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A D O'Brien

Publications and source records attributed to A D O'Brien.

At least 73 records · Page 4Linked to original sources
PubMed1988

Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay.

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five lines had kappa light chains. These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies. The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value.

Animals↗
PubMed1987

Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli.

A nucleotide sequence analysis was performed on the Shiga-like toxin I genes previously cloned from the Escherichia coli bacteriophage 933J. Two structural genes designated slt-I A and slt-I B were found to be oriented on a single transcriptional unit with slt-I A preceding slt-I B. A 12 base pair gap separated the in-phase open reading frames of slt-I A and slt-I B. Putative ribosome binding sites were identified 5' to both the slt-I A and slt-I B genes. Translation of the SLT-I nucleotide sequence revealed that both the A and B subunits were synthesized with signal peptides. The molecular weight calculated for the mature A subunit was 32,211 while the molecular weight of the mature B subunit was 7690.

Amino Acid Sequence↗
PubMed1987

Production of a unique cytotoxin by Campylobacter jejuni.

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.

Animals↗
PubMed1987

Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets.

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin. The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined. The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E. coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain. Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation. All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon. No such lesions were observed in 12 piglets inoculated with three strains of E. coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT. Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative). The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea. The reason for the reduced virulence of this strain was not clear. These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E. coli O157:H7 in gnotobiotic piglets.

Animals↗
PubMed1987

A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new fimbrial antigen and for adhesion to epithelial cells.

Of 14 strains of Escherichia coli O157:H7 isolated from patients with hemorrhagic colitis or hemolytic uremic syndrome that were examined for fimbriae, the presence of plasmids, and the ability to adhere to intestinal cells, 13 possessed a 60-megadalton plasmid and were fimbriated as assessed by electron microscopy. These strains adhered to Henle 407 intestinal cells but not to HEp-2 cells or erythrocytes. Three strains were cured of the plasmid and thereafter failed to express fimbriae and lost the ability to adhere to intestinal cells. Conversely, E. coli K-12 transformed with the 60-megadalton plasmid from each of the three strains produced fimbriae and was able to adhere to intestinal cells. A single fimbrial subunit of 16 kilodaltons was observed when purified fimbriae from the transformants and from the 60-megadalton plasmid-containing E. coli O157:H7 strains were disaggregated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera raised against one preparation of the purified fimbriae reacted strongly with 12 of 14 O157:H7 isolates in an agglutination assay and with purified fimbrial preparations from five E. coli O157:H7 strains in an enzyme-linked immunosorbent assay.

Animals↗
PubMed1987

Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea.

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Macrophage defect and inflammatory cell recruitment dysfunction in Salmonella susceptible C3H/HeJ mice.

C3H/HeJ mice are homozygous for the Lpsd allele and, as a consequence, are hyporesponsive to all of the biological effects of bacterial lipopolysaccharide (LPS) that have been studied. These mice die in the early phase of infection when inoculated with virulent Salmonella. This susceptibility is also regulated by the Lpsd allele. The mechanism of Lpsd-conferred Salmonella susceptibility was evaluated in these studies. The response of C3H/HeJ mice to S. typhimurium strains of differing virulence was compared in a series of in vivo experiments to the response of: endotoxin-responsive (Lpsn) mice that carry another Salmonella susceptibility gene (Itys) and endotoxin-responsive mice that carry a Salmonella resistance gene (Ityr). The C3H/HeJ mice (genotype Lpsd/Ityr) were more resistant than Lpsn/Itys mice to strains of S. typhimurium of reduced virulence but less resistant than Lpsn/Ityr mice. In addition, C3H/HeJ macrophages cultured in vitro were less able to contain net salmonellae multiplication than were macrophages from Lpsn/Ityr mice. Moreover, histopathological findings revealed that S. typhimurium-infected Lpsn/Ityr animals recruited an abnormally low number of cells into their livers compared to either Lpsn/Ityr mice or Lpsn/Itys mice. These data suggest that the susceptibility of C3H/HeJ mice may be the result of at least two Lpsd-encoded defects: a decreased capacity of macrophages to restrict Salmonella growth and a reduced recruitment of inflammatory cells into liver.

