Identification Of Toxins Research Articles

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome.

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/ 19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (chi2 = 5.42, P < 0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in > 50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C 1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (χ 2=5.42, P<0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in >50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Development of Digoxigenin-Labeled PCR Amplicon Probes for Use in the Detection and Identification of Enteropathogenic Yersinia and Shiga Toxin–Producing Escherichia coli from Foods

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin–producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at −20°C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.

Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.

Identification of diphtheria toxin via screening as a potent cell cycle and p53-independent cytotoxin for human prostate cancer therapeutics.

Metastatic human prostate cancer requires novel therapeutic strategies in order to overcome its low proliferative rate and its resistance to conventional chemotherapeutic agents. To identify potential cytotoxin gene products for use in experimental therapeutics such as in vivo gene therapy, an in vitro screen was designed. Eight recombinant cellular toxins were tested for activity against a spectrum of metastatic human prostate cancer cell phenotypes. Pseudomonas exotoxin A, ricin, tumor necrosis factor alpha (TNF-alpha), diphtheria toxin (DT), Crotalus durissus terrificus toxin, crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin were evaluated. Comparative survival distinguished the relative potencies of these cytotoxins for irreparable prostate cancer cell death. Of the phospholipase A2 toxins, Crotalus durissus terrificus and Naja mocambique are active against the PSA secreting LNCaP cell line; however, the effect is reversible, and no other hormone refractory prostate cell line tested is sensitive. Screening identified toxin-specific differences: dose-dependent cytotoxic activity against all human prostate cancer cell lines tested was only identified for ricin and diphtheria toxin (DT) as highly potent. DT has an IC50 in the range of 20-00 pM by clonogenic survival and kills irreversibly by both apoptosis as well as nonapototic pathways. Acquisition of p53 mutant status conferred no reduction in sensitivity to DT cytotoxicity. Cell cycle arrest by aphidicolin did not protect human prostate cells from irreversible DT-induced cell death. TNF-alpha had modest cytostatic activity in the screen; however, the combination of TNF-alpha and DT resulted in marked acceleration of the time to prostate cancer cell death. The rational screening of cytotoxins allows the identification of cell cycle-independent agents of variable potency against human prostate cancer. DT-mediated cell death is cell cycle independent, and p53 independent, making it particularly attractive for application to cytoreductive gene therapy, targeted monoclonal antibodies, and prodrug delivery of toxins applied to human prostate cancer therapeutics.

Identification and characterization of novel sodium channel toxins from the sea anemone Anthopleura xanthogrammica

Six new toxins from the sea anemone Anthopleura xanthogrammica were identified using a molecular biological approach. Five of these novel isoforms resemble the 47 residue type I long polypeptides native to Anthopleura elegantissima, Anthopleura fuscoviridis and Anemonia sulcata, while one appears to be a chimera of the two previously identified 49 residue toxins native to A. xanthogrammica. Four of these toxins were expressed in bacteria, purified and characterized by ion flux assays in RT4-B and N1E-115 cell lines expressing the cardiac and neuronal Na channel isoforms, respectively. The novel 47 residue toxin isoforms form a new subclass within the A. xanthogrammica neurotoxin family, although they are related to previously described anemone toxins. One of the three 47 residue toxins characterized, PCR2-10, enhances veratridine-dependent sodium uptake, displaying a K 0.5 of 329 nM and 1354 nM in RT4-B and N1E-115 cell lines, respectively. The novel 49 residue toxin, PCR3-7, interacts with the sodium channel with even higher affinity, enhancing sodium uptake with a K 0.5 of 47 nM and 108 nM in RT4-B and N1E-115 cells, respectively.

Stereoselective Separation Combined with Mass Spectrometry: An Indispensable Tool for the Quantification and Identification of Toxins in the Ultra Trace Range

A short survey is given over the main fields of interest of the recently established research group for organic analytical chemistry at the University of Basel. Organic trace analysis and structure elucidation in the pg to ng range are carried out using high resolution gas and chromatography liquid chromatography coupled to single and multiple mass spectrometry. Important ionisation techniques are electrospray, atmospheric chemical ionisation and negative ion chemical ionisation. Beside the development of isomer and enantiomer selective HRGC capillaries and techniques, the techniques mentioned above are applied to problems in the field of environmental analytical chemistry such as the determination and structure elucidation of mycotoxins and pesticides as well as atmospheric polar pollutants. Further fields are the identification of alkaloids in plant extracts and synthetic perfume compounds in detergents.

