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Functional significance of histologic alterations induced by Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb).

In contrast to cholera enterotoxin and other Escherichia coli enterotoxins, a pig-specific, heat-stable E. coli enterotoxin (STb) causes morphologic lesions (loss of villous epithelial cells and partial villous atrophy). These lesions reflect a loss of absorptive cells and thus suggest that STb causes impaired absorption as well as inducing net secretion. The present studies assess functional significance of morphologic changes induced by STb. Net fluid movement, mucosal surface area, sucrase activity and the electrical response induced by alanine were measured in swine jejunal loops exposed to E. coli culture filtrates with and without STb. Net fluid secretion (-11.1 +/- 1.1 ml) occurred in some STb loops (secretors) and net absorption (2.7 +/- 0.3 ml) in others (nonsecretors), but net absorption occurred in all control loops (4.9 +/- 0.2 ml). The mucosal surface area of STb loops was about 20% less than that of controls (P less than 0.01). Sucrase activity was also lower (about 15%) in STb loops than in control loops (P less than 0.01). The electrical response induced by alanine in mucosa from nonsecreting STb loops did not differ from that induced in mucosa from control loops. However, the response to alanine in mucosa from secreting STb loops was reduced about 70% from that in mucosa from nonsecreting STb loops or from control loops (P less than 0.05). It is concluded that reduced sucrase activity is a functional correlate to villous atrophy induced by STb, that STb impairs alanine absorption in some loops (secretors), and that the impaired alanine absorption is independent of the decreased surface area caused by STb. Because the impaired alanine absorption occurred independent of the decreases in surface area, it is suggested that the secretory response to STb is associated with an impairment of active absorption of alanine.

Animals↗

Growth of Staphylococcus aureus and synthesis of enterotoxins in home-made yoghurt.

Staphylococcus aureus strains FRI-100, S6, FRI-137 and FRI 472 were inoculated into milk to study growth and enterotoxin production in home-made yogurts. The yogurt used as starter was progressively weakened by successive inoculations (up to four) in milk to prepare other yogurts in order to study the ability of yogurt microflora to inhibit staphylococci. After elaboration, yogurts were stored at 4 degrees C, 22 degrees C, and 37 degrees C for a maximum of 21 days. Periodically, staphylococcal counts, pH and the production of enterotoxins A, B, C, and D were determined. Enterotoxins were only detected in the last batch. It was concluded that the inhibitory effect of the starter culture is not only due to the decrease of pH, but also to other factors.

Animals↗

Detection of a heat-labile enterotoxin gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes.

Two alkaline phosphatase-conjugated 24-mer oligonucleotide probes were developed to detect the heat-labile enterotoxin gene in enterotoxigenic Escherichia coli. Probes were antisense codon sequences, which are transcribed into mRNA, of the heat-labile enterotoxin gene of enterotoxigenic Escherichia coli of human origin. Using dot-blot hybridization, probes were tested with 100 clinical isolates and evaluated by a reflectance-type densitometer. Results agreed very well with those of an immunological test, the Biken test, and a 32P-labelled recombinant DNA probe. The oligonucleotide probes did not react with nucleic acids prepared from other diarrhoeagenic bacterial pathogens. Thus, the alkaline phosphatase-conjugated oligonucleotide probes seem to be highly sensitive and specific for detection of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli. Moreover, the results indicate a potential usefulness for densitometric evaluation of DNA hybridization.

Alkaline Phosphatase↗

Monospecific antibodies for detection of Clostridium difficile enterotoxin.

Tests of polyclonal antisera to homogeneous enterotoxin preparations of Clostridium difficile by immunoblotting demonstrated that cross-reactions occurred with proteins from cell lysates of nearly all Clostridium species. There was a notable reduction in the amount of crossreaction when only culture supernatants were analysed. The enterotoxin could be easily identified by its unique high molecular weight of 230,000. The findings suggest that care must be taken when immunological tests other than immunoblotting are used for detection of Clostridium difficile enterotoxin.

