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.     

Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli.

The genes for a new enterotoxin were cloned from Escherichia coli SA53. The new toxin was heat labile and activated adenylate cyclase but was not neutralized by antisera against cholera toxin or E. coli heat-labile enterotoxin. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on polyacrylamide gels that are similar to those of cholera toxin and E. coli heat-labile enterotoxin subunits. A 0.8-kilobase DNA probe for the new enterotoxin failed to hybridize with the cloned structural genes for E. coli heat-labile enterotoxin.     

Major outer membrane proteins in the phytopathogenic bacteria Erwinia carotovora subsp. carotovora and subsp. atroseptica

Abstract Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Characterization of immunogenic p230 as the toxin A of Clostridium difficile

Abstract For the identification of toxin A of Clostridium difficile , a 2-dimensional gel system was used. In its first dimension, samples were separated in the absence of reducing and dissociating agents, conditions which maintained the activity of the enterotoxin. This was followed by reduction and dissociation in the second dimension where a 230 kDa polypeptide was electroeluted. Rabbits were immunized with polyacrylamide gel slices containing entrapped native toxin A and the denatured 230 kDa protein. As revealed by immunoblotting, neutralizing antisera derived from native protein samples recognized the native toxin, the denatured 230 kDa protein and another polypeptide of about M _r 35 000. Using both types of antisera as probes the pI of the enterotoxin was about 5.9. Preliminary evidence suggests that the enterotoxin is a multimeric protein of 230 kDa and 35 kDa subunits.     

Cross-neutralization reactions of Escherichia coli and Vibrio cholerae enterotoxins as studied by rabbit skin inoculation test.

Neutralization of enterotoxins of Vibrio cholerae 569 B and Escherichia coli 10407 by antitoxins to V. cholerae 569 B, E. coli 334, 408-3 and 10407 was studies by intradermal inoculation test in the rabbit. Neutralization of V. cholerae enterotoxin by homologous as well as heterologous antisera of E. coli was observed, except that there was no neutralization of the enterotoxin by antiserum to E. coli 408-3 enterotoxin. Neutralization of E. coli enterotoxin to a varied extent by homologous as well as all heterologous antisera, including that of V. cholerae 569 B antitoxin, was also observed.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Immunochemical characteristics of the subunits of cholera enterotoxin and of thermolabile Escherichia coli enterotoxins of various origins

Antigenic determinants of subunits A and B of cholera enterotoxin (CT), heat-labile enterotoxin from the human E. coli strain (hLT) and heat-labile enterotoxin from the porcine E. coli strain (pLT) were analysed by Ouchterlony double gel-diffusion test against antisera to B subunits of three toxins and antisera to three holotoxins. The results have shown the existence of the following antigenic determinants: in subunits B-1. antigenic determinants, common for B subunits of all three enterotoxins-B(chp); 2. group antigenic determinants, common for B subunits of two toxins in the pair-B(ch), B(hp); 3. antigenic determinants, unique for B subunits of each CT, hLT, pLT-(B(c), B(h), B(p); in subunits A.-1. antigenic determinants, common for A subunits of all three enterotoxins-A.(chp); 2. group antigenic determinants, common for A subunits of two enterotoxins (hLT and pLT/-A(hp); 3. antigenic determinants, unique for A subunit of CT-A(c). On the basis of these results antigenic formulas for subunits of CT, hLT, pLT were proposed.     

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.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

Relationship Between Staphylococcal Antiserum Titer and Zone Development on Immune Serum Plates

A workable relationship was established between the standard serum titers of staphylococcal immune antisera and the development of precipitin zones on serum agar around colonies of staphylococcal strains producing homologous antigens (enterotoxins). The standard titer of a serum is defined as the reciprocal of that serum dilution which, with 10 mug of pure enterotoxin per ml, will give a precipitin zone 10 mm in length in single gel-diffusion tubes after 7 days of incubation at 25 C. A numerical scale was set up for determining the intensity of precipitin zones on serum agar. A reading of 3 was considered optimum. This correlated well with a standard serum titer of 25, when 1 ml of such a serum was used per 20 ml of medium per serum plate. From this relationship, the minimum volume of serum required to give optimum precipitin zone development can be calculated.     

Raising Antibodies to Staphylococcal Enterotoxins A and B

A method is described for raising specific antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) by intravenous inoculation of rabbits with small doses of enterotoxin diluted in sterile physiological saline. A course of six injections over a period of two weeks was given on four occasions. After the third course, 3/5 rabbits given SEA had a titre ± 1/50 and 4/6 given SEB had a titre of 1/50. Titres were not appreciably enhanced by the fourth series of injections. Only minor non-specific reactions which would require no absorption were found at the end of the series. It is believed that the findings in this study would be reproducible and that the method is likely to be suitable for raising antisera to other enterotoxins. Reference is also made to preliminary experiments in which latex sensitized with SEA or SEB was inoculated intravenously into rabbits.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.     

