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Assay methods for Clostridium perfringens type A enterotoxin.

Enterotoxin produced by a sporulating culture of Clostridium perfringens type A NCTC 8798 was purified to a level of 3,500 mouse mean lethal doses per mg of nitrogen. High-titer sera were obtained from rabbits injected with enterotoxin and used to compare the sensitivity of serological tests and bioassays for C. perfringens enterotoxin. Reversed passive hemagglutination was by far the most sensitive test, followed by microslide diffusion, single gel diffusion and electroimmunodiffusion, guinea pig skin test, mouse test, and rabbit ileal loop test.

Animals↗

Staphylococcal enterotoxins A and B: solid-phase radioimmunoassay in food.

An immunoassay employing (125)I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less.

Animals↗

Simplified method for purification of Clostridium perfringens type A enterotoxin.

Purification of Clostridium perfringens type A enterotoxin from sporulated cells was simplified. The method consisted of precipitation of the enterotoxin from the extract of sonically treated cells at 40% saturation of ammonium sulfate at pH 7, differential solubilization in 0.02 M phosphate buffer, pH 6.7, and repeated gel filtration on Sephadex G-200. The purified enterotoxin was at least 98% pure in ultracentrifugation, polyacrylamide gel electrophoresis, and agar gel double diffusion. Recovery was over 74% from the sporulated cell extract. The toxin had biological activities of at least 4,700 mouse intravenous minimal lethal doses/mg of N, 3,900 capillary permeability-increasing U/mg of N in the guinea pig skin, and 210 rabbit intestinal loop distension U/mg of N. The toxin, containing no hexose, lipid, or nucleic acid, appeared to be identical in sedimentation constant, isoelectric point, and ultraviolet absorption spectrum to the toxin purified previously by different procedures.

Ammonium Sulfate↗

Detection of staphylococcal enterotoxin in foods.

A short procedure for the extraction of staphylococcal enterotoxins from food materials has been developed. The procedure involves extraction of the food at pH 4.5, centrifugation, extraction of the supernatant with CHCl(3) (pH 7.5), extraction of the enterotoxin from the water layer with CG-50 ion exchange resin (pH 5.4 to 5.9), and treatment of the eluate with agar and concentration with Carbowax 20-M. The concentrate was extracted with CHCl(3), and the water layer was lyophilized. The dried material was dissolved in a 1% trypsin solution and placed on microslides, which were incubated 24 h at 37 C. The time required for enterotoxin analysis was 3 days with microslides and 1 day with the reversed passive hemagglutination technique.

Chemical Precipitation↗

Pyrogenic specificity of streptococcal exotoxins, staphylococcal enterotoxin, and gram-negative endotoxin.

Streptococcal exotoxin and staphylococcal enterotoxin share several biological properties, including pyrogenicity, lymphocyte mitogenicity, and enhancement of gram-negative endotoxin lethality. These analogies of the toxins prompted comparative pyrogenic studies. When American Dutch rabbits were immunized by repeated intravenous injections of small amounts of staphylococcal enterotoxin, they exhibited a decreased febrile response upon challenge when compared with control animals not previously injected. The same animals responded similarly to control groups when challenged with streptococcal exotoxin A, B, or C. No cross-reactivity was observed in reciprocal cross-tests, using animals immune to the streptococcal toxins and challenging with staphylococcal enterotoxin. No cross-reactivity between either the streptococcal or staphylococcal toxins and bacterial endotoxin was observed.

Animals↗

Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed.

Animals↗

Heat-stable Escherichia coli enterotoxin production in vivo.

Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo.

Animals↗

Mitogenicity of formalinized toxoids of staphylococcal enterotoxin B.

Staphylococcal enterotoxin B is a potent mitogen for mouse and human lymphocytes. Mitogenic activity was retained after detoxification of the enterotoxin by formaldehyde at pH 5.0, 7.5, OR 9.5. The most active toxoid (pH 7.5) was separated into a monomeric, a dimeric, and a polymeric fraction (1 x 10(5) to 3 x 10(5) molecular weight) by gel filtration, and although each fraction demonstrated mitogenic activity, the polymeric fraction was clearly the most efficacious. These data show that mitogenicity of staphylococcal enterotoxin B does not depend on toxicity. This suggests that the mitogenic and toxic activities are effected by different sites on the molecule.

