猪肠毒素大肠杆菌F4受体研究进展

猪肠毒素大肠杆菌F4(ETECF4)是引起1～2周龄仔猪黄、白痢最普遍、危害最大的大肠杆菌，ETECF4能否致病，决定于猪的小肠上皮细胞有无ETECF4受体，该受体由基因控制。本综述了ETEC F4受体的研究进展，提出了今后深入研究和抗病育种的可能途径。     

F4大肠杆菌黏附素受体易感性仔猪的体外鉴定研究

猪小肠上皮细胞表面的产肠毒素大肠杆菌(ETEC)F4受体的基因型与其粘蛋白4(MUC4)基因密切相关,根据MUC4基因内含子7能否被限制性内切酶XbaⅠ酶切,可以在基因水平上将所检测的猪分为ETEC F4易感型纯合子SS,易感型杂合子SR和抵抗型RR。为筛选对ETEC F4受体易感性和抗性仔猪用于本实验室的后续实验,本实验利用MUC4基因的多态性,对送检猪的样品进行初步的分型检测。选取MUC4基因内含子7不同等位基因型的仔猪,分离其小肠上皮细胞,并分别与野生型F4ab、F4ac、F4ad大肠杆菌,表达fae操纵子的重组大肠杆菌r F4ab、r F4ac、r F4ad进行体外黏附和黏附抑制试验。研究结果表明,上述野生菌或重组菌对SS和SR两种基因型的4周龄断奶大白仔猪小肠上皮细胞均具有较好的黏附能力,而且经过抗F4单克隆抗体处理后的细胞,对细菌的黏附数明显下降。而RR型仔猪小肠上皮细胞不能黏附上述野生菌或重组菌。本研究为体外鉴定F4受体易感性仔猪,以及为进一步研究ETEC F4的致病机理奠定了基础和平台。     

2个猪品种的ATP2B1基因表达与ETEC F4粘附性关联分析

以ATP2B1基因作为仔猪腹泻相关的候选基因,在2个品种63头猪的小肠组织中,采用实时荧光定量PCR技术对肠毒素大肠杆菌(ETEC)F4受体不同黏附表型个体进行表达量的比较分析,以验证其基因功能和作为仔猪腹泻相关候选基因的可能性.结果表明:ATP2B1基因在不同黏附表型间的表达量有显著的差异,可望作为F4ab受体的相关...     

δ差异显示法筛选仔猪ETEC F4ad受体相关基因

初生仔猪泻痢的病原菌主要是肠毒素大肠杆菌(ETEC)F4,仔猪小肠上皮细胞有无F4受体对于仔猪抗病性具有重要意义.本试验采用δ差异显示法筛选仔猪F4ad受体相关基因.将获得的δ条ESTs通过GenBank进行序列同源比对,发现其中1条与人糖基磷脂酰肌醇锚1(GPI 1)基因有91%同源性.表明该基因可作为F4ad受体候选基因进行进一步研究.     

肠道中有或无大肠杆菌K88受体的猪的性能

粘附于小肠上皮细胞的能力,是产肠毒素大肠埃希氏杆菌(ETEC)的重要毒力因素。ETEC的表面抗原(又称粘附素,如K88抗原)可促进其粘附于仔猪肠上皮。K88~+菌株是新生腹泻的重要病原,而K88~-菌株在断奶后腹泻中占优势。业已发现,K88~+大肠杆菌并不能粘附于所有猪的小肠,有些猪的肠道中缺乏K88抗原的受体,它们对K88~+ETEC的实验和自然感染均有抵抗力。肠道受体通过单一位点上的两个等位基因(Adh~s     

猪源大肠杆菌fedA基因的克隆及鉴定

断奶仔猪腹泻（post—weaning diarrhea,PWD）和猪水肿病（porcine edema disease,ED）是导致仔猪死亡的重要传染性疾病。PWD和ED分别由肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）和志贺氏毒素大肠杆菌（verotoxigenic Escherichia coli,VTEC）引起。ETEC和VTEC等病原菌除了产生毒素外,还同时具有菌毛粘附因子。F18菌毛是病原菌主要的粘附因子,介导细菌对小肠黏膜上皮细胞表面受体的粘附,在PWD和ED的发生中起着决定性的作用,是引起疾病的主要毒力因子之一。     

