抑制大肠杆菌K88的益生菌体外筛选

本研究以从断奶仔猪肠道分离的益生菌为试验菌株,将大肠杆菌K88和益生菌接种到体外培养的猪小肠上皮细胞(intestinal porcine epithelial cell-1,IPEC-1)中,测定培养2.5h上清液中的乳酸脱氢酶(lactate dehydrogenase,LDH)活性,同时将益生菌和大肠杆菌K88体外混合培养2.5h,进行平板计数,统计大肠杆菌K88菌数的变化,筛选出可以抑制大肠杆菌K88的益生菌。试验结果显示,干酪乳杆菌和凝结芽孢杆菌能显著降低IPEC-1培养上清液中的LDH活性(P<0.05),降低大肠杆菌K88对细胞的损伤;凝结芽孢杆菌能降低DMEM培养基中大肠杆菌K88的生长速度,结合模拟制粒过程及胃肠道环境的耐高温、耐酸及耐胆盐研究进行综合分析,该凝结芽胞杆菌对大肠杆菌K88有较好的抑制作用且具有良好的耐高温、耐酸及耐胆盐性能,具有作为微生态制剂菌株的应用潜力。本试验建立了能够抑制大肠杆菌K88的益生菌体外筛选技术模型。     

In vitro adhesion of K88ac+ Escherichia coli to Peyer''s patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac⁺ enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88⁺ enterotoxigenic E. coli in the porcine small intestine.     

中药联合益生菌对大肠杆菌致小鼠腹泻保护机制研究

【目的】前期研究表明中药联合益生菌对致病性大肠杆菌所致小鼠腹泻具有明显的保护作用,本研究旨在进一步探讨中药联合益生菌对致病性大肠杆菌致小鼠腹泻的保护机制。【方法】选取小鼠60只,随机分为5组,空白组、模型组、中药组、益生菌组及中药联合益生菌组,每组12只。除空白组外,其余各组小鼠通过腹腔注射1×10⁷CFU/kg大肠杆菌分离株菌悬液建立小鼠腹泻模型,空白组腹腔注射等量生理盐水。中药组灌胃给予中药液(2.5 g/kg蒲公英提取物、0.5 g/kg黄芪总黄酮及0.5 g/kg黄芪多糖),益生菌组灌胃给予枯草芽孢杆菌悬液(2×10⁸CFU/kg),中药联合益生菌组灌胃给予中药液(2.5 g/kg蒲公英提取物、0.5 g/kg黄芪总黄酮及0.5 g/kg黄芪多糖)与枯草芽孢杆菌悬液(5×10⁷CFU/kg)的混合液,1次/d,连续给药7 d。HE染色观察小肠组织病理学变化,扫描电镜观察小肠组织超微结构变化,ELISA法检测小鼠小肠匀浆和血清中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量,RT-PCR法检测小肠组...     

广西猪源益生菌的分离鉴定与生物学特性研究

为了解广西地区猪粪便与微生态发酵饲料中益生菌的存在情况及合理开发和利用猪源益生菌,本试验共采集了15份猪粪便样品和5份益生菌发酵饲料并对其进行益生菌的分离,研究益生菌的形态学特性、生理生化特性及16S rRNA分子序列,进行生长曲线、耐胆盐试验、温度敏感试验、模拟胃肠道耐受试验、耐药性试验、体外抑菌试验、小鼠安全性试验、体内/外抑菌试验,对分离菌株进行生物学特性的研究分析。结果显示,本试验共分离鉴定出6种、29株益生菌,分别为1株屎肠球菌（C2）、2株乳酸肠球菌（C1、C3）、3株植物乳杆菌（R20、R30、R37）、4株干酪乳杆菌（R41~R44）、7株枯草芽孢杆菌（K1~K7）和12株蜡样芽孢杆菌（Y1~Y12）。从中筛选出6株具有生长性能好、耐胆盐、耐高温、对胃肠道耐受、体外抑菌能力强的益生菌：乳酸肠球菌C1、屎肠球菌C2、植物乳杆菌R20、干酪乳杆菌R41、枯草芽孢杆菌K1和蜡样芽孢杆菌Y3。将筛选出的具有优良特性的菌株进行小鼠安全性试验,结果表明,在10⁹ CFU/mL浓度下灌胃6株益生菌对小鼠是安全无毒害的。与此同时,C2、R20、R41、Y3能极显著提高小鼠日增重（P<0.01）,K1能显著提高小鼠日增重（P<0.05）。体内抑菌试验结果表明,C2、R20、K1能降低沙门氏菌G21感染的小鼠的死亡率,有一定程度的体内抑菌作用。各项试验结果表明,R20和K1可作为猪源益生菌的选择菌株。     

