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Enterotoxin production in milk at 22 and 4 degrees C by Escherichia coli and Yersinia enterocolitica.

Enterotoxigenic Escherichia coli (five strains) and Yersinia enterocolitica (five strains) were cultivated in sterile milk at 22 and 4 degrees C. The bacteria grew well at both temperatures. Three strains of E. coli produced heat-labile enterotoxin in the milk at 22 degrees C as demonstrated by enzyme-linked immunosorbent assay. Heat-stable enterotoxins were not detected in milk by the infant mouse test.

Animals↗

Direct skin test in highly sensitized guinea pigs for rapid and sensitive determination of staphylococcal enterotoxin B.

The direct skin test in highly sensitized guinea pigs was developed as a rapid and extremely sensitive assay for detection of staphylococcal enterotoxin B (SEB) in foods. This report details the experimental conditions required to elicit optimal sensitization of guinea pigs to SEB. An intense and persistent immunoglobulin E (IgE) anti-SEB response was established in strain 13 guinea pigs pretreated with cyclophosphamide followed by four sensitizing doses of 10 micrograms of SEB 1 month apart. The conditions, however, optimal for eliciting IgE responses led to a sustained failure to produce antibody of the IgG1 subclass. With the use of highly sensitized guinea pigs, one can achieve a sensitivity ranging from 0.1 to 1.0 pg of purified SEB by the direct skin test for at least 7 months after the last challenge. For analysis of SEB in food extracts, the entire assay can be accomplished within 20 min with a sensitivity of 10 to 100 pg SEB per ml of prepared food samples, and the recovery of enterotoxin from spiked food products ranged between 75 and 89% of the amount added.

Biological Assay↗

Transport and processing of staphylococcal enterotoxin A.

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.

Biological Transport↗

Recovery, growth, and production of heat-stable enterotoxin by Escherichia coli after copper-induced injury.

Exposure of enterotoxigenic Escherichia coli strains to a sublethal concentration (0.75 mg/liter) of copper for 3 days at 4 degrees C induced sensitivity to deoxycholate (0.1%). When placed in a complex (brain heart infusion) or a defined amino acid salt medium, the copper-injured cells recovered their tolerance to deoxycholate in 3 and 6 h, respectively, and commenced active growth. Growth and heat-stable enterotoxin production of uninjured and copper-injured cells were studied in brain heart infusion medium. A slightly altered growth curve and an initial slow rate of toxin production were observed in injured cells when compared with those corresponding uninjured controls. However, maximum heat-stable enterotoxin levels in injured cultures were comparable to those produced by uninjured cells, suggesting that the enterotoxigenic potential of copper-injured cells was fully retained.

Animals↗

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin.

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.

Animals↗

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.

Chromatography, Affinity↗

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).

Clostridium perfringens↗

Nutritional requirements for synthesis of heat-stable enterotoxin by Yersinia enterocolitica.

A defined medium that supported the growth of and synthesis of heat-stable enterotoxin (YST) by clinical isolates of Yersinia enterocolitica at levels equivalent to those observed in a complex Trypticase soy broth-0.6% yeast extract medium was developed. The defined medium contained four amino acids (L-methionine, L-glutamic acid, glycine, and L-histidine), inorganic salts, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, and potassium gluconate as the carbon source. Methionine was required for growth by most strains of Y. enterocolitica used in this study; thus, it was not possible to determine whether it was also required for the synthesis of YST. The other 17 amino acids commonly found in proteins did not stimulate the synthesis of YST when added to the defined medium. The yield of YST observed with other carbon sources fermented by Y. enterocolitica ranged from 4- to 26-fold lower than that obtained with potassium gluconate. The divalent cations Ca2+ and Mn2+ had no effect on the synthesis of YST; however, concentrations of Fe2+ above 10 microM inhibited the synthesis of the enterotoxin. The addition of a mixture of pyrimidines containing thymine, cytosine, and uracil, each at a concentration of 2.0 mM, stimulated the synthesis of YST by 10 to 15%, whereas a mixture of adenine and guanine, each at a similar concentration, inhibited the synthesis of YST. Vitamins had no effect on the amounts of YST produced by Y. enterocolitica strains grown in the defined medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Maltodextrin stimulates growth of Bacillus cereus and synthesis of diarrheal enterotoxin in infant milk formulae.