Antibodies, Bacterial↗

Hemolytic and nonhemolytic enterotoxin genes are broadly distributed among Bacillus thuringiensis isolated from wild mammals.

The presence of cytotoxin K (cytK), nonhemolytic (NHE), and hemolytic (HBL) enterotoxin genes was investigated in 74 Bacillus thuringiensis strains recovered from the intestines of wild mammals from northeast Poland, using polymerase chain reaction amplification and Southern hybridization. All the isolates harbored genes coding for toxin(s) that could cause diarrhea. The B. thuringiensis strains containing the nhe genes were found more frequently (nheA 100%, nheB 77%, nheC 96%) than those with the hblACD (74%) and cytK (73%) genes. The presence/absence of the nheA, hblA, and cytK genes was confirmed in all of the B. thuringiensis strains by Southern hybridization. Interestingly, these experiments also indicated that the nheA locus is located on a more variable chromosome region compared with hblA and, to a lesser degree, cytK. Detection of the 41-kDa component of NHE enterotoxin by the TECRA assay revealed various protein levels by B. thuringiensis strains. These results indicate the existence of environmental B. thuringiensis strains bearing the potential virulence arsenal for the production of diarrheal toxins, and emphasize the importance of small animals in the spread of B. cereus-like enterotoxin genes in nature. However, further investigation is needed to clarify any possible involvement of environmental B. thuringiensis strains in human health issues.

Animals↗

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin.

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.

Animals↗

Guanidination and nitroguanidination of staphylococcal enterotoxin B.

Guanidination of the free amino groups of staphylococcal enterotoxin B with 3,5-dimethyl-1-guanylpyrazole converted 31-32 of 33 epsilon-amino groups and 30% of the N-terminal residue. This product, although markedly reduced in solubility, suffered no gross change in conformation and retained full biological activity. A derivative prepared by reaction with O-methylisourea with only one lysyl residue unaltered lost most of its emetic activity. Nitroguanidination with 3,5-dimethyl-1-nitroguanylpyrazole converted up to 28 of the epsilon-amino groups and essentially all of the N-terminus. This material was greatly reduced in ability to produce emesis and like the O-methylisourea prepared guanidinated enterotoxin, gave only a line of partial identity in double diffusion. The loss of activity is attributed to unfolding and it is concluded that the free amino groups of enterotoxin B do not critically participate in either its antigenic determinants or its active center for emesis.

Affinity Labels↗

A new purification procedure for Clostridium difficile enterotoxin.

Clostridium difficile produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A1 = 41,500, A2 = 16,000) and its pI is 3.5.

Chromatography, Liquid↗

Effect of isotypic and allotypic variations of MHC class II molecules on staphylococcal enterotoxin presentation to murine T cells.

Staphylococcal enterotoxins (SE) are known to be potent T cell activators, stimulating +/- proliferation and lymphokine production. These toxins have recently have been termed "superantigens" because of their ability to bind directly to class II molecules forming a ligand that interacts with particular V beta gene elements within the TCR complex. This interaction between SE and MHC class II molecules plays a central role in toxin-induced mitogenesis. In the present study we have examined the effect of polymorphism on the ability of MHC class II molecules to bind and present SE. Through the use of H-2 congenic mouse strains, it was possible to look directly at haplotype differences within the MHC and their effect on SE presentation to a panel of responsive V beta-bearing T cells. The results demonstrate that toxin presentation by class II-bearing accessory cells to murine T cells is greatly affected by polymorphisms within the H-2 complex. Toxin-pulsed accessory cells obtained from mice of an H-2k and H-2u haplotype were found to be less efficient in activating a variety of T cell clones and hybridomas. However, one T cell clone responded similarly to the enterotoxins presented on all H-2 haplotypes, suggesting that differences in responses of T cells are not simply a function of the degree of binding of these toxins to various class II molecules. Neutralization analysis with monoclonal anti-class II antibodies demonstrates that both I-A and I-E molecules play a significant role in SEA and SEB presentation to murine T cells. These results suggest that the differential activation of T cells by a particular enterotoxin may reflect a difference in recognition of an SE:class II ligand by a surface T cell receptor complex.

