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.     

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.     

Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets

Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC). Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05). Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal. Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E. coli and the number of bacteria adhering to 250 micron villus brush border was counted. Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection. Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001). The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E. coli.     

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.     

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     

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.     

A tryptophan-enriched diet improves feed intake and growth performance of susceptible weanling pigs orally challenged with Escherichia coli K88

We tested the effect of Trp addition to a standard weaning diet and oral challenge with enterotoxigenic Escherichia coli K88 (ETEC) on growth and health of piglets susceptible or nonsusceptible to the intestinal adhesion of ETEC. Sixty-four pigs weaned at 21 d of age were divided into 3 groups based on their ancestry and BW: a control group of 8 pigs fed a basal diet (B), the first challenged group of 28 pigs fed B diet (BCh), and the second challenged group of 28 pigs fed a diet with Trp (TrpCh). The Trp diet was produced by the addition of 1 g of l-Trp/kg to the basal diet. On d 5, pigs were orally challenged with 1.5 mL suspension containing 10(10) cfu ETEC/mL or placebo, and killed on d 9 or 23. Based on in vitro villus adhesion assay, the pigs (except the B group) were classified as susceptible (s(+)) or nonsusceptible (s(-)) to the intestinal ETEC adhesion. Thus, after the challenge, treatments were B, BChs(-), BChs(+), TrpChs(-), and TrpChs(+). Pigs susceptible to ETEC were 50.0% in the BChs(+) group (3 pigs lost included) and 46.4% in the TrpChs (+) group (1 pig lost included). During the first 4 d after challenge, the challenge reduced ADG (P < 0.05), and this reduction was greater in susceptible pigs (P < 0.05) than nonsusceptible ones. Tryptophan increased ADG and feed intake in susceptible pigs (P < 0.05) from challenge to d 4, but not thereafter. Tryptophan supplementation did not improve the fecal consistency and did not reduce the number of pigs positive for ETEC in feces on d 4 after the challenge. The K88-specific immunoglobulin A activity in blood serum tended to be greater in challenged pigs (P = 0.102) and was not affected by the addition of Trp. Villous height was affected by the addition of Trp and challenge in different ways, depending on the site of small intestine. The need to consider the phenotype for the adhesion of the ETEC in studies with different supply of Trp was clearly evident. When compared with practical weaning standard diets, Trp supplementation allowed susceptible pigs to partially compensate for the effects of ETEC challenge by increasing feed intake and maintaining an adequate BW growth. This is of practical importance for the formulation of diets for pigs selected for lean growth because of the presence of an association between this trait and the susceptibility to the intestinal adhesion of ETEC.     

Antimicrobial resistant Escherichia coli in the reproductive tract microbiota of cows and sows

Escherichia coli is a natural colonizer of the urogenital mucosa of healthy females; however it is one of the pathogens associated to reproductive failures in cows and sows. A better knowledge about the characteristics of native E. coli will allow us to differentiate them from pathogenic strains. Ninety autochthonous isolates from the reproductive tract of sows and cows were characterized to determine the phylogenetic profile, antibiotic resistance and virulence factors; also, comparisons between different breeding systems were performed. Vaginal colonization of E. coli was statistically higher in cows (57.5%) than sows (23.8%), and most isolates belonged to the phylogenetic group A: 79.69 and 80.77%, respectively; moreover phylo-groups B1 (12.5 and 11.54%) and D (7.81 and 7.69%) were significantly lower; however, none was classified as B2. Positive associations between virulence factors and group D were found. Isolates with antimicrobial susceptibility were associated with group A and the MDR (Multiple Drug Resistance) was related to the porcine source. These results contribute to the knowledge of extra-intestinal E. coli populations; which could affect the reproductive performance of females.     

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10⁷ cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.     

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.     

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.     

The effect of antibiotics on mannose-resistant haemagglutination by K88- and K99-positive Escherichia coli strains

Five E. coli strains carrying K99 antigen isolated from the intestines of calves which had succumbed to diarrhoea and six K88-positive strains isolated from fatal cases of diarrhoea in piglets were examined for their mannose-resistant haemagglutination (MRHA) capacity against pig erythrocytes. The bovine strains showed a geometric mean MRHA-titre of 1/18 and the porcine strains one of 1/45. Similar experiments were carried out after addition of the following antibiotics in doubling dilutions: ampicillin, chloramphenicol, colistin, dihydro-streptomycin, gentamicin, neomycin, polymyxin B and oxytetracycline. Colistin and polymyxin B had a marked concentration-dependent inhibitory effect on MRHA. Neomycin and gentamicin also inhibited MRHA but to a lesser degree. Chloramphenicol, dihydrostreptomycin and oxytetracycline showed no effect. With ampicillin, a trend was found for the ratio values to be inversely proportional to the concentration. This suggests that this antibiotic has an enhancing effect on the haemagglutination.     

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.     

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.     

产肠毒素大肠杆菌双重PCR检测方法的建立

为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314 bp;最终确定dNTP终浓度0.4 mmol/L,K88、K99的引物终浓度均为25 μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。     

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。