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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.

Animals↗

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.

Animals↗

Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells.

Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.

Amino Acid Sequence↗

The pathogenesis of Shigella diarrhea. I. Enterotoxin production by Shigella dysenteriae I.

A strain of Shigella dysenteriae 1, freshly isolated from a patient with dysentery in Guatemala in August 1969, was found to elaborate an enterotoxin into the liquid of broth cultures. Partial purification of the enterotoxin by ultrafiltration on graded polymeric membranes and Sephadex gel filtration (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) suggested an approximate molecular weight of 55,000-60,000. The partially purified toxin was heat-labile, pronase sensitive, and activated by alkaline pH, and it elicited fluid production in ligated rabbit ileal segments; it failed, however, to cause increased vascular permeability in skin. When the activities of equal weights of identically prepared Vibrio cholerae and S. dysenteriae enterotoxins were compared in the rabbit ileum the latter caused a significantly smaller volume response with increased concentrations of potassium, chloride, and protein. The previously described neurotoxic (mouse lethal) factor was also present and eluted from Sephadex G-150 with the enterotoxin. If these biological activities prove to be possessed by a single molecular species, it is suggested that it be renamed Shigella enterotoxin in recognition of the physiologically more relevant biological action.

Animals↗

Effects of cholera enterotoxin on adenosine 3',5'-monophosphate and neutrophil function. Comparison with other compounds which stimulate leukocyte adenyl cyclase.

Cholera enterotoxin caused a delayed accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in human leukocytes, associated with an increase in leukocyte adenyl cyclase activity. The action of cholera enterotoxin contrasted with that of other agents which stimulate adenyl cyclase: (a) the effects of the toxin were delayed in onset, while prostaglandin-E(1) (PGE(1)) and isoproterenol acted rapidly; (b) removal of the soluble toxin from the extracellular medium did not abolish its effects on cyclic AMP and inhibition of antigenic histamine release, while removal of PGE(1) did prevent its effects; (c) PGE(1), but not cholera enterotoxin, stimulated adenyl cyclase activity when added directly to broken cell preparations. Binding of the toxin to leukocytes was rapid and irreversible, and was followed by a gradual increase in cyclic AMP which was not prevented by cycloheximide. Cholera enterotoxin caused accumulation of cyclic AMP in purified human neutrophils as well as mono-nuclear cells, but did not prevent the extrusion of lysosomal hydrolases from phagocytic cells. The toxin only slightly inhibited the ability of human neutrophils to kill Candida albicans. Thus these results with the toxin cast doubt on previous proposals that cyclic AMP regulates these two functions of neutrophils. The unique action of cholera enterotoxin on cyclic AMP production provides a potentially useful pharmacologic tool, in addition to methylxanthines and dibutyryl cyclic AMP, for testing hypotheses relating cyclic AMP to altered function of leukocytes and, perhaps, of other mammalian cells.

Adenylyl Cyclases↗

Effect of Escherichia coli on fluid transport across canine small bowel. Mechanism and time-course with enterotoxin and whole bacterial cells.

An Escherichia coli strain isolated from a patient with severe cholera-like diarrhea elaborates a partly heat-labile enterotoxin shown to cause prompt adenyl cyclase stimulation and isotonic fluid secretion by canine jejunum. Both responses disappear upon removal of the enterotoxin. The duration of action of a submaximal dose of this E. coli enterotoxin was brief, despite sustained exposure to the jejunum, suggesting inactivation of the enterotoxin by its interaction with the mucosa. Inoculation of whole bacterial cultures of this E. coli strain into canine duodenum was followed by bacterial survival and induction of net secretion after 4-7 h. The onset of fluid production was associated with increasing gut mucosal adenyl cyclase activity. Washed bacterial cells could also produce fluid secretion. In vivo multiplication of this enterotoxin-producing E. coli was demonstrated 6-12 h after intraduodenal inoculation of approximately 10(6) organisms. This was associated with fluid secretion. Intestinal fluid production occurred without microscopic pathology in the mucosa.

Adenylyl Cyclases↗

Detection of staphylococcal enterotoxin H by an enzyme-linked immunosorbent assay.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of a newly identified staphylococcal enterotoxin H (SEH). Peroxidase was conjugated to antibodies specific to the enterotoxin. 2,2'Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)(ABTS) in hydrogen peroxide solution was used as the enzyme substrate. A standard curve of purified SEH was prepared with concentrations ranging from 1.3 to 50 ng/ml. SEH at levels equal to 2.5 ng/ml and higher were detected by this procedure. Culture supernatant from the growth of selected Staphylococcus aureus strains was analyzed by using the ELISA. SEH was produced by three of 20 strains that produced one identified enterotoxin. Ten of 21 strains, previously shown to produce substances that induced emesis in monkeys but not any known enterotoxins (A through E), were also positive for SEH production. The other 11 strains gave negative results in the ELISA, indicating that other unidentified serological types of enterotoxin exist.

