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PubMed1983

Rapid diagnosis of "Escherichia coli" heat labile enterotoxin (LT) by a coagglutination test.

Protein A positive Staphylococcus aureus cells, strain Cowan 1, were coated with a high titer anti-LT serum produced in rabbits. Purified E. coli heat labile enterotoxin (LT) was detected in a slide coagglutination (coa-LT) test with anti-LT coated staphylococci at the nanogram level (0.1-1 ng) while cholera toxin (CT) was detected only at much higher concentrations (10-50 ng). This LT immunoassay was evaluated in different modifications on enterotoxigenic E. coli (ETEC) isolated from humans and animal stool cultures. Colonies from primary stool cultures on blood agar were suspended and lysed in saline containing polymyxin B and a detergent (Triton X100), centrifuged and assayed for LT. The coa-LT and CHO-cell tests regularly detected LT among human LT producing ETEC, while ETEC strains from piglets and calves usually gave only weak reactions in the standard coa-LT test. We conclude that this coa-LT test is accurate and sensitive enough to be used in routine diagnosis of LT producing ETEC strains in stools from human patients. This test requires no special laboratory equipment and is well suited for rapid diagnosis of ETEC in small hospital laboratories and in developing countries. Heat stable enterotoxin (ST) giving a positive suckling mouse test, has been purified by hydrophobic interaction chromatography (HIC) on Octyl Sepharose. Purified ST was covalently linked to different proteins and such carrier complexes used for immunization of rabbits. The ST antibody titers obtained by various ST-carriers were compared in a ST coagglutination test (ST-coa) and in a conventional ELISA microtiter assay. The results and detection limits for ST in polymyxin-detergent cell lysates in human ETEC strains producing LT-ST or ST only will be presented.

Agglutination Tests↗
PubMed1983

[Model UF-2 mutant of Staphylococcus aureus 209 P for titrating Vibrio cholera enterotoxin].

The identical character of the action of crude V. cholerae enterotoxin on the anaerobic dehydrogenases of the UV-2 mutant of S. aureus 209 p and the surviving culture of Ehrlich's carcinoma has been revealed. The range of this action is linked with the concentration of the toxin and varies from the stimulation of cell dehydrogenases to their complete suppression. The rapid method for the titration of the enterotoxin in the dehydrogenase suppression test with the use of the bacterial model is proposed.

Animals↗
PubMed1983

[Isolation of staphylococcal enterotoxin type B and a study of its physicochemical, biological and immunological properties].

The homogeneous preparation of type B staphylococcal enterotoxin (SEB) has been obtained by its adsorption from the culture fluid on amberlite, elution, isoelectric focusing in the ampholine system and gel filtration in a column packed with Sephadex G-25. The isolated SEB component is a thermostable protein, homogeneous in its antigenic composition and according to the results of disc electrophoresis in acrylamide gel. Its molecular weight is 28,000 daltons with pI equal to 8.6 (the main component), 8.0 and 8.25 (the minor components). All the forms are immunochemically identical. The minimum enterotoxic dose of SEB for cats is 3-5 micrograms per kg of body weight. The biological activity of the enterotoxin disappears under the action of 6 M urine. Monospecific antiserum to SEB with a precipitating antibody titer of 1:64 has been obtained.

Animals↗
PubMed1984

[Determination of cholera enterotoxin in supernatants of homogenates from the wall of the small intestine in rabbits in the passive immune hemolysis reaction].

The article presents the data indicating that suckling rabbits can be used as a model, specific only for enterotoxin-producing Vibrio eltor strains. The signs characteristic of low virulence may appear as the consequence of the action of other products resulting from the vital activity of vibrios. No strains capable of producing toxin only in vivo or in vitro have been detected. The quantitative correlation of the synthesis of enterotoxin in the body of experimental animals and in vitro has been established. The passive immune hemolysis test can be used as an objective method suitable for the determination of the choleragenic potency of vibrios.

Animals↗
PubMed1984

[Occurrence and identification of enterotoxin-producing E. coli strains in milk and dairy products].

