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分离、纯化产肠毒素大肠杆菌(ETEC)K88ac菌毛蛋白，将其免疫产蛋鸡，经喷雾干燥制备抗ETEC菌毛抗原特异性卵黄抗体粉。间接ELISA法检测卵黄抗体效价，试验结果表明，喷雾干燥(140℃进气，65℃出气)对卵黄抗体活性无影响，加工前后抗体滴度均为1：1280；卵黄抗体粉在90℃干热条件下处理5min后抗体效价下降50％，而90℃湿热条件下处理5min抗体效价无明显变化，但处理15min后下降75％；经过1h的干热或湿热处理，抗体均基本失活；4℃和常温下放置半年对卵黄抗体粉活性无影响；体外抑制粘附试验表明，抗K88卵黄抗体能抑制K88对肠上皮细胞的粘附。 相似文献
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《国外畜牧学(猪与禽)》2015,(11)
<正>4新生仔猪中鸡卵黄免疫球蛋白对ETEC的被动保护作用Yokoyama等使用鸡卵黄抗体控制大肠杆菌引起的腹泻进行了一项卓越的研究。选择大肠杆菌产毒菌株(K88、K99和987P),在实验室发酵罐中扩繁、分离和纯化含有粘附素的棒状纤毛。将纤毛注射给蛋鸡,在抗体滴度较高时分离卵黄中的抗体(Ig Y)并喷雾干燥。将仔猪感染不同菌株的大肠杆菌,并使用适当的抗体。 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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抗猪大肠杆菌卵黄抗体变化规律及在体外对大肠杆菌生长的影响 总被引：4，自引：0，他引：4
试验研究抗猪大肠杆菌卵黄抗体变化规律及在体外对大肠杆菌生长和对肠黏膜细胞上粘附的影响。试验选用 30周龄海兰褐壳蛋鸡 2 4 0只 ,试验时间 80d。以K88、K99和 987P 3种大肠杆菌混合液为免疫原 ,免疫原中K88、K99和 987P的细菌数分别为 2 .5× 10 9、2 .5× 10 9、2 .5× 10 10 CFU/mL。免疫剂量为 0 .5、1.0、1.5、2 .0mL/只 ,在试验鸡胸腹部肌肉、分多点注射 (每点约 0 .2mL) ,每个免疫剂量免疫健康蛋鸡 5 0只 ,4 0只蛋鸡作对照 ,以个体为单位 ,每天收集鸡蛋 ,4℃保存。在每次免疫 5d后 ,翅静脉采血、分离血清 ,- 2 0℃保存。用盐析法和冷冻干燥法制备抗猪大肠杆菌卵黄抗体粉 ,配制不同蛋白含量的卵黄抗体溶液 ,用体外法研究卵黄抗体对大肠杆菌生长及对肠黏膜细胞粘附的影响。试验结果表明 :最佳免疫剂量为 1.0mL/只 ,卵黄与血清中的抗体水平没有差异 ;在大肠杆菌培养液中 ,添加蛋白含量为 0 .5mg/mL抗猪大肠杆菌卵黄抗体溶液时 ,大肠杆菌的生长受到抑制 (P <0 .0 5 ) ,不同卵黄抗体间没有交叉反应 ;蛋白含量为 0 .2 5mg/mL的抗猪大肠杆菌卵黄抗体溶液对大肠杆菌在肠黏膜细胞上的粘附有抑制作用 (P <0 .0 5 )。 相似文献
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《当代畜禽养殖业》2007,(9)
新生仔猪腹泻是由肠毒素性大肠杆菌(ETEC)产生一种或数种菌毛粘附素粘附于小肠黏膜上皮细胞及结膜层上而引起的。在实验中给禁食初乳的新生仔猪感染K88、K99、987P菌毛型的ETEC而导致腹泻,然后口服复方中药免疫球蛋白生物制剂进行治疗。体外抑菌研究结果表明:在大肠杆菌中加入复方中药免疫球蛋白生物制剂后,在最初1～3小时内其抑菌作用最强,3小时以后,其作用急剧降低。到5小时,起抑菌作用消失。体外粘附试验结果表明:效价为1:2560复方中药免疫球蛋白生物制剂在体外可以有效的抑制大肠杆菌在小肠上皮细胞的粘附,而且ETEC在小肠不同部位的菌毛受体并无明显的特异性。 相似文献
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仔猪腹泻源大肠杆菌贵州株K_(88)菌毛研究 总被引：2，自引：1，他引：1
通过玻片凝集试验、抗D_甘露糖微量血凝试验 (MRMH)、离体细胞粘附试验、菌体蛋白的聚丙烯酰胺凝胶电泳(SDS_PAGE)和免疫印迹、质粒DNA提取及电泳等 ,分析了仔猪腹泻源大肠杆菌贵州株 (4 0 9_11)所产K88菌毛的血凝谱、对肠上皮细胞粘附特性、菌毛蛋白亚单位分子量 ,并对其K88基因进行了初步定位。结果表明 :其所产K88菌毛的血凝谱能凝集豚鼠和鸡红细胞 ;能介导细菌粘附于仔猪离体肠上皮细胞 ,这种粘附作用能被特异的K88抗血清阻断 ;菌毛蛋白亚单位的分子量为2 35 0 0Da;K88基因位于一个分子量为 85kb(5 4× 10 6Da)的大质粒上。当菌株于 18℃生长时 ,其K88菌毛不能表达 相似文献
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鸡卵黄抗体在防治仔猪大肠杆菌性腹泻中的应用 总被引：2，自引：0，他引：2
仔猪大肠杆菌性腹泻病的致病过程主要与产肠毒素型大肠杆菌菌毛介导的粘附作用和产肠毒素密切相关，用禽菌毛抗原免疫产蛋母鸡制备卵黄抗体，在初生仔猪和断奶仔猪的人工感染实验中，口服卵黄抗体能降低腹泻程度和仔猪的死亡率；在商业性猪场的实验表明，卵黄抗体能降低提前断奶仔猪的腹泻发生率，并且有利于仔猪的增重，鸡卵黄抗体是仔猪大肠杆菌性腹泻病的一种新的防治方法，本文就国内外鸡卵黄抗体防治仔猪大肠杆菌腹泻的研究概况作了简要介绍。 相似文献
