Serotype, hemolysin production, and adherence characteristics of strains of Escherichia coli causing urinary tract infection in dogs.

Virulence factors were studied in 82 strains of Escherichia coli isolated from the urine of dogs with urinary tract infections. The most frequently expressed O antigens were 2, 4, 6, 25, and 22/83. Most strains were K nontypeable. Mannose-sensitive hemagglutination (MSH) with canine erythrocytes was observed in 71 strains and mannose-resistant hemagglutination (MRH) was observed in 32 strains. Strains that caused MSH of erythrocytes from dogs also caused MSH of erythrocytes from guinea pigs. Most strains that caused MRH of human A1P1 erythrocytes also reacted with erythrocytes of dogs. Of 22 strains (27%) that agglutinated human A1P1 erythrocytes, but not A1p erythrocytes, 17 (77%) had specificity for globo A, but did not react with the galactose alpha 1----4galactose beta disaccharide receptor. The remaining 5 strains and 2 others that simultaneously expressed an X adhesin agglutinated galactose alpha 1----4galactose beta-coated latex beads. Bacterial adherence to canine uroepithelial cells from the bladder was most often observed in strains expressing MSH, less often observed in strains expressing MRH, and least often observed in strains that failed to induce hemagglutination. Adherence of MSH strains to canine uroepithelial cells was inhibited by alpha-methyl-D-mannoside. As a group, MRH strains expressing globo-A- and galactose alpha 1----4galactose beta-specific adhesins did not have strong adherence. Strains of E coli isolated from dogs with urinary tract infections most commonly expressed type-1 fimbriae, and the main mechanism of in vitro adherence to canine uroepithelial cells involved a mannose-sensitive mechanism. Overrepresentation of globo-A-specific adhesins did not appear to be related to adherence of canine uroepithelial cells.     

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     

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea

The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.     

Occurrence of mannose resistant hemagglutinins in Escherichia coli strains isolated from porcine colibacillosis

Three-hundred and fifty-eight E. coli strains isolated from piglets were tested for the presence of hemagglutinins by the use of the active hemagglutination test with or without mannose. Additionally 86 strains from the mentioned number of strains were investigated for the presence of common fimbriae using the same method but growing the strains in media especially suited for the development of this kind of fimbriae. These 358 strains and additionally 202 E. coli strains were tested using antisera for 987P and K88 antigens. It was found, using the active hemagglutination test, that 51.4% of the strains were hemagglutinating. The hemagglutinating strains carried the K88 antigen. All these strains were isolated from new-born and weaned piglets with enterotoxic form of colibacillosis, called also E. coli diarrhea. From cases of this form of colibacillosis originated also 26.7% of the strains in which common fimbriae (type 1) were detected. This result was obtained when the BHI medium was used for cultivation. In case of TSA medium only 2.3% of strains were positive. No specific or common fimbriae were found in strains recovered from septic form of colibacillosis and oedema disease (called also enterotoxaemic form of colibacillosis). No strain of 560 examined showed the presence of fimbrial 987P antigen.     

Hemagglutinating and hydrophobic surface properties of salmonellae producing enterotoxin neutralized by cholera anti-toxin

Eleven Salmonella strains known to produce enterotoxin under aerobic culture conditions in deferrated (DF) medium at 37°C were shown to produce enterotoxin with and without aeration at 22, 28, 37 and 42°C. Heat-labile enterotoxin was generally produced with growth temperatures up to 37°C irrespective of aeration. Heat-stable enterotoxin was produced up to 42°C, mainly aerobically, as indicated by infant mouse assay (IMA), by six of the eleven strains tested. Nine strains produced heat-stable rapid permeability factor (RPF) in rabbit skin.Cholera anti-toxin neutralized reactivities of Salmonella heat-labile enterotoxin in four different biological assays. Mixed gangliosides also neutralized this activity in the cell—test systems.With guinea-pig erythrocytes, all strains underwent mannose-resistant hemagglutination (MRHA) irrespective of growth temperatures, i.e. 22 and 37°C or medium, i.e., DF, tryptose soy broth (TSB) and colonization factor antigen (CFA) agar. At both growth temperatures, CFA agar-grown cells of each strain caused MRHA of bovine erythrocytes. Excepting three Salmonella typhimurium strains, DF broth-grown cells gave MRHA of bovine, chicken and human group A erythrocytes, CFA agar-grown cells caused MRHA of chicken and human blood, whereas TSB-grown cells caused few MRHA reactions.Salmonellae producing both heat-stable, (ST) and heat-labile, (LT) enterotoxins adsorbed to Phenyl Sepharose whereas salmonellae that produced only LT enterotoxin did not.The presence of MRHA adhesions did not correlate with cell-surface hydrophobicity. However, mannose-resistant hemagglutinins may occur more commonly among salmonellae than has been previously recognized.     

