Influence of bovine intestinal fluid on the expression of K99 pili by Escherichia coli

A study was conducted to determine whether intestinal fluid collected from various portions of bovine intestine differed in its effect on production of K99 pili by Escherichia coli. The small and large intestines of 7 calves, euthanatized 4 hours after a final feeding of milk, were divided into 6 to 9 segments from which intraluminal fluids were collected. Depending on the amount of fluid collected, up to 20 E coli strains that express K99 pili were grown on media prepared from the content of each specimen and then were tested for K99 pilus expression. In general, intestinal fluid from the most proximal small intestinal segments were more suppressive to K99 pilus expression than was fluid from more distal segments of small intestine. Only about 20% of the E coli test strains expressed K99 pili when grown on medium prepared from proximal small intestinal segmental fluid, whereas greater than 90% did when grown on medium prepared from distal small intestinal segmental fluid. Fluid from the large intestine varied considerably from calf to calf in its effect on K99 pilus expression. A correlation was found between K99 pilus expression and pH of the intestinal fluid, with the lower pH values (characteristic of proximal intestinal segmental fluid) being suppressive. The correlation between K99 pilus production and the pH of the medium was verified, using defined laboratory media adjusted to various pH values. Strains of E coli grown in medium at or below pH 5.5 failed to express K99 pili, whereas the same strains when grown in medium at or above pH 6.5 expressed K99 pili in abundance.     

Enterotoxigenic K99+ Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein

Most enterotoxigenic Escherichia coli (ETEC) isolated from neonatal cattle with diarrhea (enteric colibacillosis) exhibit the colonization factor antigen, K99. The K99 pili are necessary for the bacteria to bind to a receptor, N-glycolylneuraminic acid-GM3 on the host cells in the small intestine where the bacteria multiply and secrete toxins that cause the diarrhea. When the attachment of the ETEC to host cell is inhibited, the bacteria do not accumulate sufficiently in the gut to cause disease. Since purified K99 pili block K99+ ETEC from binding to host epithelia, three recombinant K99 proteins of different sizes were developed and produced to demonstrate inhibition with in vitro competitive binding assays. The full-length recombinant protein, rK99-476 inhibited the binding of ETEC with an activity similar to that of the native purified K99, whereas the truncated recombinant K99 protein had no inhibitory activity. Thus this binding activity of rK99-476, which is specific and effective in blocking the receptors on the host cells, may be able to competitively inhibit K99+ ETEC infections in cattle.     

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.     

Prevalence of K88, K99, and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis

One hundred nineteen live neonatal pigs with diarrhea less than or equal to 2 weeks old were euthanatized, and frozen sections of their ilea were submitted to an indirect fluorescent antibody technique to identify K88, K99, and 987P pili (also referred to as F-4, F-5, and F-6 pili, respectively) in Escherichia coli. Ten-centimeter ileal sections were used to determine numbers of lactose-fermenting bacteria. Of 52 pigs in which E coli pili were found, 14 had K88 (27%), 23 had K99 (44%), 13 had 987P (25%), and 2 had K88 and K99 simultaneously (4%). Numbers of lactose-fermenting bacteria were significantly (P less than or equal to 0.05) higher in pigs with piliated E coli than in pigs without piliated E coli. Results of this study indicated that piliated E coli are a major cause of enteric disease in neonatal swine in Michigan, and that in pigs less than or equal to 2 weeks of age, K99 was the most frequently encountered pilus antigen.     

Isolation by phage display of recombinant antibodies able to block adherence of Escherichia coli mediated by the K99 colonisation factor