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为制备针对一些大型养猪场产肠毒素性大肠杆菌(enterotrxigenic Escherichia coli,ETEC)分离株的特异性卵黄抗体(egg yolk immunoglobulin,IgY),试验对从这些养殖场分离的ETEC分离株菌毛基因类型进行了PCR鉴定,纯化该分离株的菌毛蛋白免疫蛋鸡制备IgY,对该IgY的效价、特异性和体外抑菌效果进行了检测。结果表明,该分离株具有K88和987p 两种菌毛基因,纯化后的分离株菌毛具有较强的免疫原性,经3次免疫后产生的IgY对K88和987p全菌和菌毛的效价可达到1:64 000,分离株菌毛IgY能特异地与K88和987p反应,与K99、F41无交叉反应,5 mg/mL分离株菌毛IgY在体外具有很好的抑菌效果。 相似文献
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Harmsen MM van Solt CB Hoogendoorn A van Zijderveld FG Niewold TA van der Meulen J 《Veterinary microbiology》2005,111(1-2):89-98
Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs) that can be produced efficiently in microorganisms. Six F4 fimbriae specific VHHs were isolated. The VHH that was produced at the highest level by yeast, K609, was further analysed. 3.8 mg/L K609 inhibited 90% of bacterial attachment to intestinal brush borders in vitro. Perfusion of a jejunal segment with at least 4 mg/L K609 reduced the ETEC-induced fluid loss, but only to 30%. Preventive administration of a high K609 dose (150 mg/(piglet day)) to piglets that were challenge infected with ETEC resulted in less severe diarrhoea only at 4 and 5 days post-infection, but did not improve average daily weight gain, ETEC shedding and piglet survival. Thus, we have shown that an antibody fragment that effectively inhibited in vitro ETEC adhesion to intestinal brush borders poorly protected piglets against experimental ETEC infection. 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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Enterotoxigenic Escherichia coli (ETEC) in farm animals. 总被引：11，自引：0，他引：11
Animal diseases due to enterotoxigenic Escherichia coli (ETEC) typically appear as severe watery diarrhoea during the first few days of life (also a few days after weaning in pigs). ETEC adhere to the small intestinal microvilli without inducing morphological lesions and produce enterotoxins acting locally on enterocytes. This action results in the hypersecretion (of water and electrolytes) and reduced absorption. Adhesins and toxins are the two prominent virulence attributes of ETEC and the level of knowledge of these factors determines the chances for successful prevention and therapy of the disease. For animal ETEC the most common adhesins are the fimbriae (pili) on the surface: F4(K88), F5(K99), F6(987P), F41, F42, F165, F17 and F18. Enterotoxins (extracellular proteins or peptides) of animal ETEC are classified as heat-labile (LT) and heat-stable (ST) enterotoxins with further subdivisions (LTh-I, LTp-I, LTIIa, LTIIb, STaH, STaP, STb) according to antigenic and biological differences. Fimbriae and LT enterotoxins are made up of large molecular weight proteins which facilitate their utilisation