奶牛饲料中酿酒酵母菌的分离鉴定及其发酵液的体外抑菌活性研究

本试验旨在分离鉴定甘肃地区奶牛场饲料中的酿酒酵母菌,进而研究酿酒酵母菌发酵液的体外抑菌活性。通过YPD固体培养基对采集的5份饲草和5份饲料样品中的酵母菌进行分离,分别进行生化鉴定和26SrDNA测序鉴定,进而检测酿酒酵母发酵液的体外抑菌活性。结果表明,从样品中分离出4种共12株酵母菌,分别为3株酿酒酵母菌(Y1、Y7和Y11)、4株汉逊德巴利酵母菌、3株浅黄隐球酵母菌和2株戴尔有孢圆酵母菌;3株酿酒酵母菌的发酵液对奶牛乳房炎金黄色葡萄球菌、大肠杆菌和无乳链球菌的抑菌活性存在差异,其中酵母菌Y7和Y11的发酵液对3种病原菌均具有抑菌活性,而Y1的发酵液只对大肠杆菌具有抑制作用。本试验为进一步筛选性能优异的酿酒酵母菌用于微生态制剂研发奠定了基础。     

The Study of Isolation,Identification of S.cerevisiae from Dairy Feed and its Antibacterical Activity in the Fermentation Brothe in vitro

The aim of the study was to isolate and identify S.cerevisiae in the dairy feed and analyze antibacterial activity of S.cerevisiae fermentation brothe in vitro.5 forage samples and 5 feed samples were collected to isolate yeast by YPD solid medium.Biochemical identification and 26S rDNA sequence were used to identify the isolated strains.The antibacterial activity of S.cerevisiae strains was tested by cylinder plate method.A total of 12 yeast belonged to 4 species were isolated and identified, including 3 S.cerevisiae (Y1, Y7 and Y11), 4 Debaryomyces hansenii, 3 Cryptococcus flavescens and 2 Torulaspora debrueckii.The antibacterial activities of the 3 S.cerevisiae fermentation brothe to Staphylococcus aureus, E.coli and Streptococcus agalactiae were different.Y7 and Y11 fermentation brothe showed antibacterial activities to all the tested pathogens, while Y1 inhibit E.coli alone.This study laid the foundations for further research of probiotics containing S.cerevisiae with excellent performance.     

Inhibition of adhesion of F18+ Escherichia coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group H-2 antigen

Enterotoxigenic and verotoxigenic F18⁺ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae. The aim of the present study was to define the F18R nature. The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18⁺ E. coli-induced disease. The adhesion of F18⁺ E. coli to pig intestinal villous enterocytes was analysed in an in vitro assay. The adhesion of F18⁺ E. coli but not of F4ac⁺ E. coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity. Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18⁺ E. coli nor F4ac⁺ E. coli. Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18⁺ E. coli, but only partially the adhesion of F4ac⁺ E. coli. These data demonstrate that the F18 receptor contains the blood group antigen H-2 (-fuc-(1-2)-β-Gal-(1-4)-GlcNAc) as major carbohydrate.     