Enteric diseases caused by enterotoxigenic E. coli strains (ETEC) become more and more important all over the world. Frequently food is implicated as a vector. --From this the necessity arises to identify ETEC strains and/or their heatlabile (LT) and heat-stable enterotoxins (ST). The examinations carried out for the presented paper showed the following results: Detection of LT by coagglutination is a simple and rapid test and may be helpful for screening. Most suitable, however, is the application of ELISA which enables the detection of 1 ng/ml LT with a good reproducibility. For identification of ST the suckling mouse assay (SMA) yields reliable results. ELISA and SMA have proved to be useful in practice for examination of food samples. By means of these methods 157 E. coli strains predominantly isolated from milk and milk products were examined. Hereby, five ETEC strains could be identified (3.2%). For the purpose of food examination a procedure was developed which ensures a reliable and efficient identification of E. coli enterotoxin. The essential steps are enrichment in E.E.-broth, subcultivation in CAYE-2-broth, identification of LT by ELISA resp. coagglutination and the detection of ST with aid of the SMA. So, isolation of single colonies can be omitted, which is an essential advantage.

Agglutination Tests↗
PubMedDate not supplied

Detection of Escherichia coli enterotoxins by using mouse adrenal cell and suckling mouse assays: collaborative study.

The ability of 10 Escherichia coli strains to produce heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) was determined by 8 analysts in a collaborative study. The suckling mouse model and the mouse adrenal cell line (Y-1) tests were used to detect ST and LT, respectively. Cultures for assay were grown 24 h in casamino acid-yeast extract-trace salts broth at 37 degrees C in a shaker incubator at 250 rpm. Cell-free culture broth prepared by centrifugation and filtration was divided into 2 portions: One was heated for 30 min and used both for ST assay and as a heated control for LT assay; the other was used unheated for LT assay. Results were expressed as positive for ST, positive for LT, positive for ST and LT, or negative for both ST and LT; percent of correct estimates was calculated for each culture for each analyst. At the 95% confidence interval, the overall correct results were 96.3 +/- 2.9 and 95.0 +/- 3.4% for ST and LT, respectively. The test performances thus were satisfactory for detecting ST and LT produced in vitro by E. coli. The method has been adopted official first action.

Adrenal Glands↗
PubMed1983

The use of the Biken test to detect enterotoxigenic "Escherichia coli" producing heat labile enterotoxin.

For identification of enterotoxigenic Escherichia coli producing heat labile enterotoxin (LT), various assay methods have been developed. However, many of these assay methods are unsuitable for routine clinical purposes, especially in developing countries, because they need special materials or techniques or both, such as a large number of animals, stocks of special tissue culture cells, and radioisotopes. Recently, we developed a simple and reproducible assay method, Biken test, which can be widely used in clinical laboratories in both developing and developed countries (15). This method is based on the principles of the Elek test and Ouchterlony double gel diffusion test. The Biken test was used in a field study in Bangladesh with good results (14). Moreover, the Diarrheal Diseases Control Programme, WHO, organized a multi-laboratory study to evaluate the accuracy and reproducibility of the Biken test, in which 5 investigators tested an identical set of 100 unknown strains of E. coli, using the Biken test and at least one other assay method, which includes CHO cell assay, Y1 adrenal cell assay, GM1 ganglioside ELISA, DNA probe test and passive immune hemolysis. The results reported by Sutton (International Symposium on Bacterial Diarrheal Diseases, Osaka, 1982) showed that the Biken test was a reliable assay method. To evaluate further the Biken test, we tested a total of 2,229 strains of E. coli isolated from diarrheal patients. The results showed 99.0% coincidence with those obtained by CHO cell assay. The use of anti-LT antiserum gave sharper and clearer results than cholera antitoxin. Samples for heat stable enterotoxin (ST) assay were obtained from the agar plates used for the Biken test and the ST activities of these samples were compared with those of samples obtained from standard liquid cultures. Results on samples of 2,229 strains from the Biken agar plates and from liquid cultures were almost identical.

Animals↗
PubMed1984

Cytotoxin and enterotoxin production by Clostridium difficile.

30 strains of Cl. difficile isolated from faeces of patients with pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAC) and other intestinal disorders and from faeces of asymptomatic carriers were studied for production of toxins. Tissue culture assay was used for the detection of cytotoxin (toxin B) and ileal loop test for enterotoxin (toxin A). All Cl. difficile isolates from patients with PMC and AAC were found to produce cytotoxin, whereas enterotoxin was demonstrated only in approximately 70% of strains.

Animals↗
PubMed1984

[Action of Vibrio cholerae enterotoxin on microorganisms].