仔猪断奶前腹泻抗病基因育种技术的创建及应用

产肠毒素大肠杆菌（ETEC）F4ac是引起仔猪断奶前腹泻最主要的细菌性病原,仔猪小肠上皮细胞有无ETECF4ac受体是仔猪被感染时是否发病的关键。任军等（2012）利用大规模资源家系群体和远缘群体,通过全基因组连锁定位分析、目的区域的重组断点事件分析和远缘群体高通量SNP标记的关联性分析等严谨的遗传学分析手段,确定了ETECF4ac受体基因为MUC13,     

猪口服免疫F18ac~+非产肠毒素大肠杆菌疫苗候选株后小肠免疫细胞的组织形态学特征

由F4+和(或)F18+产肠毒素大肠杆菌(ETEC)引起的腹泻和肠毒血症是乳猪及断奶仔猪的多发病。本文通过对断奶仔猪小肠淋巴样细胞亚群表型进行定量分析,检测和评估了F18ac+非ETEC弱毒疫苗候选株对F4ac+ETEC感染的免疫原性及保护效力;同时还评估了左旋咪唑作为一种免疫应答调节剂(IRM)的调节效能及其与试验疫苗联用的佐剂活性。     

产肠毒素性大肠杆菌菌毛的研究进展

产肠毒素性大肠杆菌(ETEC)常引起仔猪腹泻,在世界范围内造成了严重的经济损失。ETEC的致病作用与其具有粘附性菌毛和肠毒素密切相关,ETEC菌株的菌毛可与宿主黏膜上皮细胞表面的相应受体结合,并在局部组织定居、繁殖和产生毒素,损伤小肠黏膜,使小肠吸收分泌功能失常而致腹泻。本文针对ETEC菌毛的生物学特性、表达条件、免疫原性等方面作一概述。     

K88菌毛及其蛋白亚基FaeG在防治仔猪腹泻中的应用性研究进展

肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原之一,其致病性主要与菌毛粘附素及其产生的肠毒素有关。ETEC依靠菌毛粘附素与小肠粘膜上皮细胞上的受体特异性结合,这种粘附作用可以使ETEC抵抗肠道蠕动的冲刷作用而定植,并大量繁殖。ETEC在生长过程中,产生大量的肠毒素并不断释放到肠道内,造成肠道内水和电解质比例失衡,从而引起腹泻。不同肠毒性大肠杆菌(ETEC)菌株的菌毛抗原类型不同,主要有K88、K99、987P及F41,其中以K88的流行最为普遍,因而亦尤为重要。1K88粘附素菌毛,又称纤毛或柔毛,由菌…     

A Single-chain Fragment Variable Recombinant Antibody against F5 Fimbria of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Inhibits Agglutination of Horse Red Blood Cells Induced by F5 Protein

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.     

The F4 fimbrial antigen of Escherichia coli and its receptors

F4 or K88 fimbriae are long filamentous polymeric surface proteins of enterotoxigenic Escherichia coli (ETEC), consisting of so-called major (FaeG) and minor (FaeF, FaeH, FaeC, and probably FaeI) subunits. Several serotypes of F4 have been described, namely F4ab, F4ac, and F4ad. The F4 fimbriae allow the microorganisms to adhere to F4-specific receptors present on brush borders of villous enterocytes and consequently to colonize the small intestine. Such ETEC infections are responsible for diarrhea and mortality in neonatal and recently weaned pigs. In this review emphasis is put on the morphology, genetic configuration, and biosynthesis of F4 fimbriae. Furthermore, the localization of the different a, b, c, and d epitopes, and the localization of the receptor binding site on the FaeG major subunit of F4 get ample attention. Subsequently, the F4-specific receptors are discussed. When the three variants of F4 (F4ab, F4ac, and F4ad) are considered, six porcine phenotypes can be distinguished with regard to the brush border adhesiveness: phenotype A binds all three variants, phenotype B binds F4ab and F4ac, phenotype C binds F4ab and F4ad, phenotype D binds F4ad, phenotype E binds none of the variants, and phenotype F binds F4ab. The following receptor model is described: receptor bcd is found in phenotype A pigs, receptor bc is found in phenotype A and B pigs, receptor d is found in phenotype C and D pigs, and receptor b is found in phenotype F pigs. Furthermore, the characterization of the different receptors is described in which the bcd receptor is proposed as collection of glycoproteins with molecular masses ranging from 45 to 70 kDa, the bc receptor as two glycoproteins with molecular masses of 210 an 240 kDa, respectively, the b receptor as a glycoprotein of 74 kDa, and the d receptor as a glycosphingolipid with unknown molecular mass. Finally, the importance of F4 fimbriae and their receptors in the study of mucosal immunity in pigs is discussed.     

