Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

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.     

Identification of Salmonella enterotoxin by coagglutination reaction

It has been shown that the intensity of enterotoxin production by various strains of Salmonella is different (it ranges from + to +). Thus, in addition to adhesive properties and skin permeability factors, the ability of various Salmonella strains to produce enterotoxin is one of the pathogenic factors of these microorganisms. Further biological characteristics of the infective agents will promote the detection of epidemiologically significant types of microorganisms during outbreaks of toxicoinfections.     

Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine

Mucin secretion in situ from rat intestinal loops was promoted more effectively by dialysed crude cholera filtrate than by an equivalent amount of purified enterotoxin. The filtrate could be rendered inactive by incubation with mixed gangliosides or passage through a GM1-affinity column, which indicated that the secretory action of the filtrate depended upon its enterotoxin component. In an effort to explain the greater potency of the filtrate, we established the presence of a metalloproteinase in the filtrate and demonstrated that this enzyme was capable of degrading purified rat intestinal mucin. Sufficient degradation occurred to cause a substantial decrease in viscosity (57% in 120 min). Biochemical analysis of the mucin before and after exposure to filtrate revealed a rise in the combined percentage of serine, threonine and proline (53-58%), suggesting that poorly glycosylated areas (which are less abundant in these amino acids) were being partly removed from the mucin. The carbohydrate composition was essentially unaltered. Inhibition of the filtrate metalloproteinase by Zincov and alpha 2-macroglobulin significantly (P less than 0.005) reduced the ability of cholera filtrate to degrade mucin or to stimulate mucin secretion from rat intestinal slices in vitro. Purified cholera enterotoxin added to enterotoxin-depleted filtrate was a more potent secretagogue (secretory stimulant) in intestinal loops than an equivalent amount of enterotoxin alone. We therefore propose that mucin secretion induced by cholera filtrate is caused by cholera enterotoxin, but that degradation of the protective epithelial mucus layer by a constituent metalloproteinase may assist the toxin by allowing increased access to mucosal GM1 receptor sites.     

Production of Escherichia coli Heat-labile Enterotoxin in Fermenter Dialysis Culture

Production of Escherichia coli heat-labile enterotoxin was investigated with one porcine and one human strain in three different media under different cultivation conditions. Cultivation in aerated fermenters at pH 7·0 yielded 10–20 times more enterotoxin/ml of culture fluid than cultivation in shake flasks. A trypton-yeast extract medium was optimal in fermenter cultures. Comparatively good yields of enterotoxin in fermenters were also obtained in a glucose-salts medium. Continuous feeding of glucose and salts during fermenter cultivation resulted in a lower production of enterotoxin/mg of bacterial cells. Since this decrease in specific yield could be reversed by using dialysis culture, it was concluded that inhibition of toxin formation was due to the accumulation of extracellular low molecular weight metabolites. The highest yield of enterotoxin in dialysis culture was 80 ED₅₀ ml⁻¹ (rabbit jejunal loop test) which is at least eight times more toxin than in ordinary fermenter culture and 80 times more toxin than in shake flask cultures.     

Induction of apoptosis in HT29 human intestinal epithelial cells by the cytotoxic enterotoxin of Aeromonas hydrophila.

The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen. In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA. Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate. Cytoplasmic blebbing and nuclear condensation also occurred. DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis. These results show that the cytotoxic enterotoxin of A. hydrophila can induce apoptosis in human intestinal cells in culture.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Effect of cloned Salmonella typhimurium enterotoxin on rabbit intestinal motility

Abstract We analysed the small intestine myoelectric responses of anesthetized New Zealand albino rabbits to Escherichia coli lysates containing an enterotoxin cloned from Salmonella typhimurium . Migrating action potential complex, which consisted of rapid bursts of action potentials and secretion of fluid, was observed only in ileal loops injected with the enterotoxin-containing lysate. Migrating action potential complex produced by Stn usually propagated aborally, which was typical of cholera toxin, but orad or bidirectional propagation occurred from a single point of origin when activity was intense. Gell lysates from an E. coli clone containing vectors alone, as well as proximal control segments injected with phosphate-buffered saline, gave neither a change in motility nor fluid secretion. These results show that Stn caused dramatic changes in intestinal motility and substantial fluid production.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin

Fourteen strains of Bacillus cereus isolated from different sources were examined for their ability to produce diarrhoeal enterotoxin by two commercial immunoassay kits (Oxoid BCET-RPLA and Tecra ELISA) and the microslide immunodiffusion assay. One strain that was positive in monkey feedings, as well as a number of other strains isolated from diarrhoeal outbreaks, gave positive results in the ELISA and negative results in the RPLA test systems. When tested in the microslide assay, these strains produced only one antigen which formed a line of identity with the reference toxin. The results of the control toxins provided with the kits substantiated that the two commercial assays did not detect the same antigen. Cultures positive with both assay kits were shown to produce diarrhoeal enterotoxin (by a line of identity) and other antigens in the microslide immunodiffusion assay.     

A rapid, colourimetric assay for cytotoxin activity in Campylobacter jejuni

Abstract Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.     

Identification of functionally active fragments of staphylococcal enterotoxin B.

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulin-dependent enzymes and is probably the toxicogenic part of the enterotoxin.     

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays.

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Salmonella enterotoxin (stn) gene is prevalent among strains of Salmonella enterica, but not among Salmonella bongori and other Enterobacteriaceae

Abstract All strains and serovars of Salmonella enterica such as serovar Typhimurium, Enteritidis, Dublin, Typhi, etc. were found to carry the Salmonella enterotoxin determinant stn as far as examined in PCR and hybridization studies. However, using MDCK cells for testing the toxicity of the strains under investigation, only a limited number of stn positive strains revealed phenotypically the Salmonella enterotoxin Stn. In contrast to S. enterica , other Enterobacteriaceae including Salmonella bongori were found neither genotypically nor phenotypically Stn toxin positive.     

Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks

Seventy-six Salmonella enteritidis , three Salmonella virchow and one Salmonella braedenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis: This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM₁ ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [³H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.