Influence of the Intestinal Flora on the Development of Immune Reactions in Infants

Breast-fed and artifically fed infants are in contact with the O antigen of Escherichia coli from the first days after birth. From the mother, the infant obtains antibodies against nonpathogenic E. coli strains in low titer, and the infant begins to form its own antibodies during the 2nd month of life. The transition is known to be continuous even though the transferred antibodies could not be differentiated from the infant's own antibodies. Contact with endotoxin caused sensitization which was detected by the skin test at about 2.5 months, and thereafter the skin test data correlated with the presence of serum antibodies against endotoxin. The newborn infant can be colonized with a different E. coli serotype; such an antigenic stimulus evokes the formation of antibodies sooner and at a significantly higher titer than (i) the level of maternal antibodies transferred or (ii) the infant's antibodies normally formed later on against other random E. coli serotypes.     

Evaluation of usefulness of the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to lipopolysaccharides of VTEC strains in patients suspected for Escherichia coli O104:H4 infection in Poland

The aim of the study was to investigate the presence of antibodies to lipopolysaccharides obtained by modified Boivin's method from E. coli serotype O104:H4 and O26, O103, O111, O121, O145, O157 in sera of 7 patients with acute diarrhea, suspected in clinical investigation for infection caused by E. coli O104:H4. Additionally, to determine the cut-off levels, the 75 sera from blood donors were tested. The high level of antibodies to LPS E. coli O104 was diagnosed in three patients from family outbreak caused by E. coli serotype O104:H4. In one of those patients, 7-years boy with HUS, we observed also a significant decrease of level of IgA, IgG and IgM antibodies in serum sample obtained in chronic phase of the disease. Furthermore, we showed that two others patients with clinical evidence of VTEC infection, not connected with this family outbreak, had a high level of antibodies to E. coli of other serotypes: one to O157 and one to O103. We did not observe presence of antibodies to LPS from E. coli O26, O111, O121 i O145 in the sera of tested patients. In conclusion, we confirmed that ELISA based on lipopolysaccharides obtained from different serotypes of E. coli may be helpful in laboratory diagnosis of infection caused by VTEC in humans.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Legionella pneumophila surface antigens cloned and expressed in Escherichia coli are translocated to the host cell surface and interact with specific anti-Legionella antibodies.

Escherichia coli clones that express Legionella pneumophila antigens were isolated from a plasmid genomic library, and their antigens were characterized by immunoblotting with rabbit anti-L. pneumophila sera. Because previous studies of L. pneumophila antigens by whole-cell radioimmunoprecipitation suggested that comigrating native antigens were surface localized, we conducted experiments to determine if the cloned antigens were surface expressed in E. coli. Aliquots of antisera were absorbed by intact cells of three representative antigen-producing E. coli clones, and surface-bound antibodies were acid eluted from the intact cells. Immunoblots made with selectively absorbed antisera and eluted antibodies confirmed that reactivity to the homologous cloned antigens could be specifically absorbed from the antisera and then eluted from the cells, demonstrating a surface (antibody-accessible) localization in the cloned state. Antibodies eluted from the surface of an E. coli clone that expressed a 19-kilodalton antigen reacted with the surface of L. pneumophila in a liquid-phase, whole-cell enzyme-linked immunosorbent assay. In addition, intact cells of this clone were used in an enzyme-linked immunosorbent assay to detect serum antibody. E. coli cells that express foreign antigens on their surfaces can be used to develop antigen-specific immunoassays and to affinity purify monospecific antibodies.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli

Patients with haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC) produce serum antibodies to the lipopolysaccharides (LPS) of Escherichia coli O157 and certain other E. coli serogroups. Patients may also make salivary antibodies to the LPS of E. coli O157. Serological tests based on these antibodies can be used to provide evidence of infection in the absence of culturable VTEC or the toxins they produce. Serum antibodies to LPS persist for several months following onset of disease, enabling both current and retrospective serological testing. The LPS of E. coli O157 shares epitopes with strains of Brucella abortus, Yersinia enterocolitica O9, Vibrio cholerae O1 Inaba, group N Salmonella and certain strains of Citrobacter freundii and E. hermanni. Serological tests for serum antibodies to E. coli O157 should be evaluated in the light of these cross-reactions. Serological tests to supply evidence of infection with E. coli O157 have been shown to provide a valuable adjunct to bacteriological procedures for detecting culturable VTEC and VT. The use of well characterized LPS antigens in association with the techniques of ELISA and immunoblotting provide valuable procedures for detecting evidence of infection with E. coli O157 and possibly other VTEC.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

Serological identification of infection by verocytotoxin-producing Escherichia coli

One hundred sera from patients with haemolytic ureamic syndrome were screened for antibodies to the lipopolysaccharide (LPS) of Escherichia coli serogroup 05, 026, 0115, 0128, 0145 and 0153, and Shigella dysenteriae 1. Three sera contained antibodies to the LPS of E. coli belonging to serogroup 05.     

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:H7 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7.     

Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB).

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

Preparation of recombinant glycoprotease of Pasteurella haemolytica A1 utilizing the Escherichia coliα-hemolysin secretion system

Abstract Three murine monoclonal antibodies were prepared against the recombinant glycoprotease of Pasteurella haemolytica A1 expressed in Escherichia coli . These monoclonal antibodies were able to recognize the authentic glycoprotease from P. haemolytica A1 culture supernatant. A recombinant plasmid which contained most of the glycoprotease gene of P. haemolytica A1 fused with the secretion signal sequence from hlyA of the E. coli α-hemolysin determinant was constructed. This recombinant plasmid expressed a fusion protein (Gcp-F) which was secreted into the culture supernantant by E. coli cells when the α-hemolysin secretion functions HlyB and HlyD are supplied in trans. Gcp-F could be readily recovered from the supernatant free from other cellular materials and is suitable for use in vaccine trials and challenge experiments in animals.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters.

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

Immunochemical and Enzymatic Comparisons of the Tryptophan Synthase α Subunits from Five Species of Enterobacteriaceae

The reactive surface structures of alpha subunits of tryptophan synthase from Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Aerobacter aerogenes, and Serratia marcescens were compared by measuring (i) their reactivities in micro-complement-fixation assays with antibodies directed specifically to E. coli wild-type alpha subunit, (ii) their reactivities in enzyme neutralization assays with the same antibodies, and (iii) their binding affinities for tryptophan synthase beta(2) subunits. The enzymes from the four heterologous species cross-reacted in the microcomplement-fixation assays with the anti-E. coli alpha subunit antibodies, each to a different degree. However, neutralization titers of the antibodies reacting with the various alpha subunits were comparatively similar, and the beta(2) subunit-binding and -stimulating abilities of the alpha subunits were even more closely alike. The results suggested that the tertiary structure of the beta(2) subunit-binding site of the alpha subunit has been conserved, relative to the rest of the molecule, during the evolutionary divergence of the species of Enterobacteriaceae.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Monoclonal antibodies against O and K antigens of uropathogenic Escherichia coli O4 : K12 : H− as opsonins

Abstract Monoclonal antibodies of subclasses IgG1 and IgG2b and specific for the O4 antigen of Escherichia coli 20025 (O4 : K12 : H⁻) and the capsular K12 polysaccharide of the same strain (IgM) were obtained with the hybridoma technique using spleen cells from Balb/c mice, immunized with a crude bacterial extract, and Sp2/O-Ag8 myeloma cells. The anti-O4 antibodies reacted exclusively with the O4 lipopolysaccharide and not with those from serologically O-cross reactive E. coli . The anti-K12 antibodies recognized as epitope (part of) the KDO moiety of the capsular K12 polysaccharide. Not only anti-K12, but also anti-O4 antibodies effectively phagoopsonized encapsulated E. coli 20025. The opsonized bacteria were killed in subsequent in vitro phagocytosis by human leokocytes in the presence of human serum complement.     

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.