Rapid detection and quantitation of Clostridium perfringens enterostoxin by counterimmunoelectrophoresis.

Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 microgram of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific and easy for the detection and quantitation of enterotoxin.     

Rapid detection and quantitation of Clostridium perfringens enterostoxin by counterimmunoelectrophoresis.

Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 microgram of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific and easy for the detection and quantitation of enterotoxin.     

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.     

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.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

A quantitative study of enterotoxin production by sheep milk staphylococci.

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Staphylococcal Enterotoxin C: Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of entertoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less.     

Double-antibody radioimmunoassay for staphylococcal enterotoxins A and B

A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxins A and B is described. The separation of the primary antigen-antibody complex of enterotoxin A and B was achieved with an anti-rabbit gamma globulin from goats. Radioiodinated aggregate fractions of staphylococcal enterotoxins exhibited reduced immunological activity and showed little competition with non-radioactive exterotoxin. The radioimmunoassay was successfully applied for the quantitation of enterotoxins in food.     

Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimnunoassay, using staphyloccal cells containing protein A as immunoadsorbent.

A sensitive radioimmunoassay utilizing Staphylococcus aureus cells containing protein A as a coprecipitant was developed for the detection and quantitation of staphylococcal enterotoxins A, B, C, D, and E in a variety of foods. The enterotoxins were extracted from the foods by a simple and rapid procedure. The sensitivity of the assay is 1.0 ng or less of enterotoxin per g of food.     

A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for Cl. perfringens producing small amounts of CPE.     

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.     

TonB-independent ferrioxamine B-mediated iron transport in Escherichia coli K12

Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.     

Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11

Abstract Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.     

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

Staphylococcal Enterotoxin B: Solid-Phase Radioimmunoassay

An immunoassay employing (125)I-labeled enterotoxin B and polystyrene tubes coated with specific antibody was used for assaying purified and crude enterotoxin. Antibody was adsorbed to untreated polystyrene tubes. Unlabeled enterotoxin competed with (125)I-labeled enterotoxin for antibody-combining sites. The uptake of (125)I-labeled toxin reflected the concentration of unlabeled toxin present. The test is sensitive to 1 to 5 ng of purified and crude enterotoxin B per ml, and cross-reactions with heterologous enterotoxins did not interfere with the specificity. This test possesses the combination of sensitivity and objectivity absent in current methods for assaying enterotoxin and provides a model for investigating other enterotoxin serotypes.     

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.     

Effect of Meat and Isolated Meat Proteins on the Thermal Inactivation of Staphylococcal Enterotoxin B

The thermal inactivation of staphylococcal enterotoxin B was studied in a phosphate-saline buffer, in the presence of two meat proteins, myosin and metmyoglobin (MetMb), and in a ground-beef slurry. When enterotoxin B was incubated at temperatures from 60 to 110 C, it was shown that the initial thermal inactivation at 80 C was faster than at 100 or 110C. The heating of enterotoxin B at 60, 80, and 100 C in the presence of either myosin or MetMb resulted in a rapid loss of the enterotoxin. Thermal loss of the enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B in a ground round slurry was rapid when compared to inactivation in a phosphate-saline buffer. The rapid loss of enterotoxin B in the slurry may be due to a combination of thermal inactivation and the binding of enterotoxin molecules to meat proteins.     

Identification of a new enterotoxin as enterotoxin C

Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481-1485. 1965.-Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain.     

Endogenous radiolabeling of enterotoxin from Clostridium perfringens type A on a defined medium.

Four enterotoxin-positive strains of Clostridium perfringens were tested for sporulation and enterotoxin production on defined media. The medium described by Sacks and Thompson (Appl. Environ. Microbiol. 35:405-409, 1978) gave the highest enterotoxin production and was selected for the production of endogenously labeled enterotoxin. The specific radioactivity of the enterotoxin was 16,000 dpm/microgram when the tritiated amino acids were added to the growth medium just before the inoculum. Addition of the radioactive amino acids during the growth period gave consistently lower specific radioactivity. When the enterotoxin was produced on the medium described by Duncan and Strong (Appl. Microbiol. 16:82-89, 1968), the highest specific radioactivity of the enterotoxin was found when the radioactive amino acids were added to the growth medium 4 h after inoculation. In this case, the specific activity of the enterotoxin was 10,000 dpm/microgram.