K99 fimbriae are important for intestinal colonisation by bovine strains of enterotoxigenic Escherichia coli. The mode of action of this colonisation factor is well understood and specific immune responses are protective. K99 was therefore chosen for this study as a model to test if antibodies with anti-adhesion activity could be isolated from recombinant libraries using phage display techniques. Potentially, this strategy could be used to understand better the action of bacterial colonisation factors and aid the design of therapies (e.g. vaccines, purified protein products or bacteria bearing colonisation-blocking antibodies) to inhibit bacterial adherence. The major fimbrial subunit from K99, FanC, was purified from a clinical E. coli isolate. The protein was coated to plastic immunotubes and used as a target for selection of antibodies from the Tomlinson I and J libraries of single chain (scFv) antibodies. Clones able to recognise K99 were isolated by iterative rounds of binding, elution and amplification. scFv antibodies chosen from the resulting panel were purified and their specificity confirmed by ELISA. Pre-incubation of several scFvs with bacteria expressing K99 fimbriae inhibited the agglutination of erythrocytes. Further investigation by microscopy confirmed that when E. coli expressing K99 were exposed to scFv antibodies, the binding of bacteria to erythrocytes was blocked with high efficiency. The study showed that recombinant antibodies were able to block the action of a bacterial colonisation factor and hence that phage display techniques might be applied to the identification of less well-characterised virulence factors and the analysis of their structure and function.     

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.     

Cloning and expression of a pili gene of Corynebacterium renale in Escherichia coli

A plasmid gene library of Corynebacterium renale piliated strain No. 109P+ was prepared in Escherichia coli in order to study the chemical structure of the pili of C. renale. Of 3,000 recombinant clones tested, 5 reacted with anti-pili anti-serum. The gene products of these clones reacted with anti-pili monoclonal antibodies 8/4, 5/2 and B20/3 but lacked the reactivity with 13/4. SDS-PAGE analysis revealed that the expressed protein had a molecular mass of 48 kilodalton and deletion analysis showed that the encoding region for this protein was localized within a 1.4 kilobase gene including a promoter sequence. Immunoelectron microscopy showed that mouse antibodies raised to the expressed protein bound to the entire surface of the pili of C. renale. These results indicate that the cloned gene encodes a major structural protein of C. renale pili.     

Passive immunisation of neonatal lambs against infection with enteropathogenic Escherichia coli via colostrum of ewes immunised with crude and purified K99 pili

Lambs sucking ewes immunised four to five weeks before parturition with crude preparations of K99 and purified K99 pili of single subunit composition were protected against challenge infection with heterologous enteropathogenic Escherichia coli strains. In contrast, the majority of lambs sucking sham-immunised ewes suffered severe diarrhoea and dehydration, followed by death in nearly half of the affected lambs. Protection was related to the presence of antibody in the colostral whey and lamb sera. K99-specific antibody activity in the colostral whey was found to be confined to IgM and IgG (IgG1 and IgG2) but not to the IgA class.     

Effects of exogenous amino acids on the multiplication of porcine Escherichia coli

The multiplication rates of 70 porcine Escherichia coli strains were compared in minimal medium and in medium supplemented with aspartic acid, lysine, serine and threonine, which were the amino acids taken up during multiplication of porcine E. coli in a complex medium. The effects of these amino acids singly or in combinations and the amino acids norleucine and norvaline on the growth of porcine E. coli were studied. Together, aspartic acid, threonine, serine and lysine increased the multiplication rates of 42.9% of the strains, an effect traced to aspartic acid, but they had no effect on an equal number of strains. The rest were inhibited, and this effect was traced to serine. Cysteine, threonine, leucine and phenylalanine singly inhibited some or all of the strains tested. Norleucine and to a lesser extent, norvaline greatly prolonged the lag phase of culture in minimal medium. The inhibitory effect of norleucine was reversed by only methionine, although isoleucine, leucine and valine which were more effective in norvaline inhibition, also showed limited antagonism to norleucine inhibition.     

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.     

