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选用断奶仔猪肠道中的大肠杆菌K88和猪霍乱沙门菌作为指示菌。杀菌性能的测定采用最低抑菌浓度（MIC）和最小杀菌浓度（MBC）,并绘制杀菌曲线。原子吸收光谱仪测定Cu2＋在肉汤和生理盐水中的释放量;透射电镜观察细菌细胞壁的变化;全自动生化分析仪测定细菌胞内酶的活性;通过K＋电极测定K＋释放量;采用SP-2型溶氧仪测定菌体悬浮液中的溶氧量。结果显示,载铜蒙脱石对E.coli K88的MIC为64mg/L,MBC为256mg/mL;对S.choleraesuis的MIC为128mg/L,MBC为512mg/L。而蒙脱石未显抗菌活性。透射电镜下观察细菌与载铜蒙脱石作用后形态发生了变化,细菌细胞膜受损,内容物外漏;细菌胞内酶活性结果显示,胞内酶的大量外漏,与对照组相比,均差异极显著（P〈0.01）;大量自由的K＋从细菌细胞内释放出来;呼吸代谢结果显示,载铜蒙脱石抑制了细菌呼吸代谢的三羧酸循环途径。结果表明,载铜蒙脱石具有强大的杀菌作用。 相似文献
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为了探讨载铜蒙脱石(MMT-Cu)对大肠杆菌K88和猪霍乱沙门菌的黏附作用,试验采用Ca-co-2细胞培养模型,观察被标记的大肠杆菌K88、猪霍乱沙门菌对载铜蒙脱石的黏附作用;并在培养液中加入载铜蒙脱石,计算其对细菌黏附的阻断率,测定胞内乳酸脱氢酶(LDH)的释放情况。结果表明:测试菌与Caco-2细胞的黏附率分别为13.23%和10.78%;载铜蒙脱石对病原菌黏附Caco-2细胞均有不同程度的阻断作用,对大肠杆菌K88、猪霍乱沙门菌的阻断率分别为67.38%和60.45%,并能极显著降低LDH的释放(P<0.01)。说明载铜蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位,可作为一种消化道黏膜保护剂。 相似文献
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以Caco-2细胞作为体外模型研究芽孢杆菌对细胞黏附、细胞存活率及细胞膜完整性的影响.采用荧光标记法探究芽孢杆菌、大肠杆菌K88和猪霍乱沙门菌对Caco-2细胞的黏附特性,并通过排斥、竞争和置换试验测定其对病原菌黏附的阻断作用.采用台盼蓝染色法测定Caco-2细胞存活率和细胞培养上清乳酸脱氢酶(LDH)活性来反映细胞膜的完整性.结果表明:芽孢杆菌对Caco-2细胞的黏附率均低于病原菌、且对病原菌的黏附无阻断作用,大部分芽孢杆菌不会影响细胞存活率及细胞膜的完整性,对细胞无毒. 相似文献
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从铜在动物体内的含量与分布及生物化学功能入手，阐述高铜对动物肠道微生物的作用机制，重点介绍高铜对仔猪肠道大肠杆菌的致死性、高铜对细菌细胞壁的影响、高铜对细菌细胞内K^＋的影响、高铜对细菌呼吸代谢的影响，为今后微量元素铜对仔猪肠道内环境的研究提供参考。 相似文献
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重组大肠杆菌K88ab和K99蛋白制备的鸡卵黄抗体的效力评价 总被引：1，自引：0，他引：1
为探索以重组大肠杆菌K88ab和K99蛋白为免疫原制备鸡卵黄抗体的可行性,本研究利用基因工程技术,在大肠杆菌中高效表达重组K88ab和K99蛋白。分别以重组蛋白和提取的天然菌毛为免疫原制备抗K88ab和K99菌毛的鸡卵黄抗体,并对抗体效价、特异性以及抗体的预防和治疗效果进行了检测和比较。试管凝集反应结果显示,以重组蛋白为免疫原制备的鸡卵黄抗体效价最高可达1∶2 560,与用天然菌毛为免疫原制备的鸡卵黄抗体的效价基本一致。该抗体分别与K88ab+和K99+大肠杆菌发生特异性凝集,并能分别有效抑制K88ab+和K99+大肠杆菌对新生仔猪肠上皮细胞的吸附。临床应用结果显示,本实验制备的卵黄抗体对仔猪黄痢和仔猪白痢的预防有效率为100%,保护率为91%;治疗有效率为100%,治愈率为87%。实验结果表明该卵黄抗体对仔猪黄痢和仔猪白痢具有良好的预防和治疗效果。 相似文献
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为研究K88ab/K88ad菌毛对细胞的黏附作用,本研究分别以产肠毒素E.coli (ETEC) K88ab C83901株和K88ad C83903株基因组DNA为模板,采用PCR技术扩增这两种K88菌毛操纵子fae基因(均约7.9 kb).将其分别克隆于表达质粒pBR322中构建pBR-K88ab和pBR-K88ad重组质粒,并将其分别转化至不含任何菌毛的E.coli SE5000株中.该重组菌能够分别与鼠抗K88菌毛阳性血清和抗K88菌毛单克隆抗体(MAb)产生凝集反应;在电镜下观察到重组菌表面大量表达K88菌毛.采用热抽提法提取其体外表达的K88ab和K88ad菌毛,SDS-PAGE电泳检测结果显示,菌毛蛋白的分子量约为26 ku.玻板凝集试验和western blot结果表明:重组表达的K88ab及K88ad菌毛与K88+参考株菌毛均能够被抗K88菌毛阳性血清和MAb识别.以猪小肠上皮细胞系IPEC-J2为模型进行黏附和黏附抑制试验,结果表明表达K88菌毛的重组菌及K88+参考株均能够黏附于IPEC-J2上皮细胞表面;而且阳性血清和MAb能够有效抑制重组菌或K88+参考株对猪小肠上皮细胞系的黏附结合. 相似文献
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嗜酸乳杆菌和两歧双歧杆菌体外粘附Caco-2细胞及其对病原菌粘附性能的影响 总被引：1，自引：0，他引：1
