A consideration to immune doses of staphlococcal enterotoxin B to rabbits

Immune doses of staphylococcal enterotoxin B to rabbits were studied by comparing the efficiency of various immunization schedules. It was experienced that even two subcutaneous injections each with such a small dose as 10 mug of enterotoxin B, the primary one with Freund's complete adjuvant and a booster one without adjuvant, could stimulate a rabbit to develop the antibody to a satisfactorily high titer determined by agar gel diffusion and passive hemagglutination.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.     

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

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.     

Hemagglutinating activity of the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

Abstract The hemagglutinating activity of the heat-labile enterotoxin (LT_p) isolated from porcine enterotoxigenic Escherichia coli was studied by hemagglutination inhibition. The hemagglutinating activity of LT_p was enhanced 64–512-fold with pronase- and neuraminidase-treated human erythrocytes although both intact human and sheep erythrocytes were not agglutinated by LT_p at the highest concentration used. No enhancement was found in hemagglutination of neuraminidase-treated sheep erythrocytes by LT_p. Hemagglutination of pronase-treated human type A erythrocytes induced by LT_p was inhibited by melibiose and galactose among mono-, di-, and polysaccharides used as inhibitors. Galactose was a slightly better inhibitor than melibiose. These findings suggest that LT_p is a bacterial lectin specific for galactose.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

Characteristics of the heat-stable enterotoxin of NAG vibrios

Cell-free culture filtrate boiled for 15 minutes has been found to retain its biological activity in various experimental models used for the determination of the toxicogenicity of cholera vibrio filtrates. During gel filtration of the concentrated filrate o. NAG vibrio, strain NO. 9852, through Sephadex G-75 toxic activity could be detected in the free volume of the column, which was indicative of the fact that the molecular weight of the thermostable enterotoxin was about 70,000 daltons and greater. The methods of gel diffusion and aggregated hemagglutination have been used to show that the thermostable enterotoxin of NAG vibrio No. 9852 is immunologically unrelated to cholerogen. Some data obtained in experimental models suggest that the thermostable enterotoxin probably differs from cholera enterotoxin in the mechanism of its action.     

Hemagglutinating activity of vibrio cholerae enterotoxin

Abstract The hemagglutinating activity and carbohydrate specificity of cholera toxin (cholera enterotoxin) was studied using hemagglutination and hemagglutination inhibition. Hemagglutination was obtained with cholera toxin at >108 μg/ml for human types A, B, and O erythrocytes, >216 μg/ml for chicken erythrocytes, and >865 μg/ml for sheep erythrocytes. When the erythrocytes were treated with either neuraminidase or pronase, the hemagglutinating activity of cholera toxin was enhanced about 8- to 32-fold. Hemagglutination of pronase-treated human type B erythrocytes induced by cholera toxin was inhibited by lactose, galactose, melibiose and l -arabinose. Lactose was the most effective of the mono-, di-, and polysaccharides used as inhibitors, being a slightly better inhibitor than galactose, and much more potent than melibiose. These results suggest that cholera toxin is a bacterial lectin specific for galactose and/or lactose.     

Haemagglutinin production by enterotoxigenic strains of Clostridium perfringens type A

Thirty-nine enterotoxigenic cultures of Clostridium perfringens type A were studied for enterotoxin and haemagglutinin production. Enterotoxin was quantitated by sandwich ELISA and DOT-ELISA techniques and haemagglutinin titres were determined using sheep and human erythrocytes. Haemagglutinins from only six cultures reacted against both sheep and human erythrocytes; a further 13 reacted only against human erythrocytes, and another five only against sheep cells.The authors are with the Department of Veterinary Public Health and Epidemiology, Ranchi Veterinary College, Birsa Agricultural University, Ranchi-834007 (Bihar), India.     

Identification and purification of a new staphylococcal enterotoxin, H.

A staphylococcal enterotoxin which elicited an emetic response in monkeys but did not share antigenic determinants with any of the identified enterotoxins was identified and purified from Staphylococcus aureus FRI-569. The emetic activity of this new enterotoxin was neutralized only by antibodies specific to it and not by antibodies to enterotoxins A, B, C, D, and E or toxic shock syndrome toxin 1. Immunodiffusion assays did not detect cross-reactivity between this new and all the other identified enterotoxins. The purification procedure involved removal of the enterotoxin from culture supernatant fluids by batch adsorption with CG-50 resin, CM-Sepharose FL ion-exchange chromatography, and Sephacryl 100 HR and Bio-Gel P-30 gel filtration. The molecular weight of this enterotoxin, 27,300, determined by gel filtration on Sephacryl 100 HR agreed with the molecular weight, 28,500, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent migration of this enterotoxin determined by SDS-PAGE did not shift in the presence of a disulfide reducing agent, indicating that it is composed of a single-chain protein. The N-terminal amino acid sequence of the enterotoxin was determined to be Glu-Asp-Leu-His-Asp-Lys-Ser-Glu-Leu-Thr-Asp-Leu-Ala-Leu-Ala-Asn-Ala-Tyr- Gly- Gln-Tyr-Asn-His-Pro-Phe-Ile-Lys-Glu-Asn-Ile, which did not match the N-terminal sequences of any known proteins. The isoelectric point of the enterotoxin determined by isoelectric focusing was about 5.7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved Reversed Passive Hemagglutination for Simple and Rapid Detection of Staphylococcal Enterotoxins A~E in Food