One hundred reconstituted milk-based infant formulae (IMF) representative of 10 leading brands available in many European Economic Community countries were examined for Bacillus cereus and for the presence of diarrheal enterotoxin. Sixty-three reconstituted IMF supported growth of the organism after 14 h at 25 degrees C, and in 4 of these, which contained maltodextrin, enterotoxin was detected. Reconstituted IMF (and basal synthetic media) supplemented with > or = 0.1% maltodextrin supported both growth of B. cereus and diarrheal toxin production when incubated for 14 h or more at 25 degrees C.

Bacillus cereus↗

Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex.

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.

Amino Acid Sequence↗

Clinical chemistry of staphylococcal enterotoxin poisoning in monkeys.

Clinical chemistry values were examined in 90 monkeys administered a purified preparation of staphylococcal enterotoxin, type B, intravenously. These studies showed an early release of epinephrine accompanied by a mild increase in blood glucose. This was followed by progressively developing prolonged hypoglycemia. An early increase in bloodurea nitrogen occurred, presumably as a result of both prerenal azotemia and functional renal failure seen in association with the observed hypotension. Serum protein, Ca, and Cl concentrations decreased with time. Pi levels increased, whereas Na and K concentrations in serum remained unchanged. Serum enzyme concentrations were unchanged, with the exception of serum glutamic oxaloacetic transaminase, which rose rapidly when compared with prechallenge control observations or with values from sham-challenged monkeys. These changes were statistically significant. These results suggested that enterotoxin administered intravenously produced early change in glucose metabolism, possibly related initially to catecholamine release and later to increased utilization of glucose and metabolic acidosis. Other findings were compatible with tissue breakdown at as yet undetermined locations and with loss of endothelial membrane integrity, as evidenced by loss of protein from the vascular space.

Animals↗

Rapid, sensitive assay for staphylococcal enterotoxin and a comparison of serological methods.

Reversed passive hemagglutination was used to assay enterotoxin in culture filtrates and in food samples. With cells tanned and then sensitized with antitoxin globulin and preserved with either formaldehyde or pyruvic aldehyde, as little as 0.0007 mug of enterotoxin was detectable. The results of hemagglutination tests compared well with those obtained by quantitative precipitin tests or by immunodiffusion, but hemagglutination was 50 to 100 times more sensitive than the immunodiffusion technique. In addition, results of the hemagglutination test were available within a few hours, and neither elimination of interfering proteins from food extracts nor concentration of the sample, both of which are necessary for immunodiffusion, was required for this procedure.

Antitoxins↗

Capillary tube immunological assay for staphylococcal enterotoxins.

A simple assay is reported in which 1 mug of staphylococcal enterotoxins A, B, and D per ml was detected in less than 1 hr. Interfacial reaction of antisera and enterotoxin solutions in a 1-ml internal diameter capillary tube allowed rapid detection of sera type.

Antigen-Antibody Complex↗

Analysis of staphylococcal enterotoxin B by the polyacrylamide electrophoresis technique.

The electrophoretic mobility of enterotoxin B was investigated through the use of the disc electrophoresis technique. Ideal patterns were developed with a 7.5% acrylamide gel system (pH 4.3). The toxin can be separated and identified from other complex proteins such as serum or suspect samples of foods by this technique. The technique can be used as an assay method for the toxin as well as to elucidate physical changes in the toxin due to temperature. The method should not be considered exclusive for enterotoxin B.

Animals↗

Regulation of staphylococcal enterotoxin B: effect of anaerobic shock.

The metabolism of glucose during enterotoxin B synthesis in Staphylococcus aureus S-6 was examined under anaerobic conditions in the presence and absence of nitrate. The repression of enterotoxin synthesis which occurs during the oxidative metabolism of glucose was relieved after a shift to anaerobic conditions; glucose was then converted primarily to lactic acid and was metabolized more rapidly, presumably to obtain the equivalent amount of energy available aerobically. A greater proportion of oxidized end products and evidently more energy per glucose molecule was produced in the presence of oxygen. Thus, available energy as judged by a change in the type and proportion of end products appears to be related to the degree of toxin repression. As expected, the addition of nitrate during anaerobic glucose metabolism prevented derepression of toxin synthesis.

Acetates↗