- Volume 23, Issue 3, 1987
Volume 23, Issue 3, 1987
- Article
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In-vitro and in-vivo studies of a cytotoxin from Campylobacter jejuni
More LessSummaryStudies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54–68% was noted. CT production was lost after 5–7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be > 30 000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.
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The effects of Clostridium difficile crude toxins and purified toxin A on stripped rabbit ileal mucosa in Ussing chambers
More LessSummaryClostridium difficile crude toxins and purified toxin A had similar effects on stripped rabbit ileal mucosa in Ussing chambers. Both toxin preparations caused secretion of sodium and chloride ions by increasing serosa to mucosa (s→m) fluxes. Transmural potential difference and resistance decreased after toxin treatment. Onset of changes in electrical measurements and ion fluxes coincided with onset of histological changes. The response to theophylline was greatly reduced in toxintreated tissue compared with control tissue.
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The effects of Clostridium difficile toxins A and B on membrane integrity and protein synthesis in intestinal cells in vivo and in vitro and in McCoy cells in vitro
More LessSummaryClostridium difficile toxins A and B inhibited protein synthesis in McCoy tissue-culture cells but not in intestinal cells in vitro or in vivo. Toxins A and B had no effect on membrane permeability of either intestinal cells or McCoy cells.
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Biological mode of action of Clostridium difficile toxin A: a novel enterotoxin
More LessSummaryAntibody neutralisation and toxin A elution experiments showed that toxin A uptake from rabbit intestinal lumen was a continuous process. The kinetics of the ileal and colonic responses were significantly different; a much longer incubation (4 h) with toxin was required for colon, compared with 45 min for the ileum, to induce fluid accumulation at 12 h. Fluid secretion was induced only when toxin had gained access to deeper tissues, probably achieved by several toxin uptake-tissue damage cycles. Toxin A induced haemorrhage in both ileal and colonic tissues. In ileum, the villus architecture was severely damaged and this gave rise to protein-rich bloody luminal fluid. In the colon, although colonocytes were removed, the basement membrane remained intact; this resulted in a tissue-localised haemorrhage and a protein-low watery ultrafiltered luminal fluid. Toxin A is thus a novel type of histotoxic enterotoxin.
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Comparison of five assays for the heat-labile enterotoxin of Escherichia coli
More LessSummaryEnzyme-linked immunosorbent assay (ELISA), DNA-DNA hybridisation, Vero cell assay, the Biken test and a new membrane-filter method were compared in the detection of heat-labile enterotoxin (LT) of Escherichia coli. Six subcultures of each of 50 strains of E. coli from the Biken collection were evaluated “blind” in the laboratory. The combined results of the most reproducible tests (ELISA and DNA-DNA hybridisation) were used to calculate the sensitivity and specificity of the other assays. The Vero-cell assay had a high sensitivity (98%) but a lower specificity (91%). The Biken and membrane-filter assays had sensitivities of 58-71% and 77-84% respectively, depending on the type of antiserum used. Only one false positive result was obtained with the Biken test; specificity of the membrane-filter assay was 94-95%. The membrane-filter assay, with anti-cholera toxin, is specific and reasonably sensitive. It has particular advantages over DNA-DNA hybridisation and the Biken test, and it may prove suitable for screening large numbers of E. coli isolates in epidemiological studies in developing countries.
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Studies on the Vibrio cholerae mucinase complex. II. Specific neuraminidase activity measured histochemically in a goblet cell assay
More LessSummaryThe activity of neuraminidase prepared from the mucinase complex of Vibrio cholerae was measured by a new, semi-quantitative goblet-cell assay. The counts of normal, alcianophilic, sialomucin-containing goblet cells (purple-stained) and neutral mucosubstance-containing goblet cells (magenta-stained) in serial sections of ileum were compared before and after neuraminidase treatment. The procedure provides a more natural assessment of the action of V. cholerae neuraminidase on the viscous intestinal mucus.
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Interaction of Campylobacter pyloridis with human immune defence mechanisms
More LessSummaryThe in-vitro susceptibility to host immune defence mechanisms of Campylobacter pyloridis was investigated. C. pyloridis was sensitive to antibodydependent complement-mediated bactericidal activity of serum. The bacteria were phagocytosed and efficiently killed by polymorphonuclear neutrophils in the presence of serum opsonins. Serum opsonin depletion studies indicated that an intact classical complement pathway was required for optimal phagocytic killing.
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Regulation of cell-mediated immunity in mice immunised with Salmonella enteritidis
More LessSummaryThe effects of diverse factors, such as route of immunisation, composition of immunogen and administration of interferon inducer, on the expression of cellmediated immune responses against Salmonella enteritidis were investigated in BALB/c and Swiss white mice. Immunisation with live cells of S. enteritidis by the intraperitoneal route (ip) generated both delayed type hypersensitivity (DTH) and protective cell-mediated immunity (CMI). However, the two responses showed diametrically opposite time kinetics. The decline and disappearance by 9 weeks after ip immunisation of DTH and the rise of protective immunity in the same period suggested the possibility that the two responses were mediated by different subsets of T cells. Immunisation by the intradermal (id) route with a sonicate of S. enteritidis generated only DTH; id immunisation also suppressed the development of the protective response following ip immunisation with live S. enteritidis. Both responses were not seen when T cells were eliminated with anti-T cell serum. Oral immunisation with live cells of S. enteritidis induced excellent CMI expressing both DTH and protective responses. On the other hand, oral immunisation with the sonicate of S. enteritidis not only did not induce CMI, but also prevented the development of the DTH and protective response to ip immunisation with live S. enteritidis.