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunoglobulins of anti-A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti-A~E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin-producing strains. No non-specific reactions with various ingredients in foods nor cross-reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 μg/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%. Under routine laboratory practice, detection and identification of enterotoxins from incriminated foods of five food poisoning outbreaks were performed by RPHA within 3 hr after reception of the specimens. Among them, three were determined to be enterotoxin A food poisoning, one to be toxin C and the rest to be intoxication of A and D. The concentration of the toxins was between 0.014 and 3.65 μg per gram of food.     

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enteroxigenic Escherichia coli

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.     

Effect of pH, Protein Concentration, and Ionic Strength on Heat Inactivation of Staphylococcal Enterotoxin B

Heat treatment of highly purified staphylococcal enterotoxin B causes a more rapid loss of immunological activity at 70 to 80 C than at 90 to 100 C. Toxicological results based on intravenous injection of dogs paralleled the results obtained by immunological means (single gel diffusion). The loss of immunological activity did not follow first-order kinetics. Results are given on the effects on heat inactivation of changing pH, ionic strength, and initial concentration of enterotoxin. Disc-gel electrophoresis of purified enterotoxin B showed a major and minor band. The minor band was a size isomer of the major band.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

The identification and purification of multiple forms of theta-haemolysin (theta-toxin) of Clostridium perfringens type A.

The theta-haemolysin of Clostridium perfringens was purified from culture supernatant fluids of type A strains by fractional ammonium sulphate precipitation and isoelectric focusing in narrow pH 5 to 8 gradients. Four components detected on electrofocusing were designated theta-1(pI6-8to6-9),theta-2(pI6-5to6-6),theta-3(pI6-1to6-3) and theta-4(pI5-7to5-9). Specific activities ranged from 0-4 x 10-6 to 1-2 x 10-6 haemolytic units/mg protein and 2950 to 3600 LD-50/mg protein. Each haemolytic component was activated by cysteine hydrochloride, and inactivated by cholesterol, by addition of sheep erythrocyte ghosts and by heating at 60 degrees C for 10 min; mouse erythrocytes were more resistant than sheep erythrocytes to haemolysis. A reaction of identity was obtained between components in gel diffusion. Sodium dodecyl sulphate polyacrylamide discgel electrophoresis gave molecular weights in the range 59,000 to 62,000 for each component. A similar value was obtained for theta-1 on density gradient ultracentrifugation. Although the multiple forms were free of 11 factors present in culture supernatants, crossed immunoelectrophoresis and disc gel electrophoresis revealed minor contaminants. These studies reveal that theta-haemolysin has physical properties in common with other oxygen-labile haemolysins.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

Detection of staphylococcal enterotoxin in foods.

A method has been developed for the detection of staphylococcal enterotoxin A in the boiled rice extract. The procedure utilized was the batch adsorption of enterotoxin from the cell-free culture supernatant by CG-50 ion exchange resin at pH 5.6. The enterotoxin was eluted by various concentrations of elution solution with different pH values. The lyophilized eluate was dissolved in Phosphate Buffer Saline (PBS) solution and analyzed with a quantitative double diffusion method. The desorption of enterotoxin from ion exchange resin appeared to be less effective by increasing the concentration of elution solution than by elevating the pH value of elution solution. The pH below 6.2 seemed to lose the ability to elute the enterotoxin from ion exchanger but enough to elimate non-specific extra proteins. The quantitative double diffusion method was able to detect enterotoxin in food with approximation in quantitation.     

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.     

Identification of Staphylococcal Hemolysins by an Electrophoretic Localization Technique

A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha-hemolysin but lysed only rabbit cells. This latter lysin was tentatively named alpha(1)-lysin. This strain of S. aureus also produced beta-hemolysin which migrated 36 mm towards the cathode and lysed sheep cells. beta-Hemolysin produced by some strains of S. aureus showed considerable tailing during electrophoresis, whereas beta-hemolysin produced by other strains of S. aureus migrated as a well-defined peak. A lysin migrating 11 mm towards the anode was probably delta-lysin. It was, however, not produced in sufficient concentration when the cultures were grown in semisolid medium.