Animals↗

Staphylococcal enterotoxin D production by Staphylococcus aureus FRI 100.

Staphylococcus aureus FRI 100 is commonly used as a control strain for staphylococcal enterotoxin A (SEA) assays. When FRI 100 was used in PCR-based enterotoxin detection methods, the strain gave a positive result for both SEA and staphylococcal enterotoxin D (SED). Production of SED was confirmed by testing concentrated and unconcentrated culture supernatants with the TECRA staphylococcal enterotoxin visual immunoassay. SED was detected after 24 h of growth in Trypticase soy broth. Primers were created to amplify the entire sed gene by PCR for subsequent sequencing. The sequenced gene showed high similarity to a previously sequenced sed gene. The SED-like gene in FRI 100 exhibited four point mutations and two deletions. Changes in the FRI 100 open reading frame altered the primary structure of the SED-like protein, allowing for coding of only the first 150 amino acids followed by a stop codon. Because the SED active site is at the proximal end, where there was no change in DNA sequence, we conclude FRI 100 produces a variant form of SED. It is necessary to note that, when using FRI 100 as an SEA control strain, it does produce a variant of the SED protein, which exhibits immunological activity, and the sed-like gene is detected by commonly used PCR primers. This phenomenon may be an important general consideration when using PCR to characterize strains of toxin-producing S. aureus. S. aureus enterotoxin-positive PCR results should be confirmed by immunological techniques.

Amino Acid Sequence↗

Adenylate cyclase in intestinal crypt and villus cells: stimulation by cholera enterotoxin and prostaglandin E1.

The secretory responses to cholera enterotoxin and prostaglandin E1 (PGE1) are dependent upon elevation of the intracellular levels of cyclic AMP. Although several previous reports have suggested that intestinal secretion due to cholera enterotoxin and PGE1 may be confined to the crypt cells, this matter has been incompletely resolved. These studies were undertaken to define the activity of adenylate cyclase in villus and crypt cells from rabbit and rat intestine, and to determine the influence of enterotoxin and PGE1 on this activity, Mucosal fractions were prepared from rabbit ileum with a planing device, and from rat distal small intestine by a vibration technique. In both species base line adenylate cyclase activity was greater in crypt than in villus cells. After exposure to cholera enterotoxin in vivo, adenylate cyclase activity was enhanced in all fractions prepared from rabbit ileum, and the response was most marked in villus cells. Furthermore, adenylate cyclase in membranes prepared from both rat villus and crypt intestinal cells was responsive to the in vitro addition of PGE1. The results of these studies indicate that both villus and crypt cells contain one of the important components required for the cyclic AMP-mediated secretory response, namely, a cholera enterotoxin and PGE1-sensitive adenylate cyclase activity. Since an increased level of cyclic AMP alone may not be sufficient to evoke a secretory response, these studies do not clarify the extent to which each of these major cell types may participate in cyclic AMP-mediated secretion.

Adenylyl Cyclases↗

[Evidence of enterotoxins and toxic shock syndrome toxin (TSST-1) in strains of Staphylococcus aureus isolated from various biological materials].

Among 413 strains of S. aureus isolated from patients with chronic staphylococcal infections, from haemocultures in bacteriaemia and septic conditions, from gynaecological materials and nasal plugs of healthy carriers the authors detected the production of one or several types of enterotoxins in 124 strains (30.0%), production of TSST-1 in 19 strains (4.6%) and the concurrent production of enterotoxins and TSST-1 in 38 strains (9.2%). The highest ratio of toxigenic strains of S. aureus was found in departments (51.4%) and from hospitalized patients in blood cultures in bacteriaemias and septic conditions (41.2%). In a group of 184 strains from patients with chronic staphylococcal infection toxin production was proved in 49 strains (26.6%). Of 74 strains of S. aureus isolated from healthy carriers there were 16 toxigenic strains (21.6%). The most frequent type of enterotoxins were enterotoxins type A (26.7%) and C (24.4%). 21 strains (24.4%) produced more than one type of enterotoxins.

Bacterial Toxins↗

[Detection of enterotoxin genes in Bacillus thuringiensis by PCR].

The presence of hemolysin HblA (hblA), enterotoxin BceT (bceT) and enterotoxin S (entS) genes from 45 strains of B. thuringiensis, 4 strains of B. cereus and B. sphaericus have been detected respectively by multiple PCR. The results showed that 95.6% B. thuringiensis strains contain the B component of hblA gene, 91.1% and 93.3% of them contain bceT and entS genes sequences respectively. The enterotoxin productions in all strains have also been analysis using two commercial immunoassay kits(TECRA and RPLA) and it has proved that most of B. thuringiensis stains and the positive B. cereus strain can produce entero toxins during their growth. However, the two hblA sequence positive stains, DBT007 was negative when tested both by RPLA and Tecra, T24 001 was negative when assayed by Tecra and positive by RPLA. One hblA sequence negative strain Dmu39 was negative when tested by RPLA but positive by Tecra. No enterotoxin and enterotoxin gene could be detected in B. thuringiensis DBT248 and the B. sphaericus strains. The results suggest that the potential risk of using B. thuringiensis, as biopesticide needs to be further evaluated.