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.     

Inactivated Salmonella expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination

We examined the potential of inactivated Salmonella strains to induce protective antibodies against two adhesins of pathogenic Escherichia coli. The receptor-binding domains of the F17a-G adhesin of F17a fimbriae and of the FimH adhesin of type 1 fimbriae were fused to the translocator domain of the autotransporter AIDA-I. An IgG response against F17a-G or FimH was induced after immunization of mice with acetone-inactivated Salmonella displaying the corresponding fimbrial receptor-binding domain. These sera inhibit in vitro agglutination of erythrocytes by E. coli carrying these fimbriae. Our results demonstrate that induced and subsequently acetone-inactivated Salmonella are useful delivery vehicles for the stimulation of an IgG antibody response against heterologous antigens.     

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.     

Curli expression by Escherichia coli strains isolated from bovine mastitis

The aim of the study was to determine the expression of mannose-sensitive and mannose-resistant adhesins by agglutination of cattle, sheep, goat, rabbit, horse, and chicken red blood cell assay, and curli fimbriae by Congo red binding assay among 341 E. coli strains isolated from 51 milk samples of clinically recognized bovine mastitis. Curli fimbriae expression within biofilms created on an inert surface was also investigated. To determine whether curli fimbriae are expressed both in conditions optimal for their production and in conditions resembling the host organism, the study was conducted in anaerobic atmosphere at 37 degrees C, and at room temperature in aerobic atmosphere. The results demonstrated that although the E. coli isolates examined were deprived of mannose-sensitive and mannose-resistant adhesins they were able to produce curli fimbriae in both aerobic and anaerobic conditions at room and higher temperature, indicating that these adhesins may be involved in the pathogenesis of bovine mastitis.     

Humoral Antibody Responses of Swine Infected Experimentally with Group E Streptococcus I. Whole Cell Agglutinin Response

Reference streptococcal antisera and sera collected from swine infected experimentally (by intranasal inoculation or contact exposure) with group E Streptococcus (GES) were studied in a tube agglutination system using whole GES cells.

Specificity studies revealed common group specific antigen among GES serotypes I and III, GES strains devoid of type specific antigen (untypable by ring precipitin testing) and group P and group U Streptococcus. The group specific antigens were not agglutinated by GES type specific antisera or by group specific antisera against Streptococcus groups A, B, C, D, F, G, H, K, L, M, N, or O. Results of the study suggested that GES serotypes I and III are invalid; i.e., they are devoid of type specific antigen.

Groug E Streptococcus type specific antigens II, IV, and V were agglutinated significantly only by their homologous antisera.

Experimentally infected swine developed significant titers against both the group and type specific antigen of GES. Antibodies appeared from three to eight weeks postexposure and persisted for the duration of the experiment (six months). The potential utilization of the whole cell agglutination (WCA) test for detection of GES carrier swine is discussed.

     

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli. strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-IIv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strauns enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.     

Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli

In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh+ strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

An examination of Escherichia coli strains isolated from cases of bovine mastitis for possible virulence determinants

Ninety-five strains of Escherichia coli isolated from cases of bovine mastitis were examined for the possession of some of the possible virulence determinants. Ten strains which caused haemagglutination of bovine and ovine erythrocytes were considered to be fimbriated and an additional strain caused agglutination of chicken erythrocytes. Colicines were produced by fifteen strains and in three strains the colicine was identified as Col V. Forty-one of 71 strains that were examined serologically possessed capsular or envelope K antigens.     

Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes

Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test. K88ac always gave negative HA results with porcine red cells. However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera. K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.     

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.     

Moraxella bovis hemagglutinins: effect of carbohydrates, heating and erythrocytes.

Several properties of the adhesins of eight isolates of Moraxella bovis recovered from cattle suffering from infectious keratoconjunctivitis, were studied. Adhesions were detected through autoagglutination in saline and hemagglutination. Autoagglutinating strains agglutinated red blood cells of the chicken, rabbit, sheep and swine, but not those of the guinea pig. The adhesins were not inhibited by D-mannose or D-galactose and resisted heating at 100 degrees C for 15 minutes. Magnesium chloride at a final concentration of 10% inhibited autoagglutination and hemagglutination. The value of the hemagglutination test for monitoring synthesis of fimbriae by M. bovis, is discussed.     

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.     

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.     

Phenotypic expression of K88 adhesin alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41

F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens. Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium. K99 antigen could be detected in each strain bearing K99 plasmid. Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen. Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all. Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen. None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20°C as found for K99 and F41 ETEC natural strains, respectively. These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains. In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.