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Verotoxin (VT), which is immunologically unrelated to VT1 (Shiga-like toxin I), was purified from the culture filtrate of Escherichia coli hemorrhagic colitis serogroup O157:H7 strain 3657 by copper ion chelate affinity chromatography followed by anion-exchange chromatography. The isoelectric point by sucrose density gradient isoelectric focusing was 5.0, the molecular weight by gel filtration on Superose 12 was about 60,000, and the 50% cytopathic dose for Vero cells was about 1 pg. This toxin was found by immunological methods to be the predominant VT in E. coli O157 isolates associated with illness in North America, with 38 of 42 strains tested producing this toxin, 20 in combination with VT1. VT from strain 3657 is immunologically identical to the described Shiga-like toxin II (VT2) of E. coli strains (from the United States) K-12(pEB1) and C600(933W) but only partially related to VT of strain E32511 (from the United Kingdom), the first to be named VT2.

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A simple and rapid method is described for detecting as little as 1 ng each of staphylococcal enterotoxins A, B, and C2 in 100 mL samples of food extracts. Samples were fractionated on copper chelate Sepharose 6B and the recovery of added staphylococcal enterotoxin was measured by the reversed passive latex agglutination test. An approximate 100 fold concentration of toxin was obtained. The method should prove useful as a partial purification and concentration step in the analysis of foods incriminated in food poisoning outbreaks.

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An external quality assessment survey for staphylococcal enterotoxin A, B, and C2 determinations was performed in the collaborative study. Three analysts in two laboratories took part. Three types of food, cheese, salami, and pasta, were artificially contaminated with either one toxin only or all three toxins. A total of 378 analyses were performed. The group mean of the analytical values corresponded fairly well to the given enterotoxin concentrations. However, a considerable interlaboratory imprecision was found despite analyses being performed with the same reagents and methodology.

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Staphylococcus aureus growth, thermostable nuclease (TNase) and enterotoxin production in inoculated canned salmon incubated at 22 ± 1°C for 4 d were dependent on the size of inoculum, and on the amount of oxygen present in the headspace; under nitrogen with an inoculum of 7 cfu/can, 102-103 cfu/g, no TNase and traces of enterotoxins (A, B, C2) were observed; under oxygen with the same inoculum ≥109 cfu/g, ≥6.0 µg TNase and up to 5.2 µg total enterotoxins (A, B, and C2)/100 g of salmon were observed. Values were intermediate under atmospheric air. After 1 week, 2 months and 4-24 months of incubation of salmon under nitrogen, S. aureus cfus were 108, 106 and 104-105 per g; TNase ranged from trace amounts to 20 µg/100 g and total enterotoxins from <1.0 µg to 6.2 µg/100 g. In canned sardines stored from 1 d to 12 months at 22 ± 1°C, levels were 109 cfu/g and 3.7-3.9 µg total enterotoxins/100 g; after 1 week, counts declined to 105 cfu/g but total enterotoxins remained relatively stable in some cans with up to 6.2 µg/100 g of sardines after 12 months. TNase varied from <1.0 µg to 20 µg/100 g of salmon with 109 and 105 cfu/g, respectively. In sardines, similar variation in TNase was observed and there was no correlation between TNase, enterotoxins and cfu/g. After 2 d to 24 months, carbon dioxide, an acidic smell and unacceptable odors were detectable over the headspace of S. aureus contaminated salmon and sardines, but not all persons who sniffed the contaminated products could recognize off-odors that would warn them against consuming the food. To prevent canned foods from causing staphylococcal illness, the conditions allowing post-process contamination should be eliminated by the producer and distributor of the products.

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The results from two studies are reported of the effects on mental performance of omitting breakfast. The objective of the first study was to compare the performances of schoolchildren who habitually ate or did not eat breakfast. In the second study the effects of omitting breakfast by those accustomed to eating the morning meal were investigated. 2. Mental performance was assessed by two short-term memory tests (a simple cancellation test in which paired letters were marked on a page or random letters) and a memory-search test in which lines containing a group of specified letters were marked, a series of numerical additions, and an attention-demanding test (in which specified statements had to be verified). 3. Neither study revealed differences attributable to the omission or consumption of breakfast.

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A simple solid-phase radioimmunoassay was developed for detecting staphylococcal enterotoxin A (SEA) in food. The method detected 85-100% SEA added to extracts of 6 kinds of foods, and 52-100% SEA added directly to foods at a concentration of 1 ng/g. Assay sensitivity is about 0.3 ng toxin/mL extract. The method is specific for SEA; results are only marginally affected by staphylococcal enterotoxins B and C2 present at concentrations as high as 500 ng/mL extract.

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Five heat-labile, partially purified toxic products of Escherichia coli were distinguished by isoelectric focusing, molecular weight, and neutralization with homologous and heterologous antisera. Only two affected the morphology of Y-1 cells, induced fluid accumulation in rabbit ileal loops, and stimulated production of cyclic AMP in Vero cells; these two did not cross-neutralize and only one showed cross-neutralization with cholera antitoxin. The remaining three products were cytotoxic for Vero cells.

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Isoelectric focusing of a heat-labile cytotoxin of Escherichia coli H30 revealed the presence of two molecular variants, pI 7.2 and a comparatively small quantity of pI 6.8. Predominant component pI 7.2 had a molecular weight of 28,000, induced some fluid accumulation in rabbit ileal loops, and showed no morphological response in Y-1 cells but a strong cytotoxic effect on Vero cells.

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Solid-phase radioassay was applied to the determination of protein A after release from staphylococcal cells with lysostaphin. Assays can be completed in 60-90 min.

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The gastric juice of a patient showing symptoms of staphylococcal food poisoning was examined by a radioimmunoassay for the presence of enterotoxins. Assays gave markedly higher results at 35 degrees than at 5 degrees. The source for this discrepancy was attributed to interference due to trypsin activity on the basis of (1) the demonstration of hydrolysis of p-toluenesulfonyl-L-arginine methyl ester by the specimen, (2) inhibition of this activity by trypsin inhibitor from lima bean, and (3) lowered values produced for enterotoxins in gastric juice when the inhibitor was included in the assay system.

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A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxin C2 is described. The assay procedure employs anti-rabbit gamma globulin, prepared in goats, to precipitate the antigen-antibody complex of enterotoxin C2 and anti-enterotoxin C2. The test is sensitive to 100 pg of enterotoxin.

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A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.

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The last three steps described in the preceding communication Robern, H., Stavric, S. and Dickie, N. (1975) Biochim. Biophys. Acta 000,000-000) were utilized successfully for the purification of enterotoxin A. The enterotoxin appeared homogeneous by gel chromatography and in the analytical ultracentrifuge but showed two bands on polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.12 mug/kg of animal weight. After treatment with beta-mercaptoethanol, urea and sodium dodecylsulfate enterotoxin A formed one band corresponding to a molecular weight of about 27700 on sodium dodecylsulfate gel electrophoresis. Molecular-weight estimation by gel filtration gave a value of 26500. The amino acid composition data indicate that enterotoxin A consists of 241 amino acid residues.

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