发酵乳杆菌和凝结芽孢杆菌对产气荚膜梭菌感染肉鸡生长性能和肠道健康的影响

试验以产气荚膜梭菌感染肉仔鸡建立坏死性肠炎模型,研究发酵乳杆菌和凝结芽孢杆菌对感染肉鸡生长性能和肠道健康的影响。将336只1日龄爱拔益加肉仔鸡随机分成4个处理组,每个处理6个重复。4个处理组分别为对照组、感染组、感染＋LF组（日粮添加1×10⁹ CFU/kg发酵乳杆菌）、感染＋BC组（日粮添加1×10¹⁰ CFU/kg凝结芽孢杆菌）。所有感染肉鸡在14~21 d经口接种A型产气荚膜梭菌。试验期为28 d。结果显示：与对照组相比,灌服产气荚膜梭菌显著降低22~28 d肉鸡的日均采食量（P<0.05）,显著增加28 d十二指肠和空肠损伤评分（P<0.05）,显著提高21 d肉鸡盲肠的产气荚膜梭菌和大肠杆菌数量,显著降低28 d回肠产气荚膜梭菌数量（P<0.05）。与感染组相比,日粮添加发酵乳杆菌显著提高21 d肉鸡回肠的乳杆菌属数量（P<0.05）,显著降低21 d盲肠大肠杆菌的数量（P<0.05）;日粮添加凝结芽孢杆菌显著降低28 d肉鸡十二指肠损伤评分（P<0.05）,显著降低21 d十二指肠隐窝深度（P<0.05）,显著提高21 d空肠绒毛高度与隐窝深度的比值（P<0.05）,显著降低21 d肉鸡回肠和盲肠的大肠杆菌数量（P<0.05）,显著提高28 d肉鸡回肠乳杆菌属数量（P<0.05）。综上所述,日粮中添加发酵乳杆菌或凝结芽孢杆菌均对感染肉鸡的肠道微生物具有调控作用;其中添加凝结芽孢杆菌有利于肠道绒毛发育,缓解肠道损伤,一定程度上提高了肉鸡的生长性能,而发酵乳杆菌没有缓解肠道损伤的作用。     

Discovery,Isolation,Identification and Characterization of Novel Non-ribosomal Peptide Antibacterial Active Substances in Bacillus

The aim of this study was to search for novel non-ribosomal peptide antimicrobial substances based on the screening of bacterial secondary metabolites.The bacteria isolated from soil,sea water and common marine organisms in Yantai coastal area were isolated and purified.E.coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria,and E.coli B2 (bla_NDM-5+mcr-1) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were used as indicator for secondary screening.The genome was extracted and the PCR products were sequenced to determine the active species.The secondary metabolites of bacteria were extracted by organic extraction,purified by gel chromatography and preparative liquid chromatography,and purity was detected by analytical liquid chromatography.The results of sequencing showed that the active strain belonged to Bacillus amyloliquefaciens sp.,and was named as Bacillus amyloliquefaciens 9-14 (active bacterium 9-14).The results of antibacterial test showed that the metabolites of active bacterium 9-14 had high inhibitory effect on Staphylococcus aureus ATCC 29213,MRSA T144,E.coli ATCC 25922 and E.coli B2.The metabolites of active bacterium 9-14 were cyclic lipopeptides composed of amino acid chains,which belonged to the derivatives of ibuprofen.The biological characteristics and antibacterial spectrum of the metabolites of active bacterium 9-14 were studied.The results showed that the metabolites of active bacterium 9-14 had good thermal stability and acid-base stability.And the antibacterial activity of the antibacterial substance treated with trypsin,pepsin,protease K and papain was not significantly weakened and had good stability.The antibacterial substance also had inhibitory effect on Staphylococcus aureus and E.coli,but had no activity against Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterococcus faecalis and Bacillus cereus.In this study,a new antibacterial substance was obtained,which could be used as the precursor of antibacterial drugs,and could provide certain reference for food safety and disease control.     