in vaccines and their detection using immunodiagnostic systems. The adhesive fimbriae and enterotoxins of animal ETEC are plasmid determined (except F41 and F17). Virulence gene probes (DNA hybridisation, PCR) are specific and sensitive diagnostic and epidemiologic tools for the detection of ETEC. Based on genetic typing, the ETEC, in spite of limited serogroups, seem to represent a population of E. coli with a diverse genetic background. 相似文献
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猪肠毒性大肠杆菌 (ETEC)是仔猪黄痢、白痢等腹泻疾病的主要病原菌 ,发病率和死亡率分别占总发病率和死亡率的 5 6 .2 %和 2 4 .7%。按其特异菌毛产生的粘附素不同有 K88、K99、987P等多种类型 ,K88又可分为 ab、ac、ad血清型 ,现已知 ETEC K88能否致病 ,取决于宿主小肠粘膜上皮细胞刷状缘有无受体 ,并受制于 1对等位基因 ,有受体 (敏感型 )为显性 (S) ,无受体 (抗性型 )为隐性 (s)。该位点位于 1 3号染色体长臂 3.1区段 ,与转铁蛋白 (Tf)连锁 (相距7.4 c M) ,且 Tf基因频率与 K88抗性存在一定关系 ,抗性型与敏感型的小肠粘液理化性质也存在差异 ,据此 ,有可能采用生化及分子生物技术筛选抗性遗传标记 ,开展抗病育种。 相似文献
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肠毒素性大肠杆菌菌毛对产蛋鸡免疫性的研究 总被引：4，自引：1，他引：3
采用热抽提法提取4种肠毒素性大肠杆菌菌毛蛋白。K88、K99、F41和987p菌毛蛋白分别制成弗氏佐剂苗;K88还制成白油佐剂苗,氢氧化铝胶苗和蜂胶佐剂苗;另将4种菌毛等比例混合制成弗氏佐剂苗。分别对产蛋鸡进行免疫,用微量凝集反应和血凝抑制试验检测卵黄抗体效价。结果表明,K88菌毛较其他3种菌毛免疫性好,诱导抗体效价最高而且能长时间维持;987p菌毛能快速诱导抗体的产生,但整体效价低。K88不同佐剂苗中,铝胶佐剂能较快地诱导抗体的产生,蜂胶佐剂苗抗体持续时间短,弗氏佐剂能诱导高效价的抗体产生而且能长时间持续。 相似文献
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Bhaskaran S Jay CM Berghman LR Wagner GG Waghela SD 《Veterinary research communications》2005,29(6):463-476
Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae
is the initial step in the establishment of colibacillosis. Prevention of ETEC F5+ adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside
that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful
as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of
the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can
be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis. 相似文献
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Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs 总被引：8，自引：0，他引：8
H U Bertschinger M Bachmann C Mettler A Pospischil E M Schraner M Stamm T Sydler P Wild 《Veterinary microbiology》1990,25(2-3):267-281
Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy. 相似文献
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仔猪腹泻是全球规模化猪场最常见的疾病之一,给养猪业带来了巨大的损失。产肠毒素大肠杆菌（ETEC）是引起仔猪腹泻的主要病原菌,黏附素和肠毒素是其重要的致病因子,ETEC通过黏附素定植于小肠上皮细胞,在增殖过程中不断产生肠毒素,引起大量水和电解质进入肠腔,导致仔猪腹泻。目前,疫苗免疫是预防ETEC最有效的方法,然而许多商品化大肠杆菌疫苗的临床免疫效果不佳,且地域局限性明显。因此,研制安全、高效、广谱的ETEC疫苗对养猪业具有重要意义。近年来,许多新型试验性ETEC疫苗被相继报道,如ETEC菌毛黏附素和肠毒素疫苗;一些新开发的疫苗,如亚单位疫苗、菌影疫苗、植物载体疫苗和囊泡疫苗等,具有不同的优势,在不同的动物试验中均表现出良好的免疫保护效果。文章简述了国内外学者在ETEC疫苗领域的研究进展,对猪源ETEC各类疫苗的优劣及应用研究情况进行了综述,以期为今后猪源ETEC疫苗的研究提供参考。 相似文献