In vitro adhesion of K88ab-, K88ac- and K88ad-positive Escherichia coli to intestinal villi, to buccal cells and to erythrocytes of weaned piglets

The phenotype of 21 weaned piglets, concerning adhesion of Escherichia coli possessing K88ab, K88ac or K88ad fimbriae to pig cells, was determined in an in vitro assay. Comparison was made with adhesion of these three K88 variant strains to buccal mucosal epithelial cells and to erythrocytes (haemagglutination) in the same piglets. Whereas adhesion of the three K88 variant strains to intestinal villi was piglet specific, buccal cell adhesion (BCA) and haemagglutination (HA) were not. The K88ab strain was weakly adhesive or non-adhesive in the BCA and negative in the HA test. K88ac strains consistently gave negative and K88ad consistently gave positive results in both assays. After washing the bacteria with phosphate-buffered saline, the K88ab strain revealed a positive HA test. Neither the BCA, nor HA test can be used to determine the pig intestinal adhesive phenotype.     

Metabolic changes in red cells in response to adhesion of porcine K88 fimbriated Escherichia coli

Red cells glycolytic enzymes attached and nonattached to K88⁺ Escherichia coli were assayed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase activities, were measured. E. coli with K88ab fimbriae, E. coli with K88ac fimbriae, and isolated K88ab fimbriae were investigated for their effect on the above enzymes. Different changes were obtained with K88ab + bacteria compared with K88ac + bacteria. Purified fimbriae gave a third set of responses.     

铜离子对引起仔猪腹泻的大肠杆菌K88杀菌机理的研究

采用K 电极对大肠杆菌K88细胞内的K 释放进行测定;采用氧电极法对其呼吸耗氧量进行测定;采用透射电子显微镜观察细胞壁结构的完整性,从而探讨硫酸铜(CuSO4)的杀菌机理.研究表明:Cu2 在微摩尔水平时就能有效抑制微生物的呼吸代谢途径.比较了典型抑制剂对硫酸铜的叠加率,得出硫酸铜主要抑制呼吸代谢的HMP途径.而且Cu2 还可诱导K 从细胞内释放出来,使细菌死亡.但它是一个可逆的过程,营养基质和EDTA可阻止细胞内K 的释放.在透射电镜下观察细菌细胞壁的完整性被破坏.因此,硫酸铜在肠道中可有效杀死引起腹泻的病原菌大肠杆菌K88.     

Inhibition of adhesion of Escherichia coli K88 antigen by mammary secretions of susceptible and resistant sows

Anti-adhesive activities of colostrum and milk from genetically susceptible sows, which protected their susceptible offspring in an outbreak of neonatal diarrhoea caused by K88-positive Escherichia coli, were compared with the activities in mammary secretions of resistant dams that did not protect their susceptible progeny. There was significantly more anti-adhesive antibody in the secretions of susceptible sows than in resistant sows, both during the disease period, and 1 year later. Fractionation of colostrum by gel filtration and ion-exchange chromatography led to identification of the anti-adhesive antibodies as including both IgA and IgM.     

Fat globule membrane of sow milk as a target for adhesion of K88-positive Escherichia coli

Membrane adhesion of K88-positive Escherichia coli was studied on intestinal brush-border membranes on 237 Finnish Landrace pigs. Forty-one per cent of the brush-border membrane preparations aggregated E. coli (positive adhesion). Similar dualism of adherence/nonadherence was observed on sow milk fat globule membranes. Washed milk fat globules (washed cream) can be used as a convenient source of material for adhesion studies. Bacterial adherence on to milk fat globules is evident as agglutination of the globules (dark-field microscopy). By this procedure the sows can be typed according to their receptor phenotype. This simple principle of fat globule agglutination due to receptors for K88-positive E. coli might be complicated by SigA-mediated bacterial adherence. Fat globule membranes were shown to contain SigA, which may act as a mediator of bacterial adherence onto fat globules. The significance of this adhesive property of milk fat globule might be to provide alternative receptors for E. coli thus preventing bacterial adhesion on to gastro-intestinal epithelium of the offspring. Sow milk fat globules can be used for typing E. coli for membrane adhesiveness. The adhesiveness of the strains showed a good correlation with the presence of the K88 antigen, as well as the hydrophobicity of the bacterial strain as determined by an association on Phenyl-Sepharose beads.     