Caprine enterotoxaemia associated with cerebral microangiopathy

Cerebral microangiopathy is described in naturally occurring clostridial enterotoxaemia in two goats. The two cases were selected from the files of two diagnostic laboratories in Australia, on the basis that...

Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.

Liquid chromatography with fluorimetric, mass spectrometric and tandem mass spectrometric detection for the investigation of the seafood-toxin-producing phytoplankton, Dinophysis acuta

The diarrhoeic shellfish poisoning (DSP) toxins, okadaic acid (OA) and its isomer, dinophysistoxin-2 (DTX-2), were determined in the marine phytoplankton, Dinophysis acuta, harvested in Ireland. Unialgal samples (22–100 cells) were extracted and derivatised using 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP) and analysed by liquid chromatography (LC). Isocratic elution on a C18 reversed-phase column, with fluorimetric detection, was used to determine OA (58±7 pg/cell) and DTX-2 (78±14 pg/cell). The detection limit was 0.1 ng OA/20 μl injection using ADAM. Gradient LC, using a polymeric bonded phase, successfully separated mixtures containing both the ADAM and BAP derivatised toxins. Identification of DSP toxins was confirmed using isocratic micro LC with tandem mass spectrometric (μLC–MS–MS) analysis of the free toxins and μLC–MS of the BAP-derivatised toxins with an ionspray (IS) interface, coupled to an atmospheric pressure ionisation (API) source. Collision induced dissociation (CID) ion mass spectra of the protonated molecule, [M+H]+, at m/z 805 for OA and DTX-2, identified three diagnostic fragment ions for each analyte which were used for selected reaction monitoring (SRM) LC–MS–MS analysis. The detection limit for OA and DTX-2 was 0.025 ng/0.2 μl injected. These studies showed that D. acuta was the progenitor of DTX-2 in shellfish.

Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks

Following four outbreaks of paralytic shellfish poisoning on Kodiak Island, Alaska, during 1994, medical records of ill persons were reviewed and interviews were conducted. Urine and serum specimens were analyzed at three independent laboratories using four different saxitoxin binding assays. High-performance liquid chromatography was used to determine the presence of specific toxin congeners. Among 11 ill persons, three required mechanical ventilation and one died. Mean peak systolic and diastolic blood pressure measurements were 172 (range 128–247) and 102 (range 78–165) mmHg, respectively, and blood pressure measurements corresponded with ingested toxin dose. All four different laboratory methodologies detected toxin in serum at 2.8–47 nM during acute illness and toxin in urine at 65–372 nM after acute symptom resolution. The composition of specific paralytic shellfish poisons differed between mussels and human biological specimens, suggesting that human metabolism of toxins had occurred. The results of this study indicate that saxitoxin analogues may cause severe hypertension. In addition, we demonstrate that saxitoxins can be detected in human biological specimens, that nanomolar serum toxin levels may cause serious illness and that human metabolism of toxin may occur. Clearance of paralytic shellfish poisons from serum was evident within 24 hr and urine was identified as a major route of toxin excretion in humans.

Genotypic identification of staphylococcal enterotoxins and toxic shock syndrome toxin 1 by the enzymatic detection of polymerase chain reaction-amplified DNA

The enzymatic detection of a polymerase chain reaction product (ED-PCR), a new detection method of PCR-amplified DNA, was evaluated for the identification of staphylococcal enterotoxin (SE) and toxic shock syndrome toxin 1 (TSST-1) genes. A total of 61 Staphylococcus aureus strains, including reference strains and strains isolated from clinical specimens and food poisoning outbreaks, were examined by ED-PCR and by reverse passive latex agglutination (RPLA) phenotypic identification. There was 100% agreement between the genotypic and phenotypic identification of SEA, SEB, SEC, SEE strains and TSST-1. In the case of SED, however, 4 strains were positive by ED-PCR and negative by RPLA. ED-PCR offers an accurate alternative to traditional immunoassays or conventional PCR using electrophoresis for the detection of SE and TSST-1 production yielding results that are more precise than with older techniques.

Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan.

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.

