肠杆菌K88),每组6头。试验第5天进行攻毒,第7天屠宰取样,观察空肠组织形态学变化,并检测血清中炎性细胞因子含量及空肠免疫相关基因的相对表达量。结果显示,与对照组相比,STa与K88组仔猪空肠绒毛明显萎缩变短,有明显损伤甚至脱落;STa、LMG194及K88组血清中IL-10、IL-8和IL-6浓度均显著升高(P<0.05),LMG194和K88组血液和肠道iFABP浓度均显著降低(P<0.05),STa和K88组血清中TNF-α浓度显著降低、NF-κB浓度显著升高,LMG194组NF-κB浓度显著降低、TNF-α浓度显著升高(P<0.05);STa和LMG194组CCL2和CXCL9基因表达水平显著上调(P<0.05),VNN1基因表达水平显著下调(P<0.05),STa和LMG194组IFN-γ组基因水平分别显著上升和下降(P<0.05);K88组CXCL9和IFN-γ基因表达水平显著下调(P<0.05),VNN1基因表达水平显著上调(P>0.05),CCL2基因表达水平无明显差异(P>0.05)。以上结果表明,表达Ⅰ型耐热肠毒素STa的重组大肠杆菌几乎具有与K88一样的毒性,可导致7日龄仔猪小肠黏膜受损,诱发严重的炎症反应,导致仔猪腹泻,可作为仔猪腹泻模型的候选菌株。     

Cytokine mRNA expression in porcine cell lines stimulated by enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is an extracellular bacterium that causes post-weaning diarrhoea (PWD) in piglets with different severity of clinical signs. The pathogenesis of ETEC is ascribed to the effect of enterotoxins. ETEC colonizes ileum and probably can penetrate the epithelium and stimulate macrophages. The aim of study was to examine whether there is any difference in cytokine response in vitro produced by two porcine cell lines, intestinal epithelial cell line (IPI-2I) and macrophage cell line (3D4/31) after stimulation with different serotypes of ETEC associated with different clinical course of PWD in piglets. Three serotypes, O149:K88 (F4), O147:F18 and O8:K88, were used. We observed that all the used serotypes were unable to induce IL-8 and TNF-alpha mRNA expression in IPI-2I cell line as measured by the real-time RT-PCR. In 3D4/31 cell line, we detected differences in cytokine response among the used serotypes. The highest IL-8 and TNF-alpha mRNA expression in 3D4/31 was detected after stimulation with serotype O149:K88 frequently associated with hemorrhagic gastroenteritis.     

TGF-β1在布鲁氏菌致炎过程中的作用及其与NLRP3炎症小体的相关性研究

建立牛布鲁氏菌2308感染小鼠巨噬细胞RAW264.7模型,用RT-qPCR检测感染细胞中TGF-β1在mRNA水平的变化;ELISA检测布鲁氏菌感染细胞后培养上清中TGF-β1、IL-1β和IL-18的释放量;Western blot分析TGF-β1蛋白水平的变化.布鲁氏菌侵染重组外源TGF-β1(rTGF-β1)预...     

Effects of casein glycomacropeptide supplementation on growth performance,intestinal morphology,intestinal barrier permeability and inflammatory responses in Escherichia coli K88 challenged piglets

Casein glycomacropeptide(CGMP) is a bioactive peptide derived from milk with multiple functions. This study was aimed at evaluating the effects of CGMP as a potential feed additive on growth performance,intestinal morphology, intestinal barrier permeability and inflammatory responses of Escherichia coli K88(E. coli K88) challenged piglets. Eighteen weaning piglets were randomly assigned to three groups.Control group and K88 challenged group received a basal diet, and CGMP treated group received the basal diet supplemented with 1% of CGMP powder. The trail lasted for 12 days, K88 was orally administered to the piglets of K88 challenged group and CGMP treated group on days 8-10. The results showed that the diet containing 1% CGMP significantly alleviated the decrease in average daily gain(P 0.05),increase in pathogenic bacteria amounts in intestinal contents(P 0.05), intestinal morphology(P 0.05) and barrier permeability damage(P 0.05), and acute inflammatory response(P 0.05)induced by E. coli K88 infection. In conclusion, CGMP supplementation in the diet protected the weaning piglets against E. coli K88 infection.     