Detection of K88 and K99 fimbrial antigens on Escherichia coli by coagglutination

Coagglutination was used to detect K88 and K99 fimbrial antigens on Escherichia coli, and results were compared to an enzyme immuno assay (EIA). When pili suspensions were tested by both methods, 28 of 66 cultures were shown to have K88 and 11 of 31 cultures had K99 antigens. No pili suspensions were positive by coagglutination that were not positive by EIA. Testing of cell suspensions gave equivalent results to pili suspensions for K99 when tested by coagglutination. Two cell suspensions reacted with the K88 coagglutination which could not be confirmed by testing of pili suspensions, while a further 20 out of 43 cultures gave equivalent results with both cell and pili suspensions for K88 when tested by coagglutination.     

Inheritance of K88-mediated adhesion of Escherichia coli to jejunal brush borders in pigs: A genetic analysis

The transmission and genetic organization of the adhesion of the serological variants of the K88 adhesin in the jejunum of the pig were investigated. The results of 28 matings of 5 boars with 15 sows are presented. On the basis of previous studies it has been accepted that the presence of specific receptor sites for K88ab and K88ac depends on a gene locus with 2 alleles S and s. The presence of additional receptor sites for K88ad is now presumed to depend on a separate locus with the alleles D and d. The expression of the alleles of the S and D loci is not always complete and is likely to be influenced by epistatic genes. Inhibition or modification of the expression of the receptor sites for K88 can result in intermediate phenotypes.     

Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.     

Enterococcus faecium NCIMB 10415 administration improves the intestinal health and immunity in neonatal piglets infected by enterotoxigenic Escherichia coli K88

Background: This study aimed to investigate the effects of oral administration of Enterococcus faecium NCIMB10415(E. faecium) on intestinal development, immunological parameters and gut microbiota of neonatal piglets challenged with enterotoxigenic Escherichia coli K88(ETEC). A total of 96 1-day-old sow-reared piglets were randomly assigned to 2 groups, with 48 piglets in each group. The piglets were from 16 litters(6 piglets each litter),and 3 piglets each litter were allocated to the E. faecium-supplemented(PRO) group, while the other 3 piglets were allocated to the control(CON) group. After colostrum intake, piglets in the PRO group were orally administrated with 3 × 10~9 CFU E. faecium per day for a period of one week. On day 8, one piglet per litter from each group was challenged(CON+ETEC, PRO+ETEC) or not(CON-ETEC, PRO-ETEC) with ETEC in a 2 × 2 factorial arrangement of treatments. On day 10(2 days after challenge), blood and tissue samples were obtained from piglets.Results: Before ETEC challenge, there were no significant differences for the average daily gain(ADG) and fecal score between the two groups of piglets. After ETEC challenge, the challenged piglets had greater fecal score compared to the non-challenged piglets, whereas E. faecium administration was able to decrease the fecal score.Piglets challenged with ETEC had shorter villous height, deeper crypt depth, and reduced number of goblet cells in the jejunum and decreased m RNA abundance of claudin-1 in the ileum, whereas increased the percentage of lymphocytes, concentrations of IL-1β in the plasma and TNF-α in the ileal mucosa, as well as increased the m RNA abundances of innate immunity-related genes in the ileum tissue. These deleterious effects caused by ETEC were partly alleviated by feeding E. faecium. In addition, piglets in PRO-ETEC group had decreased the percentage of CD8~+T cells of the peripheral blood when compared to those in CON-ETEC group. Moreover, E. faecium administration increased Verrucomicrobia at phylum level and decreased Bilophila at genus level.Conclusions: These results suggest that oral administration of E. faecium alleviated the intestinal injury and diarrhea severity of neonatal piglets challenged by ETEC, partly through improving the intestinal microbiota and immune response. This offers a potential strategy of dietary intervention against intestinal impairment by ETEC in neonatal piglets.     