Bacillus↗

First proficiency testing to evaluate the ability of European Union National Reference Laboratories to detect staphylococcal enterotoxins in milk products.

The European Commission has designed a network of European Union-National Reference Laboratories (EU-NRLs), coordinated by a Community Reference Laboratory (CRL), for control of hygiene of milk and milk products (Council Directive 92/46/ECC). As a common contaminant of milk and milk products such as cheese, staphylococcal enterotoxins are often involved in human outbreaks and should be monitored regularly. The main tasks of the EU-CRLs were to select and transfer to the EU-NRLs a reference method for detection of enterotoxins, and to set up proficiency testing to evaluate the competency of the European laboratory network. The first interlaboratory exercise was performed on samples of freeze-dried cheese inoculated with 2 levels of staphylococcal enterotoxins (0.1 and 0.25 ng/g) and on an uninoculated control. These levels were chosen considering the EU regulation for staphylococcal enterotoxins in milk and milk products and the limit of detection of the enzyme-linked immunosorbent assay test recommended in the reference method. The trial was conducted according to the recommendations of ISO Guide 43. Results produced by laboratories were compiled and compared through statistical analysis. Except for data from 2 laboratories for the uninoculated control and cheese inoculated at 0.1 ng/g, all laboratories produced satisfactory results, showing the ability of the EU-NRL network to monitor the enterotoxin contaminant.

Algorithms↗

[Assessment of the probability of encountering staphylococcal enterotoxins in lactic acid cheese packaged in laminates].

Immunoassay methods were used to identify the presence of staphylococcal enterotoxins in lactic acid cheese vacuum and non-vacuum packed. There was assessed the probability of encountering staphylococcal enterotoxin in cheese dependent on different systems of packaging, count of staphylococcal cells, intensiveness of coagulase synthesis and tightness of packaging. The presence of enterotoxin was identified in 5% of researched samples of products stored for 14 days. The influence of packaging system and tightness on presence of enterotoxin was observed. The probability of presence of staphylococcal and enterotoxin in relation to researched factors was presented by the mathematical models.

Animals↗

Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products.

A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning. Two new cpe-specific labeled DNA probes were evaluated using Southern and dot blot hybridization. Bacterial DNA was amplified by a duplex PCR procedure. The results showed that 40 enterotoxin producing C. perfringens strains generated two bands of amplicons with sizes of 420 and 280 bp, whereas 40 non-enterotoxin producing strains produced a single band of 280 bp on agarose gel-electrophoresis. No bands were observed from 32 strains of Clostridium spp and other bacteria. Southern blot analysis using either cpe-specific DNA or oligonucleotide probe showed hybridization specifically to the 420 bp band in enterotoxin-positive C. perfringens. On the dot blot membrane, both cpe-specific DNA and oligonucleotide probes were able to hybridize specifically with the corresponding DNA templates but with different efficacy (100% vs 91.1%).

Blotting, Southern↗

[The occurrence and the importance of enterotoxins from strains of Escherichia coli from bovine mastitis].

Baby mouse and Y1 cell culturing tests were conducted into 117 Escherichia (E.) coli strains from mastitis of cattle to clear up enterotoxin formation. The rate of the latter was found to be relatively low, with enterotoxins recorded from only 7 strains (6%). No correlations were established between enterotoxin formation, on the one hand, and antibiotic resistance as well as biochemical activities of E. coli isolates, on the other. The question still remains open, if enterotoxin-forming strains derive their udder pathogenicity from endotoxins or with support by enterotoxins.

Adrenal Gland Neoplasms↗

Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea.

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.

Animals↗

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.

Animals↗

Complete amino acid sequence of staphylococcal enterotoxin A.

The amino acid sequence of staphylococcal enterotoxin A is presented. Staphylococcal enterotoxin A is a single-chain polypeptide which consists of 233 amino acid residues with a molecular weight of 27,078 and has the amino acid composition Cys2, Asp17, Asn19, Thr16, Ser13, Glu15, Gln12, Pro4, Gly15, Ala7, Val13, Met2, Ile10, Leu23, Tyr18, Phe8, His6, Lys24, Arg7, Trp2, with serine as both amino- and carboxyl-terminal amino acids. Automated sequence analysis of intact enterotoxin A, as well as characterization of the peptides obtained from cyanogen bromide treatment and trypsin and chymotrypsin digestion, led to the elucidation of the complete primary structure of this protein. Less structural homology is observed among staphylococcal enterotoxins A, B (Huang, I-Y., and Bergdoll, M. S. (1970) J. Biol. Chem. 245, 3518-3525), and C1 (Schmidt, J. J., and Spero, L. (1983) J. Biol. Chem. 258, 6300-6306) than that seen between enterotoxins B and C1.

Amino Acid Sequence↗