Induction of interferon by the administration of poly I: poly C for four consecutive days after id immunisation with killed S. enteritidis suppressed the generation of DTH.
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Demonstration by immuno-electronmicroscopy of antigenic heterogeneity among P fimbriae of strains of Escherichia coli
More LessSummaryThe identification of P fimbriae on urinary strains of Escherichia coli isolated from urine was made (i) by observation of the patterns of mannose-resistant and eluting haemagglutination of erythrocytes of seven animal species (and including those of human phenotype), and (ii) by haemagglutination-inhibition tests with hydatid-cyst fluid known to contain an analogue of P-fimbrial receptor. In tests with five different, pure P-fimbrial antisera prepared in rabbits, agglutinin titres of 37 P-fimbriate strains revealed differences in their reactivity; immuno-electronmicroscopy studies with the same five antisera showed that P-fimbriate strains were markedly different in the extent to which their P fimbriae were coated with antibody. The antigenic heterogeneity observed among P fimbriae is discussed with regard to the development of P-fimbrial vaccines.
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A simple statistical approach that represents the frequency distribution of plasmids in clinical isolates of the enterobacteria
More LessSummaryThe frequency distribution of plasmids in a representative collection of Escherichia coli and other enterobacteria was compared with the frequencies predicted by the Poisson distribution. The distribution of E. coli plasmids did not differ significantly (p>0.2) whereas the difference between observed and predicted distributions of plasmids in combined populations of other enterobacteria was significant (p<0.001). Previous studies had suggested that plasmid-free strains contributed disproportionately to the overall frequency distribution. Therefore, plasmid-free strains were excluded and the frequency distribution of plasmids in plasmid-containing strains compared with frequencies predicted by a modified Poisson distribution conditional upon n ≠ 0. The results obtained showed that the frequency distribution of plasmids in E. coli and in other enterobacteria did not differ significantly from the predicted distribution (p>0.2 and p>0.6 respectively). The frequency distribution of plasmids in previously published studies was compared with predicted frequencies by means of (a) the Poisson distribution and (b) the modified Poisson distribution conditional upon n ≠ 0. The results indicate that the modified Poisson distribution described provides an adequate description of plasmid frequency distributions and represents the observed frequencies better than the distributions predicted directly from the Poisson formula.
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Demonstration of spirochaetes in patients with Lyme disease with a modified silver stain
More LessSummarySpirochaetes were demonstrated in material from patients with Lyme disease by short-time high-concentrate silver impregnation after treatment with amylase. Removal of mucoid material was essential to visualise Borrelia burgdorferi. Lyme spirochaetes were demonstrated in material from 23 patients with Lyme disease—erythema chronicum migrans (ECM) 10, lymphadenosis benigna cutis (LABC) 7, arthritis 4 and Bannwarth’s syndrome 2. Spirochaetes were localised in the subepidermal zone, peri- and intravascularly, and in collagen fibres in ECM and LABC, and beneath the synovial lining cells in arthritis, producing marked vascular changes with fibrosis and synovial hyperplasia. Spirochaetes were also demonstrated in CSF from a patient with Bannwarth’s syndrome.
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Accumulation of a precursor of subunit S1 of pertussis toxin in cell envelopes of Bordetella pertussis in response to the membrane perturbant phenethyl alcohol
More LessSummaryExponential cultures of Bordetella pertussis strain 18334 were treated with the membrane-perturbing agent phenethyl alcohol which, at a concentration of 0.075% v/v, blocked the synthesis of mature subunit S1 of pertussis toxin as revealed by Western blotting. It also caused the accumulation of a precursor, pS1, with an estimated mol. wt of 32 × 103, that was located in the cytoplasmic membrane. These findings suggested that subunit S1 of pertussis toxin was exported in a signal peptidedependent manner.
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Disseminated Candida infection syndrome in heroin addicts—dominance of a single Candida albicans biotype
More LessSummaryAmong 21 intravenous heroin abusers with cutaneous and ocular manifestations of disseminated Candida infection, a single C. albicans strain type (serotype A, biotype 15 3/7) was isolated from skin lesions in 14 cases. This suggests that central contamination of the heroin with C. albicans is less likely to be the source of infection than an endogenous source, and that one particular strain type is either better adapted than others to grow in the lemon juice used as a heroin solvent, or more likely than others to cause the specific pathology seen in these patients.
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Characterisation of anaerobic curved rods (Mobiluncus spp.) isolated from the urogenital tract
More LessSummaryThirty-two strains of anaerobic curved rods isolated from vaginal secretions and one isolated from seminal fluid were examined. Growth of all strains on solid media was superior to growth in liquid media, and at 37°C they grew both anaerobically and in O2 5% in N2; they also grew anaerobically at 33°C but not at 42°C. No growth factors were identified, but strains grew more profusely atpH values above 5–0. The strains were screened in 80 biochemical tests, and for their susceptibility to 30 different antimicrobial agents. Most of the tests did not differentiate between the strains, but they were divided into four groups on the basis of cell morphology, metronidazole susceptibility, β-galactosidase activity and arginine and hippurate hydrolysis. Group 1 consisted of 19 strains conforming to the species M. curtisi; group 2 consisted of five strains conforming to the species M. mulieris; group 3 consisted of five strains that resembled M. curtisi morphologically, and group 4 consisted of four strains that resembled M. mulieris morphologically, but the strains in the latter two groups reacted differently in at least one of the three major differential biochemical tests. Of three strains of M. curtisi and three of M. mulieris chosen at random, one of M. mulieris had a SDS-PAGE and fast-protein liquid chromatography protein profile indistinguishable from that of M. curtisi. We conclude that further efforts are required to clarify the taxonomic status of the genus Mobiluncus.
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