嗜酸乳杆菌发酵中药对产肠毒素大肠杆菌K88所致腹泻的防治作用

本试验旨在研究嗜酸乳杆菌发酵中药对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）K88所致腹泻小鼠的影响。选取6~8周龄巴比西小鼠（BALB/c）100只,分为4组,分别为空白对照组（Ⅰ组）、大肠杆菌感染模型组（Ⅱ组）、中药发酵组（Ⅲ组）及中药水提组（Ⅳ组）,其中Ⅱ、Ⅲ和Ⅳ组小鼠均用"抗生素+大肠杆菌"模式造模。Ⅰ、Ⅱ组饲喂不含任何药物的基础日粮,Ⅲ、Ⅳ组分别在基础日粮中添加10%中药发酵液、10%中药水提液。统计分析各组小鼠腹泻率、腹泻指数、免疫器官指数、肠道菌群及细胞因子IFN-γ、IL-4的含量。结果显示,ETEC K88感染小鼠36 h后,与Ⅱ组相比,Ⅲ、Ⅳ组小鼠腹泻指数（P < 0.05）与腹泻率降低,且Ⅲ组腹泻指数显著低于Ⅳ组（P < 0.05）,Ⅲ、Ⅳ组盲肠内大肠杆菌数量较Ⅱ组降低,双歧杆菌及乳酸菌数量提高,且Ⅲ组效果更优;Ⅲ、Ⅳ组的免疫器官指数均高于Ⅰ和Ⅱ组,且与Ⅱ组相比,Ⅲ组的脾脏指数、胸腺指数及肝脏指数分别提高了48.37%、29.57%、36.88%,且差异均显著（P < 0.05）;Ⅲ、Ⅳ组IFN-γ/IL-4比值较Ⅱ组提高,且Ⅲ组高于Ⅳ组。综上所述,乳酸菌发酵中药液具有降低腹泻小鼠腹泻率及腹泻指数,提高免疫器官指数,维持肠道菌群平衡及快速消除体内炎症反应等作用,可为开发防制ETEC所致的仔猪腹泻产品提供参考。     

产肠毒素性大肠杆菌K88与K99菌毛蛋白的串联表达

根据GenBank中发表的E.coli K88、K99基因序列,分别设计合成1对引物.利用PCR技术,以大肠杆菌C83907和C83644的质粒为模板分别扩增不含信号肽的K88及K99基因.通过分离、纯化、限制性核酸内切酶酶切,连接和转化,构建了含K88-K99串联表达载体的重组菌株BL21(DE3)(pETK88CK99).结果显示,经酶切,PCR鉴定和DNA序列分析,证实了构建的重组质粒pETK88CK99中含有K88K99融合基因,且基因序列和阅读框架均正确.经过SDS-PAGE分析,串联表达蛋白含量占菌体蛋白的40%左右,经Western blotting检测,该串联表达蛋白能被大肠杆菌K88、K99标准血清识别.结果表明,构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基因工程疫苗的候选菌株.     

Effect of subinhibitory concentrations of tiamulin on the haemagglutinating properties of fimbriated Escherichia coli (K88, K99)

The haemagglutinating properties (HA) of Escherichia coli possessing the fimbrial antigens K88 and K99 were significantly affected by tiamulin. HA for K88-positive strains was reduced at 1/10 to 1/40 minimum inhibitory concentration (MIC), while the corresponding values for the K99 strains were 1/2 to 1/10 MIC. Tiamulin concentrations of 3 to 12 micrograms ml-1 increased salt aggregation test values from between 1.4 and 1.5, to more than 2.     