Animals↗

Histopathological effect of Clostridium perfringens enterotoxin in the rabbit ileum.

Highly purified enterotoxin from Clostridium perfringens was found to have histopathological activity in the rabbit ileum. Unlike the action of cholera, Escherichia coli, and Shigella enterotoxins, epithelium was denuded from the tips of ileal villi at concentrations of the enterotoxin necessary to induce fluid accumulation in the rabbit. Whether or not this observed histopathology is essential for the diarrheal syndrome associated with Clostridium perfringens food poisoning remains unclear.

Animals↗

Morphological and steroidogenic changes in cultured adrenal tumor cells induced by a subunit of cholera enterotoxin.

A purified subunit of the cholera enterotoxin molecule was found to have morphological and steroidogenic inducing effects similar to those induced by the native enterotoxin on monolayer tissue cultures of Y1 adrenal tumor cells, although 1,000 times more subunit than toxin (weight basis) was required for maximal effects. In contrast to the whole toxin, the effects of the active subunit could not be prevented by prior incubation with either Gm1 ganglioside or with antibodies directed against choleragenoid (the binding subunit). These results suggest that different receptor sites may exist on cells for the binding and for the active subunits of cholera enterotoxin and/or that the active toxin fragment may exert its effects after gaining access to the intracellular compartment.

Antibodies, Bacterial↗

Plasmid-mediated properties of a heat-stable enterotoxin-producing Escherichia coli associated with infantile diarrhea.

The plasmid mediation and transmissibility of heat-stable enterotoxin production and multiple antibiotic resistance have been demonstrated for Escherichia coli O78:K80:H12 epidemiologically incriminated in a hospital outbreak of infantile diarrhea. The conjugal transfer of a 67 X 10(6) - and a 30 X 10(6)-dalton plasmid was associated with the transfer of resistances and enterotoxin production, respectively. Using antibiotics to select E. coli K-12 transconjugants from a one-step bacterial cross, all of the monitored resistances were transferred concurrently, and 36% of the resistant transconjugants produced enterotoxin.

Animals↗

Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli.

Heat-stable enterotoxin (ST) produced by porcine strains of enterotoxigenic (ENT+) Escherichia coli has been purified to apparent homogeneity by sequential ultrafiltration, acetone fractionation, preparative gel electrophoresis, diethylaminoethyl Bio-Gel A ion-exchange chromatography, and Bio-Gel P-10 gel filtration. The enterotoxin, purified more than 1,500-fold, exhibited a molecular weight of 4,400, as determined by both sodium dodecyl sulfate-gel electrophoresis and gel filtration. A molecular weight of 5,100, representing 47 residues, was calculated from amino acid analysis data. The amino acid content was distinctive, with an unusually high proportion of cystines and few hydrophobic amino acids. A single amino-terminal residue, glycine, was observed. Purified ST was stable to heating (100 degrees C, 30 min) and did not lose biological activity after treatment with Pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, and phospholipase C. Periodic acid oxidation and several organic solvents (acetone, phenol, chloroform, and methanol) had no effect on the biological activity of ST. Further, purified ST was stable to acid treatment at pH 1.0 but lost biological activity at pH values greater than 9.0. Neither lipopolysaccharide nor lipid contamination was evident in purified preparations. A characteristic absorption spectrum was observed during the course of the purification, which shifted from a maximum at 260 nm in crude preparations to 270 nm for the purified toxin. Antiserum obtained from rabbits immunized with ST or ST coupled to bovine serum albumin neutralized the action of the enterotoxin in suckling mice; however, passive hemagglutination and hemolysis titer assays suggested that ST is a poor antigen.

Amino Acids↗

Molar growth yields and enterotoxin B production of Staphylococcus aureus S-6 with amino acids as energy sources.