The damaging of V. cholerae enterotoxin on anaerobic dehydrogenases of different species of opportunistic microorganisms has been revealed. Experiments, carried out both in vivo and in vitro, have shown that under certain conditions this enterotoxin may act as one of the factors of the intra- and interspecific antagonism of bacteria.

Animals↗
PubMed1984

[Cold resistance of S. aureus and staphylococcal enterotoxins A and C2 in ice cream].

Studied was the cold resistance of enterotoxinogenic strains of Staphylococcus aureus and of A and C2 staphylococcus enterotoxins in milk and cream icecream kept under refrigerator conditions for 7 months at -18 degrees C. The S. aureus strains were shown to have high resistance in icecream. The survival rate of the organisms depended both on the specific peculiarities of the individual strains and on the composition of the icecream mixture. Staphylococci were found to survive longer in milk icecream. A and C2 enterotoxin retained fully their activity in the two types of icecream over the entire period of storing. The data obtained is important for the elucidation of the epidemiology of alimentary staphylococcus intoxications.

Cold Temperature↗
PubMed1983

Staphylococcal enterotoxin induced mitogenesis: toxin binding and cell-cell interactions.

The binding characteristics of 125I-labelled staphylococcal enterotoxin A (125I-SEA), a T-cell mitogen, to murine lymphoid cell subpopulations were analyzed. Both T- and B-lymphocytes from murine spleens possess specific binding sites for SEA, as do T-lymphocytes from thymus. B-lymphocytes appear to have a greater capacity for binding of 125-SEA than do T-lymphocytes from either thymus or spleen. Enterotoxin did not specifically bind to thioglycollate-induced peritoneal exudate cells (PECs), used as a source of macrophages. Adherent PECs however, incorporated 125-ISEA by fluid phase endocytosis. When exposed to SEA and thoroughly washed, macrophages stimulate lymphocyte mitogenesis in spleen or thymus cell cultures not directly exposed to toxin. Maximum mitogenic stimulation took place only when both PECs and lymphocytes were exposed to SEA. The presence of splenic B-lymphocytes enhanced the mitogenic response of thymus derived T-cells to SEA. Thus, B-lymphocytes appear to contribute to SEA mitogenesis. These data suggest that mitogenic stimulation and possibly other immunological phenomena associated with SEA occur as a result of complex interactions between cellular components of the immune system.

Animals↗
PubMed1983

[Animal experimental studies of passive oral immunization against staphylococcal enterotoxin B (SEB)].

Intoxications by S. aureus enterotoxins are predominant in our country, together with enteric infections by Salmonella spp. For lack of a specific therapy the possibility was examined to neutralize orally provided toxin in vivo. For this purpose before and after the intoxication, respectively, antitoxin and normal rabbit serum as a control were also applied per os. In extensive trials SPF-mice were used as a model in spite of their insensitivity to staphylococcal toxins even with 10000 times the emetic dose for monkeys. By serological means, the rate of free toxin in stomach, intestinal contents and fecal matter after and before application of antiserum was identified as an indicator for the neutralization of the toxin in vivo. As toxin, S. aureus enterotoxin B (SEB) was used which could be identified in the samples up to three hours after oral feeding. Antiserum was obtained by immunization of rabbits with toxoid. The application of antiserum one hour after intoxication resulted in an average reduction of free toxin in the intestine of 87,5 and 86,5%, (10 independent trials) one and two hours, respectively after serum administration. In contrast, administration of antiserum one hour before experimental intoxication did not produce statistically confirmed differences between the experimental and the control animals.

Administration, Oral↗
PubMed1982

Effect of berberine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in jejunum of pigs.

Intraluminal perfusion with Escherichia coli heat-stable enterotoxin (ST) reversed water and electrolyte movements from net absorption to net secretion in porcine jejunal segments. Addition of berberine hydrochloride (3.2 X 10(-5) M) to the perfusate reduced the jejunal secretory response of water, sodium, potassium, and chloride to ST and enhanced water and electrolyte absorption in control segments. At lower concentrations (1.1 X 10(-5) M), berberine reduced the secretory response in ST-exposed segments, but only the decrease of sodium flux was significant. In the presence of berberine, the mucosal enzyme activities of adenosine triphosphatase and disaccharidases were not significantly different between control and ST-exposed segments. Doses of 1, 2, 3, 4, 5, and 10 mg of berberine were injected into ligated loops of proximal part of the jejunum with 1 ml of ST filtrate. At doses of 2 or more mg/loop, berberine was effective in reducing water and electrolyte secretions induced by ST; the effect was dose-dependent. These findings indicate that berberine may be an effective antidiarrheal agent in E coli heat-stable enterotoxin mediated secretory diarrhea and provide a basis for the frequent empirical use of berberine alkaloid and berberine-containing plants in gastroenteritis and infectious diarrhea in Asian and other countries.