Escherichia coli K88 receptor expression in intestine of disease-susceptible weaned pigs.

A challenge trial was carried out in which Escherichia coli O157 K88ac was administered to a litter of weaned pigs and the development of the disease monitored over a five-day experimental period. The eight animals in the trial were assigned to two groups depending on whether they exhibited disease symptoms. Six pigs developed diarrhoea and two appeared unaffected; these were designated as the test (or K88-susceptible) group and the control (or K88-resistant) group, respectively. The animals were euthanised and the intestine was removed and sections processed for brush border membrane vesicle preparation. Microscopic and biochemical assays were undertaken on tissue samples from each animal and a strong correlation was observed between the expression of a glycoprotein receptor complex associated with the brush border membrane and the development of disease symptoms. Further investigation revealed the presence of an analogous glycoprotein complex in the K88-resistant group which did not bind the K88-fimbriae antigen. These results suggest that genetic differences in the glycosyl moieties of the receptor complex provide the basis for disease susceptibility to K88-positive E. coli.     

检测K88~+肠毒素性大肠杆菌PCR方法的建立

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     

猪K88、K99、987P、F41埃希氏大肠杆菌高密度发酵培养技术的初步探讨

采用高密度发酵和普通深层通气两种方法培养含K88、K99、987P、F41菌毛抗原的四株猪埃希氏大肠杆菌,对其培养液进行活菌数、OD值、pH值、效价的测定。实验结果表明,运用高密度发酵方法培养,各菌活菌数可达4.1×1010~4.9×1010CFU/mL,效价为211~213;运用普通深层通气方法培养,各菌活菌数为0.51×1010~0.59×1010CFU/mL,效价为24~25,可选择高密度发酵方法替代普通深层通气方法培养,用于制备猪埃希氏大肠杆菌K88、K99、987P、F41四价菌毛提纯苗。     

Escherichia coli diarrhoea in pigs with or without the K88 receptor.

There was a high incidence of neonatal scours in 38 litters of pigs born at Compton in a four month period during 1978. The most important cause of the disease was an enteropathogenic Escherichia coli strain which possessed the K88 antigen. The Compton herd has been bred to produce pigs of three genotypes with respect to the presence or absence of the intestinal receptor for the K88 antigen. These are homozygous dominants (SS) and heterozygotes (Ss) susceptible to infection by virulent K88-positive E coli, and homozygous recessives (ss) resistant to the disease. The highest incidence of diarrhoea was in the susceptible progeny of resistant dams and susceptible sires. There was no K88 associated diarrhoea in resistant progeny or in susceptible progeny of susceptible dams.     

Effects of casein glycomacropeptide supplementation on growth performance,intestinal morphology,intestinal barrier permeability and inflammatory responses in Escherichia coli K88 challenged piglets

Casein glycomacropeptide(CGMP) is a bioactive peptide derived from milk with multiple functions. This study was aimed at evaluating the effects of CGMP as a potential feed additive on growth performance,intestinal morphology, intestinal barrier permeability and inflammatory responses of Escherichia coli K88(E. coli K88) challenged piglets. Eighteen weaning piglets were randomly assigned to three groups.Control group and K88 challenged group received a basal diet, and CGMP treated group received the basal diet supplemented with 1% of CGMP powder. The trail lasted for 12 days, K88 was orally administered to the piglets of K88 challenged group and CGMP treated group on days 8-10. The results showed that the diet containing 1% CGMP significantly alleviated the decrease in average daily gain(P 0.05),increase in pathogenic bacteria amounts in intestinal contents(P 0.05), intestinal morphology(P 0.05) and barrier permeability damage(P 0.05), and acute inflammatory response(P 0.05)induced by E. coli K88 infection. In conclusion, CGMP supplementation in the diet protected the weaning piglets against E. coli K88 infection.     

产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子fae全基因的克隆、表达及生物学活性的初步研究

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.