Alleles↗

CD28 and staphylococcal enterotoxins synergize to induce MHC-independent T-cell proliferation.

The bacterial exotoxins staphylococcal enterotoxin A and B (SEA and SEB) mediate disease through their effects on T lymphocytes. In this manuscript we have demonstrated that both SEA and SEB can directly activate purified T cells in the absence of accessory cells as determined by a transition from G0 to G1 and induction of IL-2 receptor expression. However, neither SEA nor SEB alone was sufficient to result in T-cell proliferation. The induction of T-cell proliferation by SEB or SEA required the addition of a second costimulatory signal. This could be provided by either accessory cells or monoclonal antibody stimulation of CD28. As previously reported, T-cell proliferation induced by enterotoxin in the presence of accessory cells was partially inhibited by a blocking antibody against class II MHC. In contrast, in purified T cells when costimulation was provided through CD28, proliferation was not inhibited by class II antibody, and HLA-DR expression was not detectable. In addition, costimulation through CD28 was partially resistant to the effects of cyclosporin A. These results demonstrate that CD28 costimulation is sufficient to induce proliferation of enterotoxin-activated T cells, and that this effect is independent of class II MHC expression.

Antigens, CD↗

Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.

A decapeptide highly homologous to the STa Escherichia coli heat-stable enterotoxin and to several other heat-stable enterotoxins was fused genetically to the amino-end of the B-subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa-related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide-linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti-STa antibodies. Expression of the decapeptide-CTB hybrid by non-toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single-step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non-toxic, immunogenic decapeptide-CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E. coli and other bacteria producing immunologically related heat-stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.

Amino Acid Sequence↗

Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum.

BACKGROUND/AIMS: Activation of intestinal mast cells and neurons is involved in intestinal inflammation and diarrhea. This study compared the effects of neuronal inhibitors and inhibition of intestinal sensory afferent nerves on the intestinal actions of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin. METHODS: The effects of lidocaine, hexamethonium, atropine, and long-term pretreatment of capsaicin on fluid secretion, mannitol permeability, myeloperoxidase (MPO) activity, and release of rat mast cell protease II (RMCPII) were measured in toxin A- and cholera toxin-exposed loops in vivo. RESULTS: Lidocaine, hexamethonium, and capsaicin, but not atropine, inhibited toxin A-mediated secretion and MPO activity, but only capsaicin reduced mannitol permeability. Lidocaine, but not capsaicin, reduced secretion and permeability caused by cholera toxin. Toxin A caused release of RMCPII from rat ileum in vivo and in vitro; this was inhibited by lidocaine or capsaicin, whereas cholera toxin had no effect on release of RMCPII. CONCLUSIONS: Neuronal mechanisms are important in the in vivo effects of these two enterotoxins. Capsaicin-sensitive sensory afferent neurons and mast cells are involved in the intestinal mechanism of toxin A, but not cholera toxin.

Animals↗

A double antibody sandwich enzyme-immunoassay for Clostridium perfringens type A enterotoxin detection in stool specimens.

A double antibody sandwich enzyme-immunoassay has been developed for detection of Clostridium perfringens enterotoxin. Anti-enterotoxin immunoglobulin G-alkaline phosphatase conjugates were prepared using a rapid minicolumn procedure. The assay can achieve a sensitivity of greater than or equal to 1 ng/ml with purified enterotoxin. Sensitivity for detection of cases of C. perfringens enteritis in a C. perfringens outbreak (86 individuals tested) was between 85.7 and 98.0 per cent depending upon stringency of criteria for defining positive cases. Specificity of the assay was demonstrated by the lack of positive results in 53 individuals involved in a gastroenteritis outbreak of unknown etiology.

Antibody Specificity↗

The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice.