芽孢杆菌中新型非核糖体肽类抗菌活性物质的发掘、分离鉴定及特性研究

本研究旨在基于对细菌次级代谢产物的筛选,寻找新型非核糖体肽类抗菌物质。通过对烟台沿海地区的土壤、海水及近海常见海洋生物中的细菌进行培养,分离纯化得到细菌的单克隆,以大肠杆菌（E.coli）ATCC 25922和金黄色葡萄球菌ATCC 29213为指示菌进行初步筛选,以耐多黏菌素和碳青霉烯E.coli B2（bla_NDM-5+mcr-1）和耐甲氧西林金黄色葡萄球菌（MRSA） T144作为指示菌对有活性的细菌进行二次筛选。通过提取基因组进行PCR扩增产物测序比对,确定活性菌种属。采用有机萃取法对细菌的次级代谢产物进行萃取,通过凝胶层析和制备液相色谱进行纯化,利用分析型液相色谱进行纯度检测,进—步利用质谱对纯化后的抗菌活性物质进行结构鉴定。测序结果表明,活性菌属于解淀粉酶芽孢杆菌种,将其命名为解淀粉酶芽孢杆菌9-14（活性菌9-14）。抑菌试验结果表明,活性菌9-14的代谢产物对金黄色葡萄球菌ATCC 29213、MRSA T144、E.coli ATCC 25922和E.coli B2均具有高效抑制作用。活性菌9-14代谢产物是由氨基酸链组成的环状脂肽,属于伊枯草菌素的衍生物。对活性菌9-14代谢产物的生物学特性及其抑菌谱研究发现,活性菌9-14的代谢产物具有良好的热稳定性和酸碱稳定性,该抗菌物质经胰蛋白酶、胃蛋白酶、蛋白酶K和木瓜蛋白酶处理后抗菌活性没有明显减弱,具有较好的稳定性;该抗菌物质对所用金黄色葡萄球菌和大肠杆菌同样具有抑制作用,对绿脓杆菌、肺炎克雷伯杆菌、粪肠球菌和蜡样芽孢杆菌均不表现活性。本研究得到一种新型的抗菌物质,以该抗菌物质为抗菌药物的前体,可为食品安全和疾病控制提供一定的参考依据。     

牛源大肠杆菌对秀丽隐杆线虫的致病性研究

为建立牛源大肠杆菌-秀丽隐杆线虫致病模型，本研究从广西南宁、桂林、柳州等地区收集的牛病料中分离出13株大肠杆菌，继而对这些大肠杆菌进行血清学鉴定及小鼠、秀丽隐杆线虫的致病性试验。采用玻片凝集法鉴定大肠杆菌的O血清型，并将13株大肠杆菌培养液以3.0×10⁹ CFU/mL、0.2 mL/10 g体重给小鼠腹腔注射，同时分别喂食N2野生型秀丽隐杆线虫。结果显示，大肠杆菌的O血清型为O127、O126和O44，其中O126为优势血清型（4/13）。致病性结果显示，有11株大肠杆菌能使小鼠致死（致死率为40%~100%），2株大肠杆菌对小鼠没有致死性（致死率为0）。对小鼠有强致病性的大肠杆菌，对线虫的致死率也较高，死亡率在第3~6天最为显著，半数致死时间为3~4.5 d，最长存活时间为9 d；对小鼠不致死的两株大肠杆菌对线虫的致死率也较低，线虫死亡率下降趋势缓慢，半数致死时间为5~6 d，最长存活时间为10~11 d。肠道细菌计数结果显示，大肠杆菌在线虫体内的数量与时间呈线性关系，大肠杆菌不断破坏线虫的免疫系统，从而导致了线虫的死亡。本研究结果表明，大肠杆菌对线虫和小鼠的致病力试验结果一致，说明成功建立了牛源大肠杆菌-秀丽隐杆线虫致病模型，为牛病防治与临床用药提供了依据。     