Identification of Tetrodotoxin and Paralytic Shellfish Toxins in Marine Gastropods Implicated in Food Poisoning

Identification of Tetrodotoxin and Paralytic Shellfish Toxins in Marine Gastropods Implicated in Food Poisoning

Comparative assessment of the specificity of the brine shrimp and microtox assays to hepatotoxic (microcystin‐LR‐containing) cyanobacteria

AbstractSpecific, straightforward, and rapid procedures are required for the detection, identification, and quantification of the potent low molecular weight toxins that are produced by blooms and scums of cyanobacteria (blue‐green algae) in waterbodies.Use of the Microtox bioluminescence assay and the brine shrimp (Artemia salina) has been advocated for the initial screening of cyanobacterial blooms for microcystin hepatotoxins. Inhibition of bacterial luminescence in the Microtox assay and brine shrimp mortality were determined with microcystin‐containing and nonmicrocystin‐containing cyanobacteria.Extraction and fractionation of test samples was undertaken to select and isolate microcystincontaining fractions and reduce interference from other fractions. Maximal inhibition of bacterial luminescence in the Microtox assay occurred with fractions from Microcystis strains and an Anabaena bloom that did not contain microcystins. By contrast, the bioassay of fractions using brine shrimps correlated with the distribution of microcystin‐LR in the fractionated Microcystis extracts. © 1994 by John Wiley &amp; Sons, Inc..

Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes

The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.

Ciguatera: effective control of a growing health problem

Ciguatera fish poisoning is an important public health problem wherever humans consume tropical and subtropical fish. It accounts for over half of fish-related poisonings in the United States but is uncommonly diagnosed and underreported. Produced by dinoflagellates, ciguatoxin accumulates up the food chain in herbivorous and carnivorous fishes. Cnidaria (jellyfish and related invertebrates) have not previously been associated with direct ciguatera intoxication in humans. We report the first case of ciguatera fish poisoning associated with cnidarian ingestion. A 12-year-old Tongan female presented to our Emergency Department with mid-abdominal pain, nausea, change in mental status, and new-onset movement disorder after ingestion of jellyfish imported from American Samoa. Clinical diagnosis was confirmed by strongly positive serum identification of ciguatoxin and related polyether toxins (including okadaic acid) with a rapid extraction method (REM) and highly reliable solid-phase immunobead assay (S-PIA) performed by the Food Toxicology Research Group, University of Arizona. Ciguatera pathophysiology, clinical presentation, differential diagnosis (including consideration of palytoxin poisoning), and treatment are briefly reviewed. We emphasize the growing incidence of ciguatera fish poisoning outside “high-risk” areas. In regions with immigrant populations, privately imported exotic fish may be toxin vectors. Marine species other than carnivorous fish are now suspect in human ciguatera intoxication. Reliable tests can aid in premarket fish testing, diagnosis, and follow-up of ciguatera fish poisoning. The global prevalence of marine toxins demands fishermen, consumers, and physicians maintain a high index of suspicion for ciguatera fish poisoning.

Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes.

The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.

Pseudomembranous colitis following resection for Hirschsprung's disease

Enterocolitis is the most common cause of significant morbidity and death in Hirschsprung's disease. Although most cases respond to nasogastric decompression, antibiotics, and colonic evacuation, some children have an unusually fulminant or protracted clinical course. Four cases are reported of pseudomembranous colitis (PMC) that developed 1 to 18 months (mean, 8 months) after definitive surgery for Hirschsprung's disease (Soave endorectal pull-though, 2; Duhamel procedure, 2). While all children presented with fever, abdominal distention, and diarrhea, indistinguishable from typical Hirschsprung's enterocolitis, the clinical course was fulminant in two cases, both of whom died of septic shock. Postmortem examination in both showed extensive colonic pseudomembranes despite identification of Clostridium difficile toxin and subsequent vancomycin therapy (initiated late in the clinical course). Two children in the series had protracted hospitalizations and eventually required diverting enterostomy despite recognition of C difficile toxin and treatment with enteral vancomycin, in one child necessitating multiple courses of antibiotic therapy. Awareness of the virulence of PMC associated with Hirschsprung's disease (even after definitive resection) should prompt submission of stool specimens from any child who presents with enterocolitis for both C difficile culture and toxin levels. On the basis of our experience it is our policy to initiate a prompt course of vancomycin by rectal lavage or nasogastric tube in all children with Hirschsprung's enterocolitis, pending culture results, in view of the significant morbidity and mortality exemplified by cases in this review.

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.