Heterogeneity of Staphylococcus aureus enterotoxin B as a function of growth stage: implications for surveillance of foods.

Staphylococcus aureus was grown in a fermentor under controlled conditions of pH, oxygenation, and temperature, while the higher-molecular-weight products of its growth were continuously removed across ultrafiltration membranes. These products were examined by single and double gel diffusion and immunoelectrophoresis against a variety of available anti-enterotoxin B antisera. All antisera examined were polyvalent for S. aureus antigens. However, two electrophoretically distinct proteins were the major reactants with the antisera. One of these was present in early- to mid-log-phase cultures. After mid-log growth was achieved, both were present but in continuously changing proportions. This observation was repeated with a variety of growth conditions and media. A significant part of the physicochemical heterogeneity of enterotoxin B observed over the past 20 years is thus correlated with the growth phase of the organism. Taken together, these facts are used to argue for a two-step rationale for the detection of food-borne staphylococcal disease: (i) screening for a presumptive hazard by analysis for any antigen, toxin, or enzyme of S. aureus in a foodstuff and (ii) confirmation of the hazard by identifying the presence of an enterotoxin using a combination of physicochemical and serological techniques.     

Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody.

A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification.     

Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification

Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity.     

Heterogeneity of Staphylococcus aureus enterotoxin B as a function of growth stage: implications for surveillance of foods.

Staphylococcus aureus was grown in a fermentor under controlled conditions of pH, oxygenation, and temperature, while the higher-molecular-weight products of its growth were continuously removed across ultrafiltration membranes. These products were examined by single and double gel diffusion and immunoelectrophoresis against a variety of available anti-enterotoxin B antisera. All antisera examined were polyvalent for S. aureus antigens. However, two electrophoretically distinct proteins were the major reactants with the antisera. One of these was present in early- to mid-log-phase cultures. After mid-log growth was achieved, both were present but in continuously changing proportions. This observation was repeated with a variety of growth conditions and media. A significant part of the physicochemical heterogeneity of enterotoxin B observed over the past 20 years is thus correlated with the growth phase of the organism. Taken together, these facts are used to argue for a two-step rationale for the detection of food-borne staphylococcal disease: (i) screening for a presumptive hazard by analysis for any antigen, toxin, or enzyme of S. aureus in a foodstuff and (ii) confirmation of the hazard by identifying the presence of an enterotoxin using a combination of physicochemical and serological techniques.     

Production and Evaluation of Antibody to the Heat-Stable Enterotoxin from a Human Strain of Enterotoxigenic Escherichia coli

Escherichia coli heat-stable enterotoxin was coupled to bovine serum albumin by a carbodiimide reagent. Antibody to the conjugate was produced by immunization of rabbits. Data from radioimmunoassay and infant mouse tests indicate the presence of antibody to the enterotoxin. The antisera can be used in a radioimmunoassay to measure enterotoxin in various fluids.     

Comparative structural analysis of staphylococcal enterotoxins A and E

Structural analysis of staphylococcal enterotoxins A and E, two functionally and serologically related proteins, has been carried out using circular dichroism, and tryptophan fluorescence quantum yield and quenching. Secondary structures derived from the far-UV circular dichroic spectra revealed that both enterotoxins are in predominantly beta-sheets/beta-turn structures (80-85%). Staphylococcal enterotoxin A has significantly higher alpha-helical content (10.0%) than staphylococcal enterotoxin E (6.5%). Tryptophan fluorescence spectra of both enterotoxins showed maxima at approximately 342 nm, indicating that the fluorescent tryptophan residues are in polar environments. However, the tryptophan fluorescence quantum yields indicated that tryptophan residues are approximately 41% more fluorescent in staphylococcal enterotoxin A than in staphylococcal enterotoxin E. Tryptophan fluorescence quenching by a surface quencher, I-, and a neutral quencher, acrylamide, indicated that at least 1 of the 2 tryptophan residues in both staphylococcal enterotoxins A and E is located on the outer surface of the proteins. This tryptophan residue is in significantly different environments in the two enterotoxins. Six antigenic sites are predicted from the hydrophilicity and secondary structure information; at least four sites are identical. In general, staphylococcal enterotoxins A and E have some structural similarities which are compatible with their common biological activities.