Combined rotavirus and K99 Escherichia coli infection in gnotobiotic pigs

Fifty nine 3-day-old gnotobiotic pigs were randomly assigned to 4 experimental groups: 14 pigs were orally inoculated with rotavirus (RV), 14 were orally inoculated with enterotoxigenic Escherichia coli (ETEC), 18 were orally inoculated with both agents, and 13 were controls. Pigs inoculated with RV plus ETEC were given the RV inoculum at 3 days of age and then, 24 hours later, were given the ETEC inoculum. Three pigs inoculated only with RV, 3 pigs inoculated only with ETEC, 4 pigs inoculated with RV plus ETEC, and 3 pigs in the control group were euthanatized at 5 and 7 days of age. Two pigs in each of the 4 experimental groups also were euthanatized at 9 days of age. Intestinal segments from 6 sites in the small intestine were examined by virologic, bacteriologic, and histologic procedures. For 10 days after inoculation, the remaining pigs in each group were observed clinically to monitor severity and duration of diarrhea, mortality, and shedding of RV or ETEC. Pigs inoculated with the combined RV plus ETEC inoculum developed more severe diarrhea, compared with pigs inoculated with the single agents; all dually inoculated pigs died between 3 and 6 days after inoculation. There was no mortality in pigs inoculated with either RV or ETEC. Lesions were restricted to the small intestine in pigs inoculated with RV plus ETEC and in pigs inoculated with RV or ETEC. There was no difference in the severity of the villus atrophy between the dually inoculated pigs and pigs inoculated only with RV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial adherence in mastitis caused by Escherichia coli.

The possible role of bacterial adherence in the pathogenesis of experimental mastitis in the mouse was examined with four strains of Escherichia coli. Two of these strains had a known adhesion antigen (K88) and two did not. The K88 antigen did not play a significant role in the virulence or infectivity of E. coli either in the murine or bovine mammary gland. Two E. coli strains, W1 (K88+) and J2 (K88-) were virulent in the mouse but did not adhere to epithelial cells. Both these strains produced clinical mastitis in the cow. A third strain, D282 (K88-), produced mild disease in the mouse but was avirulent in the cow. The fourth strain, 233/ID (K88+), was avirulent in both the mouse and the cow. Strains D282 and 233/1D were killed rapidly by bovine serum whilst J2 and W1 were more resistant. All strains were more sensitive than the control resistant strain E. coli P4, which is known to be highly virulent for the lactating udder.     

Adhesion of K99-positive Escherichia coli to intestinal brush borders of pigs

Ileal samples from 242 pigs, collected at 3 Michigan slaughterhouses, were studied to determine the prevalence of intestinal receptors for K99-positive Escherichia coli. A brush border adhesion test was used to identify the receptors. Of the 242 samples examined, receptors were demonstrated in 230 (95%). After storage of brush border preparations at 4 C, bacterial aggregates lacking identifiable brush border fragments were present in samples tested for adhesion, indicating that K99 receptors may be released from brush border membranes. Seemingly, most, if not all, pigs have intestinal receptors for K99 pili, and an inheritance pattern similar to that observed for K88 receptors probably does not exist for K99 receptors.     

The diagnostic value of immunoperoxidase in detecting K99+ Escherichia coli on histological sections

The small intestine of 51 calves was examined for the presence of K99+ Escherichia coli by means of both an immunoperoxidase procedure performed on paraffin sections and by the slide agglutination test after isolation. Twelve cases resulted immunoperoxidase positive (23.5%) and 8 of them were also agglutination positive. Results of the 2 diagnostic tests agreed in 46 cases (90.2%). In immunoperoxidase positive sections a thick layer of immunoreactive bacteria was seen on the luminal surface of the enterocytes. Post-mortem autolysis or prolonged fixation did not seem to affect the immunoperoxidase reactivity of the sample.     

肠毒素性大肠杆菌K99-ST1融合基因的克隆及原核表达

为有效预防肠毒素性大肠杆菌（ETEC）引起的犊牛和羔羊腹泻,将PCR扩增获得的ETECK99菌毛抗原基因和人工合成的ST1基因片段,借助pUCm—T载体,构建了重组质粒pUCm-K99-ST1,从而获得了融合基因K99-ST1;使用EcoRⅠ＋SalⅠ双酶切将该融合基因定向插入表达载体pET-30a中,成功构建了表达载体pET—K99-ST1,将其转入表达菌株BL21（DE3）中,经IPTG诱导,获得31ku的蛋白;Western—blotting结果显示,该融合蛋白可与K99阳性血清发生特异性反应。     

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。