抗大肠埃希氏菌K88ab、K88ac和K88ad特异单克隆抗体

用MCA对K88粘附素扩原结构进行分析表明：在K88ab、K88ac和K88ad三种血清到中,同一种血清型的菌株至少能表达一个共同的型特异抗原决定簇;K 88ad菌林和K88ac菌株都能稳定地表达一个K88ab菌株不县有的共同的抗原决定簇;某些抗原决定簇仅存在于同一种K88血清型的部分菌株中。     

四株大肠埃希氏菌国际参考菌株动力和鞭毛抗原检定的研究

Authors identified four strains of Escherichia coli from Collaborative Centre for reference and research on Escherichia coli-Klebsiella (WHO) in Denmark. Our results were different from original record and reference. It was shown that strain H311b and H5 were motile strains, and its H antigen respectively is H11 and H27. The strain W27 is nonmotile. H antigen of the strain H511 is H40, not H8. Antigen formula of four strains respectively is 26:60:11 (strain H311b), 81:97:27 (strain H5), 115:-:- (strain W27), 102:-:40 (strain H511).     

协同凝集法检测大肠埃希氏菌LT毒素的研究

产毒素大肠埃希氏菌（ＥＴＥＣ）是致急性腹泻、旅游者腹泻的重要病原。该菌可产生不耐热（ＬＴ）和耐热（ＳＴ）二类肠毒素。ＳＴ因分子量小,难以制备出单（多）克隆抗体,故一般免疫学检查手段难以检出。ＬＴ分子量较大,且与霍乱毒素（ＣＴ）具有共同的抗原性,故可用...     

一种快速提取禽源性大肠埃希氏菌外膜蛋白的方法

介绍一种快速提取禽源性大肠埃杀氏外膜蛋白的方法,该法是对Ｋａｐｕｒ等发表的方法的改进,全过程只需超速离心一次,比Ｋａｐｕｒ等的方法缩短了４ｈ,所得样品可直接用于禽源性大肠埃希氏菌外膜蛋白模式的测定。     

四株大肠埃希氏菌国际参考菌株动力和

作者检定了丹麦世界卫生组织大肠埃希氏菌和克雷伯氏菌研究中心的四株大肠埃希氏菌菌株,其结果不同于原来的记录和文献记载。菌株H311b和H5有动力,其H抗原分别为11和H27;菌株W27是无动力株;菌株H511的H抗原是H40,而不是原记载的H8。这四株菌株的抗原式分别为26：60：ll(H311b株),8l：97：27(H5株),115：--：--(w27株),102：一：40。     

5种大肠埃希氏菌Col质粒接合传递实验研究

以5种61株Col~ 大肠埃希氏菌为供体,与受体E.Coli K_(12)进行接合实验,结果有16株供体菌的Col质粒传递给K_(12),占25.4％。其中NPEC、ETEC、EPEC、EIEC、EAEC的传递比率分别是8/22、 4/21、2/15、1/2、1/1。按传递方式分类,52株耐药供体菌有10株的Col质粒和耐药标记(或R质粒)发生共同传递,占19.2％,伴发Col质粒传递的耐药标记有8种,其中TC、SM、COS、KM的并发传递率较高(16—20％)。不同耐药标记菌株的Col质粒传递率范围在0—66.7％。在8株敏感菌和1株耐药菌中有6株Col质粒发生单独传递,占66.7％。实验发现4个配对组的Col质粒和R(r)质粒发生分离而独自传递。本文分析了Col质粒传递方式和耐药性的关系,同时讨论了接合传递的不同方法。为阐明和研究R~ Col~ 肠道菌的演变及其对正常菌群生态平衡的影响提供遗传学依据。     

抗大肠埃希氏菌F41粘附素特异单克隆抗体的研制及其初步应用

应用杂交瘤技术获得7株能稳定分泌F41特异单克隆抗体的杂交瘤细胞系,分别命名为Ll0、B10、C32、B1、E7、E40和B49。在直接凝集试验、酶联免疫吸附试验和间接荧光试验中,这7株单克隆抗体对所试的31株肠道菌中所有F41阳性菌株都发生反应,与F41阴性菌株则无反应性。抗原竞争ELISA试验结果发现,这些单克隆抗体是针对F41粘附素上相同或十分相近的抗原决定簇。采用肢体金标记技术和免疫电镜证实,这些抗原决定簇在F41粘附素的每条纤毛上多次重复出现。体外肠吸附抑制试验表明,7株F41l特异单克隆抗体对B41M菌株具有很强的抑制能力,对B41菌株则必须用F41和K99单克隆抗体同时作用才具有完全的肠吸附抑制效果。本文用直接酶标单克隆抗体建立的酶联免疫斑点试验具有高度的特异性和灵敏性,可广泛用于现场快速诊断。     