中药联合益生菌对大肠杆菌性小鼠腹泻的保护作用

【目的】研究中药联合益生菌对大肠杆菌性小鼠腹泻的保护作用。【方法】通过正交试验筛选中药联合益生菌最佳因素配比。取60只小鼠随机分为空白组、模型组、中药组、益生菌组和中药联合益生菌组,每组12只。空白组小鼠腹腔注射生理盐水(0.2 mL/只),模型组、中药组、益生菌组和中药联合益生菌组均先腹腔注射分离菌悬液1×10⁷ CFU/kg,然后中药组按10 mL/kg灌胃中药液(其中蒲公英提取物浓度为0.25 g/mL,黄芪总黄酮和黄芪多糖浓度均为0.05 g/mL),益生菌组按4 mL/kg灌胃枯草杆菌悬液(菌含量5×10⁷ CFU/mL),中药联合益生菌组按上述量灌胃中药液和枯草杆菌混合液,每天定时灌胃给药1次,连续3 d。每日记录小鼠腹泻、体重、采食量等情况。末次给药12 h后分离心脏、肝脏、肾脏、脾脏、肺脏,称重并计算脏器系数;血液分析法检测血液学指标;取粪便及小肠内容物检测粪便隐血情况并进行细菌计数;ELISA法检测小鼠血清和小肠组织中髓过氧化物酶(MPO)活性。【结果】中药联合益生菌最佳因素配比为A₁B₂C₂D₂。与空白组相比,模型组小鼠腹泻率100%且无自愈现象,症状和剖检病变明显,腹泻指数极显著上升(P＜0.01),体重、采食量均极显著下降(P＜0.01),肝脏、脾脏器官指数均显著升高(P＜0.05),淋巴细胞比率(LYM)、中间细胞比率(MID)均显著下降(P＜0.05),粒细胞比率(GRAN)极显著上升(P＜0.01);与模型组相比,中药组和中药联合益生菌组腹泻指数极显著下降(P＜0.01),体重、采食量显著上升(P＜0.05),中药组肝脏、脾脏器官指数显著下降(P＜0.05),中药联合益生菌组脏器系数极显著下降(P＜0.01),中药组、益生菌组及中药联合益生菌组LYM显著上升(P＜0.05)、GRAN显著下降(P＜0.05)。粪便隐血检测结果显示,模型组检测呈强阳性;中药组、益生菌组呈弱阳性,中药联合益生菌组呈阴性。与空白组相比,模型组小鼠肠内大肠杆菌数、血清及小肠组织中MPO活性均极显著上升(P＜0.01);与模型组相比,中药组和中药联合益生菌组小鼠肠内大肠杆菌数、血清及小肠组织中MPO活性均显著下降(P＜0.05)。【结论】中药联合益生菌可降低腹泻指数、肝脏和脾脏器官指数,增加体重、饮食量,使粪便隐血转阴并降低肠内大肠杆菌数、血清及小肠组织中MPO活性,从而对大肠杆菌性小鼠腹泻产生协同保护作用。     

特异性识别K1荚膜大肠杆菌的噬菌体PNJ1809-36生物学特性及全基因组分析

本研究以K1荚膜大肠杆菌DE058（avian pathogenic Escherichia coli）为宿主菌,从鸡粪中分离烈性噬菌体PNJ1809-36并对其进行生物学特性的研究和基因组测序及分析。将噬菌体PNJ1809-36进行分离纯化,通过形态学观察、最佳MOI测定、一步生长曲线测定、温度和酸碱耐受性测定、体外裂解能力测定以及全基因组测序及分析来研究该噬菌体的生物学特性和基因组特征。结果显示：噬菌体PNJ1809-36能够形成清晰透明的噬菌斑,其电镜照片显示为具有收缩尾部的肌尾科噬菌体;宿主谱分析结果显示,该噬菌体能够特异性裂解具有K1荚膜的大肠杆菌;最佳MOI为0.01;一步生长曲线结果显示,其潜伏期为10 min,爆发期为40 min;噬菌体PNJ1809-36在40和50℃下存活良好,60℃ 30 min可使噬菌体完全失活;噬菌体在pH3~11之间均可存活,最适pH为6;体外裂解试验表明,该噬菌体可在6 h内完全抑制宿主菌的生长。经基因组测序与分析,噬菌体PNJ1809-36基因组全长为152 343 bp,GC含量为39.11%,含有277个开放阅读框（ORF）,11个tRNA基因。同源性分析显示其与早期发现的K1特异性的短尾噬菌体（如K1F、K1E）的同源性较低,而与噬菌体nepoznato、PVP-SE 1和phAPEC8等K1特异性噬菌体同源性较高,基因序列相似性达90%。噬菌体PNJ1809-36是一株特异性识别K1荚膜大肠杆菌的烈性噬菌体,属于肌尾科噬菌体新的系统发育分支。其针对K1荚膜的特性,提示该噬菌体在鉴定K1型大肠杆菌和治疗K1型大肠杆菌引起的疾病方面存在潜力。     