以Caco-2细胞作为体外模式,观察嗜酸乳杆菌、两歧双歧杆菌、大肠杆菌K88和猪霍乱沙门氏菌对Caco-2细胞的粘附特性,并探讨嗜酸乳杆菌和两歧双歧杆菌的抗粘附作用。结果表明,所试4种细菌菌株均表现出很强的粘附Caco-2细胞的特性;粘附抑制试验表明,无论在排斥试验、竞争试验还是置换试验,嗜酸乳杆菌或两歧双歧杆菌均能明显抑制大肠杆菌K88和猪霍乱沙门氏菌的粘附;杀菌试验表明,嗜酸乳杆菌或两歧双歧杆菌上清液均能显著抑制病原菌的生长,但上清液用胰蛋白酶、蛋白酶K、乳酸脱氢酶处理后,杀菌作用明显降低,表明杀菌作用是乳酸和蛋白质样物质共同作用的结果。 相似文献
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检测K88~+肠毒素性大肠杆菌PCR方法的建立 总被引：5，自引：0，他引：5
以K8 8菌毛结构基因保守序列为靶序列 ,设计合成了一对可扩增长 2 0 1bp的目的片段的引物 ,成功地建立了检测肠毒素性大肠杆菌(ETEC)K88菌毛基因的PCR方法。进行了PCR方法的特异性试验和敏感性试验。对K99+ ,F41 + ,987p+ 参考菌株和鼠伤寒沙门氏杆菌 ,链球菌 ,金黄色葡萄球菌和猪肺疫巴氏杆菌的检测结果均为阴性 ;该检测方法的敏感度可达 1 0个细菌。用此方法对 1 0株腹泻仔猪粪便分离物进行检测 ,结果有 2株阳性 ;与血清学检测的结果一致。结果表明此方法特异性和敏感性都很高 ,可用于临床K88+ 肠毒素性大肠杆菌病的快速诊断和流行病学调查 相似文献
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The aim of this research was to determine the antibacterial properties and mechanisms of Cu(II)-exchanged montmorillonite (MMT-Cu) in vitro. Escherichia coli ATCC K88 and Salmonella choleraesuis ATCC 50020 were chosen as indicators of intestinal tract pathogenic bacteria in weanling pigs. The antibacterial activity of MMT-Cu and MMT were evaluated by determining the minimum inhibitory concentrations (MICs) using two-fold serial dilutions in MH broth, and the amount of Cu2+ released into the broth was measured by an atomic absorption technique. The rate of oxygen consumption was measured using a SP-II-type oxygen electrode analyzer; the structural integrity of cell walls of bacteria was observed by transmission electron microscope (TEM); enzymatic activity of bacteria was examined with a semi-automatic biochemical analyzer. The results showed that MMT-Cu inhibited the growth of E. coli K88 and S. choleraesuis, and the MICs were 1024 and 2048 microg/ml, respectively. The amount of Cu2+ released into the broth was in the range 6.51-45.65 microg/ml. Nevertheless, both tested bacteria still grew in broth containing 32,768 microg/ml of MMT. Treatment with MMT-Cu could lead to significant release of intracellular enzymes from the tested bacteria. Data from oxygen consumption of bacteria showed that MMT-Cu could inhibit the TCA pathway of the bacterial respiration metabolism. These results show that MMT-Cu has an antibacterial activity. 相似文献
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载铜蒙脱石对大肠杆菌和仔猪腹泻的影响 总被引：1，自引：0，他引：1
对通过阳离子交换反应合成的载铜蒙脱石 (MMT- Cu) ,应用 XRD衍射分析显示 ,载铜反应前后 ,蒙脱石的(0 0 1)面网间距从原先的 1.5 4 4 nm增加到了 1.5 88nm ,表明铜是以水合阳离子或复合阳离子形式进入蒙脱石晶格层间位置。体外试验表明 ,MMT- Cu对大肠杆菌 K88具有较强的抗菌能力。MMT- Cu对仔猪的试验表明 :与对照组相比 ,日粮中添加 0 .2 % MMT- Cu使仔猪日增重提高了 13.89% (P<0 .0 1) ,料重比下降了 8.38% (P<0 .0 5 ) ,平均腹泻率降低了 71.80 % (P<0 .0 1) ,结肠内容物中大肠杆菌数显著下降 (P<0 .0 1) ,空肠绒毛高度及绒毛高度与腺窝深度的比率分别提高了 19.2 1% (P<0 .0 5 )和 37.10 % (P<0 .0 1)。 相似文献