Adjuvant effect of Gantrez®AN nanoparticles during oral vaccination of piglets against F4+enterotoxigenic Escherichia coli

In this study, the adjuvanticity of methylvinylether-co-maleic anhydride (Gantrez^®AN) nanoparticles (NP) was investigated in an oral immunisation experiment of pigs against F4+enterotoxigenic Escherichia coli (F4+ETEC). In addition, Wheat Germ Agglutinin (WGA)-coating of the nanoparticles was tested for enterocyte-targeting. Pigs were either vaccinated with F4 fimbriae, F4 encapsulated in Gantrez^®AN NP, F4 encapsulated in Gantrez^®AN NP coated with WGA or F4 fimbriae mixed with empty Gantrez^®AN NP. Only vaccination with the combination of F4 mixed with empty Gantrez^®AN NP improved protection against F4+ETEC infection. In addition, vaccination with this formulation also resulted in an F4-specific serum antibody response prior to F4+ETEC challenge. Encapsulation of F4 in Gantrez^®AN NP only raised the serum antibody response after F4+ETEC challenge compared to soluble F4, but did not improve protection, whereas WGA-coating almost completely abolished the serum antibody response. These data indicate that nanoparticle effects after F4 encapsulation were of lesser importance for the adjuvant effect of Gantrez^®AN NP, contrarily to the reactivity of the Gantrez^®AN polymer used to prepare the nanoparticles.     

卵黄抗体对ETEC粘附仔猪小肠上皮细胞的体外抑制作用研究

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。分别制成单价或多价的菌毛蛋白白油佐剂抗原 ,对产蛋鸡进行胸部肌肉分点注射免疫 ,初免后 2周加强免疫 1次。收集高效价卵黄抗体。用所获得各卵黄抗体对体外分离的初生仔猪小肠上皮细胞进行体外粘附抑制试验。结果表明 ,各种菌毛卵黄抗体均能特异地显著抑制相应大肠杆菌对仔猪上皮细胞的粘附 ,而对其他血清型大肠杆菌对肠上皮细胞的粘附无抑制作用     

Enteric-coated pellets of F4 fimbriae for oral vaccination of suckling piglets against enterotoxigenic Escherichia coli infections

To prevent enterotoxigenic Escherichia coli (ETEC) induced postweaning diarrhoea, the piglet needs an active mucosal immunity at the moment of weaning. In the present study, the feasibility of oral vaccination of suckling piglets against F4+ETEC infection with F4 fimbriae was studied. Furthermore, oral vaccination with enteric-coated pellets of F4 fimbriae was compared to vaccination with F4 fimbriae in solution. Therefore, piglets were orally administered 1mg F4 fimbriae in pellets or in solution during three successive days at the age of 7 and 21 days, whereas control piglets were not vaccinated. Five days postweaning (33 days of age), all animals were orally challenged with F4+ETEC. Despite the induction of an immune response upon oral administration of both F4 fimbriae in pellets as in solution, the colonisation of the small intestine by F4+ETEC upon oral challenge could not be prevented. However, a marginal but significant reduction in F4+ E. coli faecal excretion was found in the piglets vaccinated with F4 fimbriae in pellets, indicating that the use of an enteric-coat which protects the F4 fimbriae against inactivation by milk factors and degradation by enzymes and bile improves vaccination.     

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA⁺ B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA⁺ B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.     

Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea

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.     

Cholera toxin improves the F4(K88)-specific immune response following oral immunization of pigs with recombinant FaeG

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4(+)E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4(+)E. coli excretion following F4(+) ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.     

Receptor-specific binding of purified F4 to isolated villi.

Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT+Sta+STb+) were performed and the purity and yield of F4ac were compared. Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10(11) bacteria/ml). The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10(12) bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10(12) bacteria, i.e. 26.2%) and the relative contamination higher (1.0 mg per 10(12) bacteria, i.e. 73.8%). A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels. The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained. The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay. Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb.     

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro

The role of flagella in the pathogenesis of F4ac⁺ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902 ETEC strain (O8:H19:F4ac⁺ LT⁺ STa⁺ STb⁺), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac⁺ ETEC adhesion in vitro.