Influence of dietary ingredients on in vitro inflammatory response of intestinal porcine epithelial cells challenged by an enterotoxigenic Escherichia coli (K88)

Enterotoxigenic Escherichia coli (ETEC) K88 is the main bacterial cause of diarrhea in piglets around weaning and the adhesion of ETEC to the intestinal mucosa is a prerequisite step for its colonization. In this study, the adhesion of a fimbriated ETEC and a non-fimbriated E. coli (NFEC) to the intestinal cells and the activation of the innate immune system were evaluated using a porcine intestinal epithelial cell line (IPEC-J2). The impact of several feedstuffs (wheat bran (WB); casein glycomacropeptide (CGMP); mannan-oligosaccharides (MOS); locust bean extract (LB) and Aspergillus oryzae fermentation extract (AO)) on ETEC attachment and the inflammatory response were also studied. The gene expression of TLR-4; TLR-5; IL-1β; IL-8; IL-10 and TNF-α were quantified using Cyclophilin-A, as a reference gene, and related to a non-challenged treatment. The fimbriated strain was markedly better than the non-fimbriated strain at adherence to intestinal cells and inducing an inflammatory response. All the feedstuffs studied were able to reduce the adhesion of ETEC, with the greatest decrease with CGMP or MOS at highest concentration. Regarding the inflammatory response, the highest dose of WB promoted the lowest relative expression of cytokines and chemokines. All tested feedstuffs were able to reduce the adhesion of ETEC to IPEC-J2 and interfere on the innate inflammatory response; however WB should be further studied according to the beneficial results on the intestinal inflammatory process evidenced in this study.     

嗜酸乳杆菌发酵中药对产肠毒素大肠杆菌K88所致腹泻的防治作用

本试验旨在研究嗜酸乳杆菌发酵中药对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）K88所致腹泻小鼠的影响。选取6~8周龄巴比西小鼠（BALB/c）100只,分为4组,分别为空白对照组（Ⅰ组）、大肠杆菌感染模型组（Ⅱ组）、中药发酵组（Ⅲ组）及中药水提组（Ⅳ组）,其中Ⅱ、Ⅲ和Ⅳ组小鼠均用"抗生素+大肠杆菌"模式造模。Ⅰ、Ⅱ组饲喂不含任何药物的基础日粮,Ⅲ、Ⅳ组分别在基础日粮中添加10%中药发酵液、10%中药水提液。统计分析各组小鼠腹泻率、腹泻指数、免疫器官指数、肠道菌群及细胞因子IFN-γ、IL-4的含量。结果显示,ETEC K88感染小鼠36 h后,与Ⅱ组相比,Ⅲ、Ⅳ组小鼠腹泻指数（P < 0.05）与腹泻率降低,且Ⅲ组腹泻指数显著低于Ⅳ组（P < 0.05）,Ⅲ、Ⅳ组盲肠内大肠杆菌数量较Ⅱ组降低,双歧杆菌及乳酸菌数量提高,且Ⅲ组效果更优;Ⅲ、Ⅳ组的免疫器官指数均高于Ⅰ和Ⅱ组,且与Ⅱ组相比,Ⅲ组的脾脏指数、胸腺指数及肝脏指数分别提高了48.37%、29.57%、36.88%,且差异均显著（P < 0.05）;Ⅲ、Ⅳ组IFN-γ/IL-4比值较Ⅱ组提高,且Ⅲ组高于Ⅳ组。综上所述,乳酸菌发酵中药液具有降低腹泻小鼠腹泻率及腹泻指数,提高免疫器官指数,维持肠道菌群平衡及快速消除体内炎症反应等作用,可为开发防制ETEC所致的仔猪腹泻产品提供参考。     

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.