凝结芽孢杆菌在生猪养殖中的应用及其作用机理的研究进展

近年来集约化养殖已成为畜牧业的发展方向,养殖规模越来越大,特别是生猪养殖是关乎民生的支柱性产业,占整个畜牧业养殖规模的一半以上。如何在保证生猪养殖业可持续发展的基础上,提高生产效益并达成无抗养殖已成为当下的研究热点。凝结芽孢杆菌（Bacillus coagulans）作为一种新型饲用益生菌,兼具乳酸菌和芽孢杆菌的性质,既能产生乳酸,又能形成芽孢;具有改善生猪肠道健康、提高机体免疫力和抗病能力、调节肠道菌群平衡和提高动物生产性能等作用,在生猪养殖中具有很大的应用前景。作者概述了凝结芽孢杆菌的生物学特性;重点总结了凝结芽孢杆菌在无抗日粮、替抗方案、有抗日粮、与其他添加剂混合使用以及其他方面对生猪生产性能的影响;并从肠道健康与消化吸收、机体免疫力和肠道菌群平衡方面结合已有的试验结果分析了凝结芽孢杆菌对生猪生长性能改善的作用机理;提出了凝结芽孢杆菌在生猪养殖应用中存在的问题及建议,以期为进一步开发应用凝结芽孢杆菌制剂提供参考。     

Research Lactobacillus rhamnosusFermented Herbal and Bacillus subtilis Complex on Immune Performance,E.coli Infection of Broiler

This experiment was designed to study the complex of Lactobacillus rhamnosus fermented herbal and Bacillus subtilis on White Feather broiler immunity performance and impact of Escherichia coli infection.360 one-day-old broiler chickens were randomly divided into 3 groups with 4 replicates in each group and 30 chickens per replicate.The pretrial period lasted for 7 d,and the experiment lasted for 35 d.The chickens in the group Ⅰ(positive control group) and group Ⅱ(negative control group)were all only fed a basal diet,group Ⅲ was test group,by additive 1% fermented herbal preparations,groups Ⅱ and Ⅲ were intraperitoneal injection of 1 mL E.coli at 35 d,broiler mortality,immune organ index,cecalmicroflora content,immunoglobulin levels,IL-2 and IL-6 content were tested.The results showed that injection of E.coli caused massive death of chickens,group Ⅱtook up to 75.00%,it was significantly higher than groups Ⅰ and Ⅲ (P < 0.05),the mortality in group Ⅲ was significantly lower than group Ⅱ(P < 0.05),was only 23.33%.Injection of E.coli maked spleen index and thymus index of group Ⅱincreased significantly (P < 0.05),the spleen index and thymus index of groups Ⅰ and Ⅲ were no significant difference (P > 0.05).E.coli counts was significantly decreased after injectionin group Ⅲ (P < 0.05),but the number of intestinal Lactobacilli of group Ⅲ was significantly increased (P < 0.05),and inhibited the propagation of E.coli,the counts of E.coli in groups Ⅰ and Ⅲ were no significant difference (P > 0.05).At 42 d,the sIgA of the intestinal fluid in group Ⅲ were higher than that of groups Ⅰ and Ⅱ with 11.99% and 36.56%,respectively(P < 0.05).The serum IgG concentrations of group Ⅲ was higher than that of groupsⅠand Ⅱwith 14.68% and 28.15%,respectively(P < 0.05).At 42 d,the IL-2 content of group Ⅱ was the lowest,it was significantly lower than group Ⅲ(P < 0.05),the IL-6 of group Ⅲ was significantly lower than group Ⅱ(P < 0.05).     