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The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC). 相似文献
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Koh SY George S Brözel V Moxley R Francis D Kaushik RS 《Veterinary microbiology》2008,130(1-2):191-197
Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis. 相似文献
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中药制剂产后康复宁的药理研究 总被引：2，自引：0，他引：2
应用ＬＭＳ－２Ｂ二道生理记录仪及配套装置,通过免疫器官重量法和鸡红细胞（ＣＲＢＣ）作免疫原的溶血素（ＩｇＭ）测定法及体内抑菌试验法,观测了中药制剂产后康复宁对家兔离体、在位子宫和小鼠免疫功能的影响,以及对感染大肠杆菌Ｋ８８小鼠的保护作用。结果表明,产后康复宁可使兔离体子宫收缩频率显著增加（Ｐ＜０．０１）,张力显著增强（Ｐ＜０．０１）,振幅显著增高（Ｐ＜０．０１）;与麦角相比,仅５ｍｉｎ相同时间的子宫收缩频率差异明显（Ｐ＜０．０５）,其余相同时间子宫收缩频率、张力、振幅均无明显差异（Ｐ＞０．０５）。使在位子宫收缩频率明显增加（Ｐ＜０．０５）,张力明显增强（Ｐ＜０．０５）,振幅明显增高（Ｐ＜０．０５）;与麦角比较,子宫收缩频率、张力、振幅在５、１０ｍｉｎ相同时间内差异均不明显（Ｐ＞０．０５）。对免疫功能的影响,不但使小鼠脾脏显著增重（Ｐ＜０．０１）,而且溶血素明显增多（Ｐ＜０．０５）。对感染大肠杆菌Ｋ８８的小鼠有明显的保护作用（Ｐ＜０．０５）。提示产后康复宁有增强子宫收缩,促使宫内恶露排除,加速子宫复旧,增强机体免疫功能等作用。 相似文献
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Equine small intestinal brush-border membranes, from 40 adult horses were tested in vitro for the presence of receptors for the Escherichia coli adhesive antigens K88ab, K88ac and K99. Only K88-positive strains of E. coli adhered strongly to horse brush-border membranes. In contrast, a K88-negative mutant strain J2, 2 K99-positive strains and 3 E. coli strains isolated from foals failed to adhere to horse brush-border membranes. Purified K88ac pili when reacted with equine brush-border membranes inhibited to a great extent the adhesion of K88-positive E. coli. Similarly, K88-positive E. coli previously reacted with K88 antibody, did not attach to equine brush-border membranes. Oral inoculation of 4 newborn foals with strains of K88-positive enterotoxigenic E. coli, producing either heat-stable or heat-labile enterotoxin, caused diarrhoea in 1 animal. 相似文献
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Live Pasteurella haemolytica biotype A, serotype 1 isolates (n = 3) and Escherichia coli K-12, strain W3110, were reacted with bovine pulmonary lavage cell (PLC) suspensions. The comparative effects of the different bacteria on the functional and metabolic activity of alveolar macrophages (AMO) in the PLC suspensions were assessed simultaneously by use of 51Cr release, luminol-dependent chemiluminescence (LDCL), and AMO bactericidal assays. The bovine PLC responded differently to E coli, than to the 3 P haemolytica isolates in each of the 3 experimental test systems; however, responses to each of the P haemolytica isolates were not found to be significantly different. Unopsonized live P haemolytica