Animals↗
PubMed1986

Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities.

Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

Animals↗
PubMed1986

Morphologic evaluation of the effects of Shiga toxin and E coli Shiga-like toxin on the rabbit intestine.

The effects of a Shiga toxin derived from Shigella dysenteriae Type 1, Strain 60R, and a Shiga-like toxin from the enterohemorrhagic Escherichia coli O157:H7, Strain 933, were studied in the in vivo rabbit ileal loop model. The effects of both toxins were similar and resulted in severe villus blunting by 18-24 hours after exposure. With both toxins, a dose effect was noted; and the lesions, first detected at 2 hours after inoculation, became more severe over time. Both toxins appeared to act directly and selectively on the mature columnar absorptive epithelium of the intestinal villus, which resulted in the premature expulsion of these cells from the lateral villus wall, with a decrease in the villus/crypt ratio. The goblet mucous cells remained attached and frequently formed clusters on the blunt villus apices. The crypt epithelium underwent a rapid proliferation and maintained the epithelial integrity. The ultrastructural changes observed in the toxin-injured villus absorptive cells suggested that these cells underwent a process of apoptosis, rather than necrosis. These findings suggest that both toxins act in vivo in the small intestine on a specific cell population, the mature, differentiated absorptive villus epithelium.

Animals↗
PubMed1985

Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli.

The genes controlling high-level production of Shiga-like toxin (SLT) in Escherichia coli were cloned from the SLT converting phage 933J. This phage was isolated from a strain of E. coli that caused a foodborne outbreak of hemorrhagic colitis. The genes that convert normal E. coli to organisms producing high levels of toxin were cloned into the plasmid pBR328 and expressed in E. coli HB101. DNA restriction mapping, subcloning, examination of the cloned gene products by minicell analysis, neutralization, and immunoprecipitation with antibodies to SLT were used to localize the toxin converting genes and identify them as structural genes for SLT. Southern hybridization studies established that the DNA fragment carrying the cloned toxin structural genes had homology with the DNA of Shigella.

Animals↗
PubMed1985

The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.

Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers. This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.

Adhesins, Escherichia coli↗
PubMed1985

Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.

Three monoclonal antibodies, designated MAb 16E6, MAb 13C4, and MAb 19G8, were produced which recognize Shiga-like toxin (SLT) from Escherichia coli. All three monoclonal antibodies neutralized the cytotoxicity of E. coli SLT and were able to immunoprecipitate intact labeled toxin with Staphylococcus aureus protein A. The three antibodies were of the G1 heavy and kappa light chain classes. MAb 16E6 bound to the B subunit of SLT in Western blots and also neutralized the lethality of the toxin for mice and the enterotoxicity of the toxin in ligate rabbit ileal loops. The ability of MAb 16E6 to neutralize the cytotoxicity, lethality, and enterotoxicity of E. coli confirms the hypothesis that all three activities are associated with a single toxin. MAb 16E6 and MAb 13C4 also neutralized the cytotoxicity of purified Shiga toxin from Shigella dysenteriae type 1 and Shiga-like toxic activities in crude cell extracts from Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium. Thus, Shiga toxin and the SLTs from E. coli, Shigella flexneri, V. cholerae, V. parahaemolyticus, and Salmonella typhimurium share a common B subunit epitope that is involved in neutralization. MAb 13C4 has been successfully used in a colony blot assay for the detection of bacterial colonies which produce high levels of SLT. Sixty-two different strains of bacteria were tested by both the cytotoxicity and colony blot assays for the presence of SLT. The colony blot assay detected all strains of bacteria which produce greater than or equal to 10(5) 50% cytotoxic doses of SLT per ml of sonic lysate. There were no false-positive results among the 62 samples tested.

Animals↗