鼠李糖乳杆菌发酵中草药复合枯草芽孢杆菌对黄羽肉鸡生长后期免疫性能及大肠杆菌感染的研究

试验旨在研究鼠李糖乳杆菌发酵中草药复合枯草芽孢杆菌对白羽肉鸡生长后期免疫性能及大肠杆菌感染的影响。选取体重相似、健康状况基本相同的360只1日龄肉鸡随机分成3个组,每组4个重复,每个重复30只。试验预试期7 d,正试期35 d。试验Ⅰ组为阳性对照组,试验Ⅱ组为阴性对照组,均饲喂基础日粮,试验Ⅲ组在基础饲料中添加1%发酵中草药制剂,且试验Ⅱ、Ⅲ组在肉鸡35日龄时腹腔注射大肠杆菌菌悬液1 mL,连续攻毒2 d,测定肉鸡死亡率、免疫器官指数、盲肠菌群含量、免疫球蛋白水平和白介素含量。结果表明,腹腔注射大肠杆菌造成鸡大量死亡,试验Ⅱ组高达75.00%,显著高于试验Ⅰ、Ⅲ组(P < 0.05),使用发酵中草药微生态制剂后鸡死亡率显著下降(P < 0.05),仅有23.33%;注射大肠杆菌后试验Ⅲ组脾脏指数和法氏囊指数显著升高(P < 0.05),但使用发酵中草药微生态制剂后脾脏指数接近于试验Ⅰ组,而法氏囊指数稍高于试验Ⅰ组,说明发酵中草药微生态制剂可以抵抗大肠杆菌感染,脾脏和法氏囊发育不受影响;注射大肠杆菌后试验Ⅲ组大肠杆菌数量显著下降(P < 0.05),但试验Ⅲ组由于饲喂发酵中草药复合芽孢杆菌制剂,肠道乳杆菌数量显著增加,抑制大肠杆菌的繁殖,从而大肠杆菌数量与正常饲喂状态下的试验Ⅰ组趋于一致;42日龄时,盲肠内容物sIgA含量试验Ⅲ组分别高出试验Ⅰ、Ⅱ组11.99%和36.56%(P < 0.05),血清IgG含量试验Ⅲ组分别高出试验Ⅰ、Ⅱ组14.68%和28.15%(P < 0.05);42日龄时,试验Ⅱ组IL-2含量最低,显著低于试验Ⅲ组(P < 0.05),试验Ⅲ组IL-6含量显著低于试验Ⅱ组(P < 0.05)。     

Contribution of AIDA-I to the pathogenicity of a porcine diarrheagenic Escherichia coli and to intestinal colonization through biofilm formation in pigs

In order to evaluate the role of the AIDA-I of porcine diarrheagenic Escherichia coli strain PD20 serogroup O143 (AIDA-I⁺, STb⁺), a mutant strain PD20M (AIDA-I⁻, STb⁺) was generated from strain PD20 by an allelic exchange procedure. In addition, the full-length aidA gene was reintroduced into strain PD20M to generate the complemented strain PD20C (pTaidA, AIDA-I⁺, STb⁺). A non-pathogenic E. coli strain PD71 was used as negative control. Each strain was inoculated to newborn pigs via stomach tube. Severity of diarrhea was evaluated clinically and intestinal colonization was assessed by histology, immunohistochemistry (IHC), and transmission electron microscopy (TEM) including immunogold electron microscopy (IGEM). The adhesion pattern to HeLa cells, bacterial auto-aggregation and biofilm formation were evaluated in vitro. Pigs infected with strains PD20 or PD20C developed diarrhea 16 and 28 h after inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71. Histology, IHC, TEM and IGEM examinations showed heavy bacterial colonization with biofilm formation in the large intestine, and marked in vivo expression of AIDA-I protein in pigs infected with strains PD20 or PD20C in contrast to pigs infected with strains PD20M or PD71. The in vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial auto-aggregation and significant biofilm formation (p < 0.05) by the AIDA-I⁺ strains, when compared to AIDA-I⁻ strains. These results demonstrate that expression of AIDA-I is essential for intestinal colonization and in vitro bacterial autoaggregation and biofilm formation. Thus, AIDA-I may be considered a significant virulence determinant in development of diarrhea caused by porcine diarrheagenic AIDA-I⁺ E. coli PD20 in piglets.     

Study on Aerotransmission of Escherichia coli Carrying the Plasmid-mediated Quinolone Resistance Genes of Swine

To investigate the aerotransmission of Escherichia coli (E.coli) carrying the plasmid-mediated quinolone resistance (PMQR) genes,airborne E.coli were isolated from indoor air,upwind air and downwind air samples in five swine farms.Fecal samples from swine houses were randomly collected to isolate the E.coli.The sensitivities of the E.coli strains against 14 antibiotics were tested.The E.coli carrying the PMQR genes (qnr,aac(6')-Ib-cr,qepA) were identified by ERIC-PCR,and then the genetic fingerprints of E.coli were established to analyze its origins and spread toward the outside surroundings.The results showed that E.coli isolated from five swine farms showed high resistance against 12 antibiotics,such as gentamicin,kanamycin,tetracycline,streptomycin,nalidixic acid and sulfamethoxazole,and presented multi-drug resistant.Results of ERIC-PCR showed that 46.34% (19/41) of strains isolated from indoor air samples had the same origin with fecal-obtained strains,and 73.68% (14/19) of them shared the same PMQR genes with fecal-obtained strains.68.42% (26/38) of strains isolated from downwind air samples had the same origin with fecal-obtained or indoor air-obtained strains,and 65.38% (17/26) of them shared the same PMQR genes with fecal-obtained or indoor air-obtained strains.This indicated that E.coli carrying PMQR genes and originating from feces in swine houses could form aerosols to pollute the indoor air and then spread to the downwind air through air exchange (≥400 m),which could be a potential threaten to public environment and human health.     