Staphylococcus aureus S-6 was able to utilize glutamate, proline, histidine, aspartate, alanine, threonine, serine, or glycine as a major energy source, when these were added singly at a concentration of 2.5 g/liter to a base medium containing salts, vitamins, and 30 mg of each of 18 amino acids per liter. Molar growth yields (Y) for each of the eight amino acids were determined in aerobic batch cultures. Except with aspartate and serine, all molar growth yields decreased with increasing concentration of the energy source, whether or not it was present in excess. The average molar growth yields for both aspartate and serine were Y = 35 g/mol. The highest Y values obtained with the other amino acids were 79, 78, 56, 46, 58, and 21 g/mol for glutamate, proline, histidine, alanine, threonine, and glycine, respectively. Histidine was not utilized as an energy source at concentrations of less than 2 mM. Enterotoxin yields (mg of toxin per g [dry wt] of cells) higher than in the basal medium were obtained when proline, histidine, alanine, serine, and glycine were the major energy sources. When proline and alanine were the major energy sources, enterotoxin yields were not affected by the concentration, whereas toxin yields were reduced at high concentrations of the other six amino acids. The highest cell yields and Y values were obtained with glutamate, but its utilization as an energy source markedly reduced enterotoxin yields.

Amino Acids↗

Effect of minerals on staphylococcal enterotoxin B production.

Raising the magnesium level from 0.4 to 1.5 mM in a medium containing only amino acids (2.08%), salts, and vitamins increased enterotoxin B production by Staphylococcus aureus S-6 by about 80%. The level of phosphates in the medium was lowered to one-tenth (to 2.87 mM) the original amount without adversely affecting growth and enterotoxin production. The optimum level of potassium was between 15 and 45 mM. Ammonium salts were not essential in the amino acid medium. Sufficient quantities of trace metals were already present. Revising the salt mixture according to the information obtained resulted in a doubling of the enterotoxin B production.

Enterotoxins↗

Production and partial purification of Salmonella enterotoxin.

By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin.

Bacterial Toxins↗

Swiss and inbred mice in the infant mouse test for the assay of Escherichia coli thermostable enterotoxin.

Swiss and inbred mice (C3H, ASn, B10A, and C57) were used to assay different thermostable enterotoxins prepared from Escherichia coli strains 3 (human), E57 (swine), B41 (cattle), and S13 (sheep). All strains of mice detected enterotoxins, but some reacted better to a given thermostable enterotoxin preparation than other inbred strains. Since the results obtained with Swiss mice were more consistent than with inbred mice, the former are apparently more suitable in the infant mouse test.

Analysis of Variance↗

Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures.

The heat-labile enterotoxin (LT) has been isolated in homogeneous form with high specific activity from three sources: cell-free supernatant, NaCl extract, and whole-cell lysates of an enterotoxigenic Escherichia coli strain. In vitro immunological assays were used in lieu of tedious and highly variable bioassays to recognize fractions with activity. This revealed that the major portion of the LT remained adherent to columns containing agarose, from which it could be eluted quantitatively in practically homogeneous form by galactose. Isolated LT has remarkable similarities to the cholera enterotoxin (choleragen) in both subunit structure and amino acid composition, although there are also notable differences in these two enterotoxins, which are related immunologically and by mode of action. Unlike choleragen, in which the A region is totally nicked, E. coli LT, depending on its source, is activated by proteolytic processing. The activity of LT is equivalent to that of choleragen in bioassays on adrenal cells, in rabbit skin, and in rabbit ileal loops, especially when, depending on the source of material, the LT has been activated by treatment with trypsin. The whole-cell lysate is the richest source of LT.

Amino Acids↗

Escherichia coli heat-stable enterotoxin: rapid method of purification and some characteristics of the toxin.

In this study we used Escherichia coli strain F11(P155) of porcine origin. The heat-stable enterotoxin (ST) was produced with a batch fermentor under agitation (500 rpm) and forced aeration (5 liters/min) in Casamino Acids yeast extract medium containing 0.2% glucose. The pH varied from 7.2 to 7.8. The maximum amount of ST was obtained after 7 h of growth. ST was purified by ammonium sulfate precipitation, ultrafiltration on Amicon membranes, and chromatography in Bio-Gel P-4. The enterotoxin, which was purified approximately 1,000 times, was active at nanogram levels. On 20% polyacrylamide gel electrophoresis, ST exhibited an Rf of 0.6 ST was recovered from the gel slices by eluting in buffer and testing the activity in suckling mice, since no band appeared on the gel after staining with Coomasie brilliant blue R, Schiff reagent, or red oil. ST was resistant at pH 2 to 10 and at 100 degrees C for 15 min, but it was inactivated at 121 degrees C; it did not lose biological activity after treatment with pronase, lipase, or amylase. In suckling mice antiserum obtained from rabbits or goats immunized with ST neutralized the enterotoxin activity of a cell-free supernatant of purified ST.

Animals↗