Staphylococcal enterotoxin B is known to be a powerful T cell stimulant in mouse and man. In this paper we show that, for mice, this is because the protein in association with major histocompatibility complex class II molecules stimulates virtually all T cells bearing V beta 3 and V beta 8.1, 8.2, and 8.3, and few others. Neonatal mice given the enterotoxin eliminate all mature, and some immature, T cells bearing these V beta s, demonstrating that tolerance to exogenously administered antigen can be caused by clonal deletion of reactive T cells. The enterotoxin shares these "superantigenic" properties with known self-antigens in mice, Mls-1a and Mls-2a, and a B cell-derived product, a shared property that is unlikely to be coincidental or inconsequential.

Animals↗

Production of enterotoxins and thermonuclease by Staphylococcus aureus in cooked egg-noodles.

Production of enterotoxin A (SEA), enterotoxin C (SEC), and thermonuclease (TNase) by Staphylococcus aureus was determined during growth in cooked egg-noodles at different temperatures (15-37 degrees C). Both SEA and SEC and TNase were detected when greater than or equal to 4.0 x 10(7) colony forming units (cfu)/g were present. The contents of SEA, SEC, and TNase in egg-noodles mainly increased at the end of the exponential growth phase. In contrast with SEA and SEC the production of TNase always continued till the end of each experiment. Recovery rates of SEA and TNase in cooked noodles were dependent on their amounts. High amounts (64 ng SEA/g; 1 unit TNase/g) were recovered at a rate of 93% (SEA) and 54% (TNase) respectively, whereas low concentrations (1 ng SEA/g; 0.004 units TNase/g) were recovered at a rate of only 45% (SEA) and 1.1% (TNase). TNase usually is produced at all conditions which allow growth of S. aureus. Evidence of TNase was proposed for screening for staphylococcal enterotoxins (SE) in foods. But sometimes foods contain no TNase but SE. For this reason the ELISA-test which is simple and sensitive should be used for determination of SE-production in foods.

Animals↗

The effect of glucose, starch, and pH on growth, enterotoxin and haemolysin production by strains of Bacillus cereus associated with food poisoning and non-gastrointestinal infection.

Brain-Heart Infusion medium, modified by (a) 1.0% w/v glucose supplement, (b) 1.0% w/v soluble starch supplement, (c) pH adjustment to 8.8 or (d) pH adjustment to 5.0, was used to investigate the influence of glucose, starch and pH on growth, enterotoxin and haemolysin production by Bacillus cereus. The four test strains selected for comparison originated from episodes of emetic-syndrome food poisoning, diarrhoeal-syndrome food poisoning, traumatic wound infection and bovine mastitis, respectively. In the presence of either glucose or starch, growth of all strains was found to be near-optimum and accompanied by enhanced enterotoxin production. However, under the moderately acid and alkaline test conditions B. cereus strains exhibited varying degrees of growth inhibition and partial repression of enterotoxin and haemolysin production. In this respect the food-poisoning isolates proved to have greater resistance to adverse pH environments than did the non-gastrointestinal infection isolates.

Animals↗

Production of enterotoxin A and thermonuclease by Staphylococcus aureus in legumes.

Growth, enterotoxin A (SEA) and thermonuclease (TNase) production of S. aureus (Strains CP 7 and FRI 722) was determined in media produced from the following heat or irradiation sterilized legumes: peas, black beans, mung beans, adzuki beans and soybeans. Media containing the five legumes alone or in combination with Brain Heart Infusion Broth (BHI) were tested. With the exception of heat-sterilized black beans and adzuki beans, S. aureus growth was excellent in all media with cell counts after 48 h (25 degrees C) exceeding 10(8) cfu/ml. In black beans and adzuki beans cell counts were 1-2 log-cycles lower. Enterotoxin A was produced in amounts of 33 to 72 ng/ml in BHI after 48 h. Almost no toxin was produced in the four different beans following heat or irradiation treatment; in peas the toxin concentration reached 14 to 15 ng/ml. In the medium prepared from irradiated soybeans and BHI the final toxin concentration was about the same as in BHI alone. In all the other media consisting of a combination of legumes with BHI toxin concentrations were three to four times higher than in BHI alone. Production of thermonuclease showed variation and did not always correlate with enterotoxin production.

Colony Count, Microbial↗

Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA.

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.

Animals↗