猪源携带质粒介导喹诺酮类耐药基因大肠杆菌气源性传播的研究

为了调查养猪场携带质粒介导喹诺酮类耐药基因大肠杆菌的气源性传播情况,本试验分别在5个猪场舍内及舍外上风向和下风向不同距离收集空气样品,并在舍内随机采集粪便样品,分离大肠杆菌。药敏试验检测其对14种抗生素的耐药性。以质粒介导喹诺酮类耐药基因(qnr、aac(6')-Ib-cr、qepA)为指示基因,利用肠杆菌基因间重复一致序列聚合酶链式反应(ERIC-PCR)鉴定技术,分别对5个猪场不同样品中大肠杆菌的遗传相似性进行分析,评估其向舍外空气的传播情况。结果显示,5个猪场中的大肠杆菌对庆大霉素、卡那霉素、四环素、链霉素、萘啶酸、复方新诺明等12种常用抗生素耐药率较高,且呈现多重耐药。ERIC-PCR结果显示,46.34% (19/41)的舍内空气分离株与粪便分离株来源相同,其中73.68% (14/19)的分离株携带的耐药基因也相同;68.42% (26/38)的舍外空气分离株与舍内空气或粪便分离株来源相同,其中65.38% (17/26)的菌株携带相同的耐药基因。结果表明,起源于舍内粪便的携带质粒介导喹诺酮类耐药基因的大肠杆菌能形成气溶胶污染舍内空气,并借助舍内外气体交换,传播到舍外不同距离空气中(≥400 m),对养殖场周围的环境卫生及社区居民的健康形成威胁。     

撒坝猪源大肠杆菌高致病性毒力岛诱导病理损伤的超微结构特征

为探究撒坝猪源大肠杆菌（E.coli）高致病性毒力岛（HPI）诱导小鼠病理损伤的超微结构特征,本研究将实验室保存的E.coli HPI阳性株（HPI⁺）和E.coli HPI基因缺失株（^ΔHPI）进行复苏和培养,分别用E.coli HPI⁺、E.coli ^ΔHPI菌株以腹腔接种的方式感染昆明小鼠,检测菌株的半数致死量（50% lethal dose,LD₅₀）,通过HE染色和透射电镜观察并分析菌株对小鼠病理损伤的超微结构特征,利用免疫组织化学标记白细胞介素-1β（interleukin-1β,IL-1β）阳性细胞在被感染小鼠肝脏和肾脏组织中的分布,以反映E.coli HPI⁺及E.coli ^ΔHPI菌株所引起炎症水平的差异。结果显示,E.coli HPI⁺和E.coli ^ΔHPI菌株的半数致死量分别为1×10^7.39和1×10^8.62 CFU/mL;HE染色显示,E.coli感染小鼠后,可见肝脏细胞肿胀、变性,肝窦淤血,肾脏间质淤血,肾小管上皮细胞变性脱落等病理变化;超微结构变化显示,肝脏细胞的完整形态消失,胞核呈不规则形态,线粒体畸形,粗面内质网上核糖体脱落,滑面内质网增生;多数肾小管上皮细胞出现胞核固缩,部分细胞核核仁边移、体积增大,足突融合,系膜细胞间隙变宽。此外,E.coli HPI⁺感染组小鼠于肝脏、肾脏的水肿现象较E.coli ^ΔHPI菌株感染的小鼠更为明显。免疫组化结果显示,大肠杆菌感染小鼠后,IL-1β蛋白主要表达于肝细胞、中央静脉周围、肾间质细胞和肾小管上皮细胞,且E.coli HPI⁺感染组的IL-1β表达量高于E.coli^ΔHPI感染组。综上所述,撒坝猪源E.coli HPI能够调控E.coli对小鼠的致病性,HPI的调控作用可使E.coli对小鼠肝脏、肾脏造成的病变及超微结构变化更明显,并且能够增加小鼠的炎症反应。