cells adversely affected the functional and metabolic response of PLC, whereas there was no evidence of a cytotoxic (cytocidal) influence of E coli. A difference in 51Cr release for reaction mixtures containing E coli and P haemolytica was not detected at zero time; however, at each subsequent time, reaction mixtures phagocytically stimulated with P haemolytica had significantly increased amount of 51Cr release (P less than 0.05), compared with those mixtures containing E coli. Bovine AMO in the PLC suspensions were able to effectively kill E coli in vitro, but were unable to prevent survival and subsequent growth of P haemolytica. The luminol-dependent chemiluminescence profiles for reaction mixtures phagocytically stimulated with E coli provided evidence of sustained production of oxygen radicals with antimicrobial capabilities by bovine AMO in the PLC. Production of these highly reactive antimicrobial oxidants appeared initially in cultures containing P haemolytica but, subsequently, their production declined precipitously and ceased altogether. 相似文献
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以蒙脱石（MMT）为原料，通过阳离子交换反应合成载铜蒙脱石（MMT-Cu）。为探讨载铜蒙脱石对肠粘膜屏障功能的影响，采用体外培养的单层Caco-2细胞模型为研究对象，以细菌易位数量和肠绒毛损伤程度做为指标进行研究。结果表明：MMT-Cu可明显减少（P〈0．01）E．coilK88、S．choleraesuis侵入到Caco-2细胞内的数量，与对照组相比，下降了2logCFU／mL～3log CFU／mL；并能显著降低（P〈0．01）LDH的释放量。扫描电子显微镜下观察可见，加入MMT-Cu一段时间后，当E．coil K88和S．choleraesuis粘附Caco-2细胞时，微绒毛依旧排列整齐、致密，可见肠上皮细胞保持完好。结果表明，MMT-Cu对肠粘膜具有屏障作用，可作为一种消化道粘膜保护剂。 相似文献
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Prevalence of K88, K99, and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis 总被引：1，自引：0，他引：1
One hundred nineteen live neonatal pigs with diarrhea less than or equal to 2 weeks old were euthanatized, and frozen sections of their ilea were submitted to an indirect fluorescent antibody technique to identify K88, K99, and 987P pili (also referred to as F-4, F-5, and F-6 pili, respectively) in Escherichia coli. Ten-centimeter ileal sections were used to determine numbers of lactose-fermenting bacteria. Of 52 pigs in which E coli pili were found, 14 had K88 (27%), 23 had K99 (44%), 13 had 987P (25%), and 2 had K88 and K99 simultaneously (4%). Numbers of lactose-fermenting bacteria were significantly (P less than or equal to 0.05) higher in pigs with piliated E coli than in pigs without piliated E coli. Results of this study indicated that piliated E coli are a major cause of enteric disease in neonatal swine in Michigan, and that in pigs less than or equal to 2 weeks of age, K99 was the most frequently encountered pilus antigen. 相似文献