大肠埃希氏菌的分型方法及其研究进展

大肠埃希氏菌是一种条件性致病菌,致病性的大肠埃希氏菌具有高度的传染性,会严重危害健康。快速准确地测定大肠埃希氏菌的污染来源对有效缩小疫情影响范围极有帮助,从而避免对人类健康和经济贸易造成重大损失。建立简便高效的分型方法是微生物溯源的关键,常见的大肠埃希氏菌分型方法可分为表型分型和分子分型,这些分型方法各有优劣,具有不同的适用范围。本文详细介绍了大肠埃希氏菌的分型方法,并对国内外大肠埃希氏菌分型的研究进展进行综述,为致病菌溯源方法的选择提供参考依据,对防御并控制致病菌引起的流行病传播具有重要的意义。     

牛膝多糖抑制大肠埃希菌细胞粘附的实验研究

目的 :研究牛膝多糖能否影响大肠埃希菌对细胞的粘附。方法 :使用 Hela细胞进行了粘附试验及粘附抑制试验。结果 :发现牛膝多糖浓度为 0 .8mg/ml时 ,对细菌的细胞粘附抑制最为明显 ,粘附率由(2 5 7.0± 5 .2 )个细菌 /细胞 降低到 (63 .6± 3 .6)个细菌 /细胞 。结论 :牛膝多糖对大肠埃希菌的细胞粘附具有抑制作用 ,提示该多糖具有调节肠道微生态的潜在应用价值     

PCR assay for detection and differentiation of K88ab1, K88ab2, K88ac, and K88ad fimbrial adhesins inE. coli strains isolated from diarrheic piglets

Primers were designed and prepared and conditions were determined for PCR detection and differentiation of enterotoxigenic E. coli bacterial strains isolated from diarrheic pigs. Primers K88/1 and K88/2 are 25 bp oligomers that correspond to a region of genes encoding one of serological variants of the K88 antigen (K88ab(1), K88ab(2), K88ac or K88ad). A positive result of PCR is an amplificate of 792 bp in size for K88ab and K88ad variant or 786 bp for K88ac variant. The individual serological variants of genes of the K88 antigen could be differentiated by cutting the obtained PCR amplificates by restriction endonucleases. The PCR analysis of 674 E. coli strains isolated from diarrheic pigs showed that 184 strains were K88 positive. By using restriction endonucleases the K88-positive strains were in 4 cases classified as K88ab variant, 180 as K88ac variant and none contained gene for the K88ad variant. Ninety-five % coincidence with serological examination using K88ab, K88ac and K88ad specific antibodies was shown.     

Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs

Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E. coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets. The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene. Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci. Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (θ). Our results demonstrate that the two K88 receptor loci are closely linked (θ= 0.02) with a maximum lod score value (Z_m) of 46.0. In addition, they are linked to the TF locus, θ= 0.14, Z_m= 19.6 for the K88abR locus and θ= 0.16, Z_m= 17.9 for the K88acR locus. The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR. This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.     

Isolation of K88 Antigen Variants (ab,ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.     

The porcine intestinal receptor for Escherichia coli K88ab,K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen

The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree. The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses. The K88abR and K88acR loci were tightly linked with the K88abR locus localized 7·4 cM (sex average) proximal to the transferrin locus. The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4. Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E. coli vaccine.     

K88ab gene of Escherichia coli encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin.

The K88ab adhesin operon of Escherichia coli encodes for a fimbrial protein (the K88ab adhesin) which is involved in colonization of the porcine intestine. We characterized a structural gene (gene A) which is part of the K88ab adhesin operon and codes for an as yet unidentified polypeptide (pA). A mutation in gene A resulted in accumulation of K88ab adhesin subunits inside the cell. The nucleotide sequence of gene A was determined, and the deduced amino acid sequence suggested that pA is synthesized as a precursor containing a typical N-terminal signal peptide. The molecular weight of pA was calculated to be ca. 17,600. Gene A is preceded by a sequence showing homology with the consensus promoter. Fimbrial subunits from a number of E. coli strains have significant homology at their N- and C-termini. pA also contained some of these conserved sequences and showed a number of other similarities with fimbrial subunits. Therefore, it seems likely that the K88ab adhesin operon codes for a fimbrial subunit (pA) distinct from the K88ab adhesin subunit.     

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Summary Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.     

Characterization and purification of porcine small intestinal mucus receptor for Escherichia coli K88ac fimbrial adhesin

The objectives of this study were to investigate the nature of, and to purify K88ac fimbrial adhesin-specific receptors in the mucus from the small intestine of piglet. Adhesion was studied by incubating (3)H-labeled Escherichia coli with mucus that were treated with or without pronase, proteinase, trypsin or sodium metaperiodate. The results indicated that treatment with either proteolytic enzymes or sodium metaperiodate (to oxidize sugars) significantly reduced E. coli K88ac or K88+MB adhesion to the mucus, suggesting that the K88ac and K88+MB specific receptors in this preparation were, at least in part, glycoprotein in nature. The K88+MB fimbriae specific receptor was purified using affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified K88+MB specific receptor together with the above data suggested that the receptor from the mucus of the small intestine of the pig was a 80-kDa glycoprotein.     

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.