Animals↗
PubMed1980

Purification and characterization of heat-stable enterotoxin produced by a strain of E. coli pathogenic for man.

Escherichia coli heat-stable enterotoxin derived from a strain pathogenic for man has been purified 13,000-fold to apparent homogeneity. The purification scheme involved growth in a minimal medium. Amberlite XAD-2 chromatography, acetone fractionation, Sephadex G-25 filtration, DEAE-Sephacel ion exchange chromatography, and Sephadex G-25 gel filtration. This scheme resulted in a white flocculent material which was biologically active in 2.7-ng quantities. Heat-stable enterotoxin was homogeneous as determined by the following: (a) a single peak on gel filtration; (b) a single band on thin layer chromatography; (c) a single band on thin layer electrophoresis; (d) a single NH2-terminal amino acid residue, asparagine; and (e) an amino acid composition demonstrating a stoichiometric relationship among the amino acids. The molecule is composed of 10 different amino acids, a total of 18 amino acid residues, one-third of which are half-cystine. The molecule contains no detectable carbohydrate. Biological activity is promptly lost on treatment with the reducing reagents, 2-mercaptoethanol or dithiothreitol, or after performic acid oxidation, suggesting the presence of disulfide bridges which are required for biological activity.

Amino Acids↗
PubMed1980

[Possibility of obtaining the anaform of E. coli enterotoxin].

The nontoxic (on the model of raw edema in white mice) anaform of E. coli enterotoxin was obtained under the action of heat and formalin. In the process of detoxication the charge of the enterotoxin molecules was changed. The toxoid thus obtained preserved the antigenic activity of the initial preparation and, when used for the immunization of rabbits, stimulated the production of active antitoxic sera.

Animals↗
PubMed1982

[Use of stationary-growth broth cultures for obtaining Escherichia enterotoxins. I. Study of the preparations from reference strains of enterotoxigenic Escherichia].

Experiments with standard enterotoxigenic E. coli (ETEC) strains 06:H16, 015:H11, 078:H11, 0148:H28 and 26 nonenterotoxigenic enterobacterial strains used for control were carried out. As a result, the preparation, simple in manufacture and containing thermolabile enterotoxin (TLE) and thermostable enterotoxin (TSE), was obtained: the supernatant fluid (SF) of 48-hour ETEC culture grown at 37 degrees C in flasks with Hottinger's broth without shaking. the possibility of using SF obtained by the centrifugation of the cultures 2 times for 1 hour at 8,000-10,000 r. p. m. was shown. The comparison with the control preparation indicated that the SF thus obtained possessed high biological activity and induced specific reactions in the paw edema test in mice for detecting TLE and in the anal test in suckling mice for detecting TSE.

Animals↗
PubMed1982

[Use of stationary-growth broth cultures for obtaining Escherichia enterotoxins. II. Study of Escherichia preparations isolated from diarrhea patients].

The preparation, simple in manufacture and consisting of the supernatant fluid (SF) of 48-hour cultures grown in flasks with Hottinger's broth without shaking, was shown to be suitable for the detection of enterotoxigenic E. coli (ETEC) in diarrhea patients. SF of the cultures of 1206 E. coli strains isolated from 809 adults, together with control preparations, were studied by means of anal test in suckling mice, used to detect thermostable enterotoxin (TSE), and the paw edema test, used to detect thermolabile enterotoxin (TLE). These SF contained highly active TSE and having specific action. The use of SF ensured the high frequency of ETEC detection in patients with different intestinal diseases (in 262 out of 430 patients), placed in the same hospital. The specificity of these results and the relation of the isolated ETEC to the diseases diagnosed in the patients were confirmed by the fact that the frequency of ETEC detection was significantly less in healthy persons (in 22 out of 242 subjects).

Adult↗